Crossover Trial of Purified Gluten and Whole Wheat (Gluten With Amylase-trypsin Inhibitors) as Triggers of Gastrointestinal Symptoms in Patients With Irritable Bowel Syndrome
Phase: NA
Conditions: Irritable Bowel Syndrome
Sponsor: McMaster University

Study No 09.25.NRC 
CONFIDENTIAL 
Page 1 of 77 
 
                                                                        
 
 
STUDY PROTOCOL 
 
 
Project HiREB ID: 4367 
ClinicalTrials.gov Trial No: NCT03664531 
 
 
Dietary triggers of gastrointestinal symptoms in IBS patients 
 
 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 2 of 77 
RESPONSIBLE PERSONNEL 
Principal Investigator 
Name: Premysl BERCIK 
Title: MD, Associate Professor, 
Department of Medicine. 
Function: Principal Investigator. 
Institution: McMaster University 
Address: 1280 Main Street West, HSC 
3N9, Hamilton, ON L8S4K1 
Tel: +1 905 521 2100/ ext: 73495 
Fax: +1 (905) 521 4958 
bercikp@mcmaster.ca 
 
 
………...….......……….… 
Date 
 
 
........................................ 
Signature 
Co-Investigator 
 
Name: Elena VERDU 
Title: MD, Associate Professor 
Department of Medicine.    
Institution: McMaster University 
Address: 1280 Main Street West, HSC 
3N8, Hamilton, ON L8S4K1 
Tel: +1 905 527 4323/46043 
Email: verdue@mcmaster.ca 
 
 
………………………… 
Date 
 
 
..................................... 
Signature 
Co-Investigator 
Name: Stephen M. COLLINS 
Title: MD, University Professor 
Department of Medicine 
Institution: McMaster Universtity 
Medical Centre 
Address: 1200 Main Street West, HSC 
4W8, Hamilton, ON L8N 3Z5 
Canada Tel: +1 905 525 2100/8452 
Email: scollins@mcmaster.ca 
 
 
………………………… 
Date 
 
 
..................................... 
Signature 
Co-Investigator 
 
Name: Paul MOAYYEDI  
Title: MD, Professor  
Department of Medicine. 
Institution: McMaster University Health 
Sciences Centre  
Department of Medicine 
Hamilton, ON 
+1 905.525.2100 / 76764 
Email: moayyep@mcmaster.ca 
 
 
………………………… 
Date 
 
 
..................................... 
Signature 
Co-Investigator 
 
Name: M. Ines PINTO SANCHEZ 
Title: Assistant Professor 
Department of Medicine 
Institution: McMaster Universtity 
Medical Centre 
Address: 1280 Main Street West, 
Hamilton, Canada. 
Tel: +1 905 525 9140/ 21875 
Email: pintosm@mcmaster.ca 
 
 
………………………… 
Date 
 
 
..................................... 
Signature 
 
 
Co-Investigator 
 
Name: Andrea NARDELLI 
Title: MD, Research fellow 
 
 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 3 of 77 
Department of Medicine 
Institution: McMaster University Medical 
Centre 
Address: 1280 Main Street West, 
Hamilton, Canada. 
Tel: +1 905 525 9140/ 21875 
Email: nardela@mcmaster.ca 
………………………… 
Date 
 
 
..................................... 
Signature 
Co-Investigator 
Name: Detlef SCHUPPAN 
Title: MD, Professor 
Department of Medicine 
Institution: Johannes Gutenberg 
University 
Address: Langenbeckstrabe 1D, Mainz, 
Germany. 
Tel: +49 6131 17-7356 
Email: detlef.schuppan@unimedizin-
mainz.de 
 
 
………………………… 
Date 
 
 
..................................... 
Signature 
Co-Investigator 
Name: Gaston RUEDA 
Title: MD, Research Fellow 
Department of Medicine 
Institution: McMaster University Medical 
Centre 
Address: 1280 Main Street West, 
Hamilton, Canada. 
Tel: +1 905 525 9140/ 21267 
Email: tongasrueda@gmail.com 
 
 
………………………… 
Date 
 
 
..................................... 
Signature 
Co-Investigator 
Name: Pedro MIRANDA 
Title: PhD, Research Fellow 
Department of Medicine 
Institution: McMaster University Medical 
Centre 
Address: 1200 Main Street West, 
Hamilton, Canada 
Tel: +1 (905) 515 6989 
Email: mirandp@mcmaster.ca 
 
 
………………………… 
Date 
 
 
..................................... 
Signature 
 
Co-Investigator 
Name: Rajka BOROJEVIC 
Title: Research Assistant 
Department of Medicine 
Institution: McMaster University Medical 
Centre 
Address: 1280 Main Street West, 
Hamilton, Canada. 
Tel: +1 (905) 525 9149 
Email: borojevi@mcmaster.ca 
 
 
………………………… 
Date 
 
 
..................................... 
Signature 
 
Study Coordinator  
Name: Caroline SEILER 
Title: PhD Candidate 
Department of Medical Sciences 
Institution: McMaster University Medical 
Centre 
 
 
………………………… 
Date 
 
 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 4 of 77 
Address: 1280 Main Street West, 
Hamilton, Canada. 
Tel: +1 905 525 9140/ 21875 
Email: seilercl@mcmaster.ca 
..................................... 
Signature 
 

Study No 09.25.NRC 
CONFIDENTIAL 
Page 5 of 77 
SYNOPSIS 
Dietary triggers of gastrointestinal symptoms in IBS patients 
Principal Investigator: Premysl BERCIK 
Study center: McMaster University Medical Centre, Hamilton, Canada. 
 
Primary objective and outcome: 
The primary objective of this clinical trial is to evaluate the effect of two possible dietary 
triggers (whole wheat flour and non-purified gluten containing amylase trypsin inhibitors 
(ATIs) and purified gluten) on gastrointestinal symptoms in IBS patients who have been on 
a gluten-free diet for 3 or more weeks. 
Secondary objectives and outcomes are to evaluate the effect of dietary triggers on 
ü IBS symptoms (IBS-SSS) 
ü Stool frequency and consistency (Bristol stool scale) 
ü General GI symptoms (PROMIS scales for bloating & gas, diarrhea, 
constipation, and belly pain) 
ü Anxiety, depression, and stress (DASS21) 
ü Somatic symptoms (PHQ-15) 
ü Gastrointestinal transit time (SHAPE study) 
ü Gut microbiota composition (16S rRNA) 
ü Systemic immune activation (isolated PBMC assay) 
ü Daily diet pictures (MealLogger) 
ü IBS symptom duration (IBS-SSS) 
ü Long-term gluten-free diet adherence 
ü Psychological and GI symptoms after learning diet triggers and genetic 
results 
Design: 
This is a prospective, randomized, placebo-controlled, crossover design. 
Number of patients (to be enrolled / to be analyzed): 
40 patients will be enrolled such that the final number of patients analyzed will be at least 
33 patients (+20% dropout)  
Description of subjects and main criteria for inclusion: 
Patients aged 18+, diagnosed with IBS (Rome IV criteria) whose symptoms previously 
improved while on a gluten-free diet. 
Intervention: 
Test product: muesli bars containing 1) whole wheat flour and non-purified gluten 
containing ATIs (10 g), 2) purified gluten (10 g) 
Placebo: gluten-free, vegan, low FODMAPs muesli bars 
Amount, dosage, route of administration, duration of treatment: 
Muesli bars will be administered daily for a period of 7 days, followed by a washout period 
of 14 days 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 6 of 77 
Funding: 
This study is supported by the Canadian Digestive Health Foundation and a Society for the 
Study of Celiac Disease (SSCD) grant for Non-Celiac Gluten/Wheat Sensitivity sponsored 
by the Nestle Research Center, Nestec SA to Drs. Bercik and Pinto-Sanchez.  
 
LEGEND  
 
Gluten free diet (GFD); wheat free diet (WFD); irritable bowel syndrome (IBS); 
gastrointestinal (GI); anti-gliadin antibodies IgG (AGA IgG); Fermentable, Oligo-, Di-, 
Monosaccharides And Polyols (FODMAP); gluten-related diseases (GRD)

June 2022- Version 6 
                                                                                                     Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
 
Page 7 of 77 
BACKGROUND 
Irritable bowel syndrome (IBS) is a common, poorly understood condition 
characterized by abdominal discomfort or pain that is associated with bowel activity 
changes1,2. Multiple factors, such as dietary, psychological, visceral hypersensitivity and/or 
motility disturbances, contribute to its pathology3. Various dietary approaches, including the 
gluten-free diet (GFD) and wheat-free diet (WFD), have been used on IBS patients with 
varying effects4. 
Gluten, a prolamin or storage protein of wheat, rye, and barley, is composed of the 
proteins gliadin and glutenin5,6. It is commonly consumed at around 10 g/d in Western 
countries and is implicated in various gluten related disorders, such as celiac disease or non-
celiac gluten sensitivity 7–9.  
Sensitivity to gluten has been proposed as one possible cause of functional 
gastrointestinal (GI) symptoms in patients diagnosed with IBS10,11. In fact, many IBS patients 
believe that certain foods, specifically gluten, trigger their symptoms12,13, prompting them to 
start a GFD. Clinical studies have associated gluten to intestinal and extraintestinal symptoms, 
even without the autoimmune activity characteristic of celiac disease14,15. Another study found 
that a diarrhea-predominant subgroup of IBS patients who produce anti-gliadin antibodies 
(AGA) or are HLA DQ2 genotype carriers are more likely to respond to a GFD16.  
However, gluten cannot be considered in isolation when investigating IBS. Removal of 
dietary wheat clinically improves symptoms in some IBS patients, indicating that only a 
proportion of patients may truly be gluten sensitive13,17. One key component of wheat, also 
found in rye and barley, that may shed light on NCWS is the group of at least 11 mono-, di-, 
and tetrameric wheat proteins known as amylase trypsin inhibitors (ATIs)18,19. ATIs function 
by inhibiting amylase and trypsin enzymes in mammals and insects, indicating a protective 
effect in wheat19. They are found in higher quantities in modern hexaploidy wheat compared to 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 8 of 77 
traditional spelt, emmer, einkorn, or durum wheat species19. Preclinical studies demonstrated 
that ATIs can cause intestinal inflammation by activating innate immune pathways, 
specifically through toll-like receptor 4 (TLR-4)20. On average, ATIs are consumed at 0.5-1.5 g 
per day18. It is unknown what amount is needed to provoke a significant immune reaction, thus 
the Salerno criterion of gluten flour with at least 0.3 g of ATIs/8 g of gluten will be used in this 
study21.  
There is a need for well-designed RCTs to disentangle the effects of these specific 
dietary components and better understand the role of gluten and ATIs in IBS pathophysiology. 
It is possible that gluten itself may not be responsible for IBS symptoms in some patients, and 
that instead ATIs act independently from or as an adjuvant to gluten to produce symptoms. 
Therefore, we designed a crossover RCT to investigate the effects of purified gluten versus 
non-purified gluten and whole wheat flour containing other wheat components on intestinal 
and extraintestinal symptoms in IBS patients.  
 
RATIONALE 
IBS is an extremely common condition affecting up to 30% of the general population. 
It is estimated that 12-14% of primary care visits and 28% of patients referred to 
gastroenterologists are diagnosed with IBS, making this illness a larger reason for consultancy 
than diabetes, hypertension, or asthma22–24. Not only is IBS associated with more visits to 
physicians, but patients also undergo more diagnostic tests, consume more medications, miss 
more workdays, and are hospitalized more frequently with higher overall financial and social 
costs than patients without IBS22,25. 
The GFD has gained considerable popularity in the general population22,26. Between 
2004 and 2011 the market for gluten-free products grew at a compound annual growth rate of 
28%, with annual sales expected to reach $4.7 billion by 202027 (Appendix 1). As of 
September 2017, Amazon.com listed 150 579 entries for the topic “gluten-free.” A Google 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 9 of 77 
search at the same time for “gluten-free diet” produced more than 4.1 million results. The 
number-one reason consumers cite for buying gluten-free products is that they are perceived to 
be healthier than their gluten-containing counterparts26. 
Despite the perceived benefits of a GFD by IBS patients, the role of diet in this 
population is still unclear. Wheat-derived non-digestible carbohydrates have been reported to 
have beneficial effects like: a) decreased postprandial glycemia and insulinemia28,29; b) 
reduced fasting triglycerides30 and blood pressure31; c) improved immune status32 and vitamin 
and mineral absorption33,34; and d) beneficial changes in gut microbiota composition35,36. Thus, 
a GFD is questionable in people without celiac disease and is imprudent dietary advice for the 
general population5. Even for celiac patients who must follow a GFD indefinitely, it is difficult 
and is associated with social restrictions affecting their quality of life37. Additionally, 
avoidance diets can pose a significant socioeconomic challenge for non-celiac patients since 
the costs are not covered by government subsidy. Although food costs vary depending on the 
selection, overall gluten-free products were reported to be 240% more expensive compared to 
gluten-containing food38. Consequently, it is crucial to have a better understanding of the 
effects of the diet in patients suffering from IBS and to develop a rational approach to its 
treatment39. 
OBJECTIVES 
 
Main objective: 
To evaluate whether non-purified gluten and whole wheat flour (with ATIs) versus purified 
gluten worsen symptoms in IBS patients who previously improved with a GFD (Appendix 2).  
Secondary objectives:  
To evaluate the effects of non-purified gluten and whole wheat flour (with ATIs) and purified 
gluten on: 
ü IBS symptoms (IBS-SSS) (Appendix 2) 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 10 of 77 
ü General GI symptoms (PROMIS scales for belly pain, constipation, diarrhea, 
gas, and bloating) (Appendices 3-6) 
ü Stool frequency and consistency (Bristol stool scale) (Appendices 7 and 8) 
ü Anxiety, depression, and stress (DASS-21) (Appendix 9) 
ü Somatic symptoms (PHQ-15) (Appendix 10) 
ü Gastrointestinal transit time (SHAPE study) (Appendix 11) 
ü Gut microbiota composition (16S rRNA Illumina) 
ü Systemic immune reactivity (isolated PMBC assay) (Appendices 12 and 13) 
ü Diet composition (diet pictures using MealLogger app) 
ü IBS symptom duration (IBS-SSS) 
ü Long-term adherence to gluten-free diet 
ü Psychological and GI symptom impacts after learning genetic background and 
diet triggers (unblinding participants to diet triggers) 
TRIAL DESIGN 
This is a randomized controlled trial with a double-blind crossover design (Appendix 14). 
Crossover designs are most appropriate in studies where the effects of the treatment(s) are 
short-lived and reversible and are best suited to trials related to symptomatic but chronic 
conditions or diseases like IBS40. This type of design was considered appropriate since it 
estimates not only between groups, but within-patient differences in treatment effects. Because 
each patient receives all treatments, identical prognostic factors are expected, increasing the 
power of the results41. However, while considering this specific design it is important that the 
intervention during the first period must not carry over in the second period42. This design was 
implemented successfully in a similar study13 and the carryover effect was not a concern after 
one week of washout. This study will use a two-week washout to ensure there is no carryover 
effect. 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 11 of 77 
Subjects, groups, and centers 
All in-person visits will be carried out at McMaster University Medical Centre, Hamilton, 
Canada. Due to the COVID-19 pandemic, however, McMaster University may or may not 
permit in-person research visits. If in-person research visits are not permitted, participants will 
have all study visits completed remotely via telephone, email, Signal, and/or the Zoom video 
conferencing platform. Patients will be required to be on a GFD 3 weeks prior to starting the 
study. Patients will receive two different dietary challenges, as well as placebo challenge, in a 
period of 7 weeks. The total duration of the study will be 8-9 weeks. According to previous 
experience, we expect that the study will be completed within a 2-year period. 
Study population 
Inclusion Criteria: 
Adults aged 18+ with IBS diagnosis based on Rome IV criteria who previously improved 
while on a GFD, are able to comply with the study procedures (according to the investigator’s 
own judgment), and have signed the Study Informed Consent will be considered for the study. 
Exclusion Criteria: 
Subjects representing one or more of the following criteria will be excluded from participation: 
• Concurrent systemic disease and/or laboratory abnormalities considered by 
investigators to be risky or that could interfere with data collection. 
• Concurrent organic benign GI pathology (i.e. celiac disease, inflammatory bowel 
disease, etc.) other than benign polyps, diverticulosis, hemorrhoids, lipoma, and 
melanosis coli. 
• History of active cancer in the last 5 years, other than skin basal cells cancer. 
• Pregnant or breastfeeding women. 
• Patients currently participating or having participated in a trial within the past month. 
• Patients currently receiving antibiotics or have received them 1 month before V0. 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 12 of 77 
• Use of new medications 4 weeks or less prior to the study. Those on a consistent dose 
for over 4 weeks are acceptable. 
• Major abdominal surgery with the exception of hernia repair, appendectomy, Caesarian 
section, tubal ligation and cholecystectomy, hysterectomy, and hemorrhoidectomy. 
• Note: Participants who previously tested positive for COVID-19 may be allowed to 
participate in the study, but they will be required to wait at least two additional weeks 
after becoming asymptomatic or until they receive one negative COVID-19 test before 
participating in the study. 
Intervention 
All patients will be instructed to maintain a strict GFD in alignment with Canadian Celiac 
Association guidelines (Appendix 15)43. One session with the study coordinator at the 
beginning and the end of the study will be scheduled with this purpose. Habitual diet will be 
characterized by the study coordinator using a validated food frequency questionnaire44,45 
(Appendix 16). During the first visit (Study V1), patients will receive instructions to maintain a 
strict GFD and will be provided with a printed or PDF version of a list of allowed gluten free 
products and most common brand marks. GFD adherence will be reviewed at each of the 
following visits using a validated questionnaire46 and will be categorized as Compliant 
(“Excellent”, as expressed in the questionnaire), Partially Compliant (“good, fair, poor, very 
poor”), or Non-Compliant (Appendix 17)46. GFD compliance will also be assessed by 
measuring gluten immunogenic peptides in stool at each visit (iVYDAL GIP-S kit; Biomedal 
S.L., Sevilla, Spain).  
Randomization 
Participants will be randomized to 7-day dietary challenges of muesli bars containing either A, 
B, or C (commercial whole wheat flour and non-purified gluten containing ATIs, purified 
gluten, or a pure muesli bar (placebo)) separated by a washout period of 14 days. Participants 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 13 of 77 
will be divided into six arms, so that they are randomized to have each of the three challenges 
(Table 1). A challenge period and washout were chosen based on previous studies involving a 
gluten challenge47–50. The sequence of the treatments will be randomly generated using a 
computer-based pseudo-random number generator (randomizeBE package in R version 3.5.0). 
Patients will receive one of challenges A, B, or C consisting of muesli bars (gluten-free, low 
FODMAPs, vegan) with either 10 g of commercial gluten and whole wheat flour containing 
ATIs (G5004-Sigma Lab, ON, Canada and Whole Wheat All-Purpose Flour, Robin Hood, ON, 
Canada), 10 g of purified gluten (the commercial gluten Sigma will be purified to remove 80-
90% of the ATIs using the method from Schuppan in Appendix 18), or a pure muesli bar 
(placebo). The muesli bars will be made at McMaster University using a modified recipe by 
Monash University (Appendix 19)51.  
The identity of the specific product will be blinded to subjects, investigators, and 
statistician. All muesli bars will have the same appearance, taste, and smell to preserve 
blindness. Muesli bar production and packaging will be performed by the study coordinator 
and coded by a person otherwise uninvolved in the study. Allocation will be concealed for 
participants and investigators throughout the study. 
Patients will be requested to take 1 bar per day (similar amount of gluten/ATIs normal 
consumption7,8) during the treatment period. Subjects will be asked to bring remaining muesli 
bars back to the investigator at the end of the period. Compliance will be calculated as a 
percentage and recorded in the Case Report Form (CRF). All products received and dispensed 
by the investigator will be inventoried and accounted for throughout the trial period. Unused 
product will be destroyed on site at the end of the study.  
Table 1.  
 
Challenge 1 
Challenge 2  
Challenge 3 
Arm 1 
Gluten & whole wheat 
Purified gluten 
Placebo 
Arm 2 
Gluten & whole wheat 
Placebo 
Purified gluten 
Arm 3 
Purified 
Gluten & whole wheat 
Placebo 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 14 of 77 
Arm 4 
Purified 
Placebo 
Gluten & whole wheat 
Arm 5 
Placebo 
Gluten & whole wheat 
Purified gluten 
Arm 6 
Placebo 
Purified gluten 
Gluten & whole wheat 
 
Post-study follow-up of participants 
Due to restrictions from COVID-19 lockdowns, a large proportion of study participants were 
unable to complete the original study protocol including blood sampling for immune 
parameters and genetic susceptibility for celiac disease. Further, many study participants 
expressed interest in learning about their personal study results, including which diet 
challenges triggered symptoms and their celiac disease genetic susceptibility. However, it is 
unknown how disclosing diet challenge information impacts IBS patient behaviour and 
symptoms. Therefore, study participants who completed the study will be offered the 
opportunity for follow-up in which they will: 1) have the opportunity to come to McMaster to 
provide blood samples for genetic and immune assessments, if they completed the study 
virtually; and 2) receive their study results. Subjects who already completed blood sampling 
will not be invited to complete additional blood sampling but will only be asked whether they 
would like to receive their study results in this follow-up study. 
Once we obtain consent for this follow-up, participants who completed the study virtually will 
have the option to come in-person to McMaster to provide blood samples for immune and 
genetic assessment. After all the blood samples are analyzed for anti-gliadin antibodies and 
celiac genotype, all previous study participants who completed the study at least 6 months 
previously will be asked to complete two online follow-up visits. At the first follow-up visit, 
we will ask follow-up questions on their diet adherence and practices after completing the 
study, as well as whether they had any significant changes in health since the study 
completion. We will also ask participants to complete the same surveys they completed during 
the study (including IBS-SSS, Bristol, PROMIS, DASS-21, and PHQ-15) at this baseline visit. 
Once these surveys are completed, we will send them a personalized study results form that 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 15 of 77 
includes their clinical reactions to the study interventions, their blood anti-gliadin antibodies, 
and whether they have a celiac-prone genotype. Follow-up surveys would then be emailed to 
participants one month after disclosing this information to assess impacts on symptoms, diet 
adherence, and participant perceptions. 
Concomitant diets and treatments 
Any medication or treatment initiated during the course of the trial must be recorded in the 
clinical chart. Participants will be encouraged to maintain their dietary habits and medications. 
In case of any change in medication prescribed by the treating physician during the study 
period, the research team will discuss the decision to exclude them from the study. General 
dietary changes will be assessed by the study coordinator at each visit. 
Unauthorized concomitant diets/treatments/medications 
The following treatments may interfere with the primary outcome as considered by the 
investigator, but may be allowed if the dose is stable and has not been changed in the 4 weeks 
before the study commences: 
a) Antidiarrheals, antispasmodics, laxatives, or antiemetic drugs (i.e. Prochlorperazine 
(Compazine), Promethazine (Phenergan), or Trimethobezamide (Tigan)); b) Anxiolytics, 
antidepressants, opioids (i.e.: morphine or codeine). These drugs will be only permitted if they 
are used in stable doses during the last 3 months and will not to be changed during the whole 
study period; c) Antibiotics should not be used in a 1-month period before V0 and during the 
whole study. 
Outcomes and measurements 
Primary outcome: Worsening of IBS symptoms of ≥ 5052 points on the Irritable Bowel 
Syndrome Symptom Severity Score (IBS-SSS) (Appendix 2) after dietary challenges. 
Secondary outcomes: 
a) Increase in IBS symptoms. The IBS-SSS will also be evaluated as a continuous variable.  

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 16 of 77 
b) Increase in general GI symptoms (PROMIS scales): The Patient-Reported Outcomes 
Measurement Information System (PROMIS) is a standardized set of patient-reported 
outcomes (PROs) by the National Institutes of Health (NIH). This study will use the PROMIS 
scales to evaluate constipation (9 questions), diarrhea (6 questions), belly pain (5 questions), 
and gas and bloating (13 questions)53.  
c) Changes in GI Motility. 1) Orocecal Transit: The colonic SHAPE study54 is a common 
technique used to measure colonic transit using radiopaque markers. Using a modified version 
of the Evans Method on the recommendation of the Department of Radiology, the patients will 
ingest one SITZMARKS capsule (each capsule contains 24 markers) 2 days (48 hours) prior to 
the study visit, at which point a plain abdominal film (x-ray) will be taken. The Evans Method 
is only able to detect delayed or normal gastrointestinal transit55. Therefore, this modified 
method will be used to detect delayed, normal, and expedited gastrointestinal transit. Fertile 
women will provide urine samples prior to the x-ray so that an instant pregnancy test can be 
done because x-rays pose a risk to developing fetuses. The urine pregnancy test will be 
performed by the study coordinator at the beginning of the study visit at McMaster University 
Medical Centre. Urine collection instructions are included in Appendix 20. If 20-24 (>83.3%) 
of the markers are passed by 48 hours, colonic transit is expedited. If 4-20 (16.7-83.3%) of the 
markers are passed by 48 hours, colonic transit is grossly normal. If 0-4 (<16.7%) of the 
markers are passed by 48 hours, the colonic transit is delayed. Paired statistical tests will be 
conducted to account for within-participant variation. More information is in Appendix 11. 
Due to the COVID-19 pandemic, in-person research visits may be restricted. Therefore, the 
colonic SHAPE study will only be conducted during in-person visits. 
d) Determination of anti-gliadin antibodies (AGA IgA and IgG) and tissue transglutaminase 
IgA antibodies. Analyses will be done by the central laboratory at McMaster University 
Medical Centre for each participant visit (V1-V6). Due to the COVID-19 pandemic, in-person 
research visits may be restricted. Therefore, blood samples will only be taken at in-person 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 17 of 77 
visits. If participants completed the study virtually, during follow-up before disclosing their 
study results, they will have the option to provide a blood sample for this assessment.  
e) Blood chemistry and hematology will be done for the baseline visit (V1). CBC with 
automated differential, ESR, sodium, potassium, urea, fasting glucose, creatinine, total protein, 
AST, ALT, GGT, alkaline phosphatase, lactate dehydrogenase, total bilirubin, PT in INR, and 
PTT are the blood markers that will be measured. All analyses will be done by the central 
laboratory at McMaster University Medical Centre. Due to the COVID-19 pandemic, in-person 
research visits may be restricted. Blood samples will only be taken at in-person visits. If 
participants completed the study virtually, during follow-up before disclosing their study 
results, they will have the option to provide a blood sample for this assessment. 
f) Changes in GI microbiota. To evaluate changes in gut microbiota as a possible mechanism 
of symptom generation, we will require that patients collect anaerobically one stool sample for 
each visit. All sample collection details will be explained to the patient during V1 and a written 
explanation will be also provided (Appendix 21). Analysis will be performed by 16S rRNA 
Illumina.  
g) Genotype HLA DQ2/8. Will be performed using sequence-specific primer polymerase chain 
reaction (PCR) (DQ-CD Typing kit, BioDiaGene, s.r.l., Palermo, Italy)56 at baseline. Due to 
the COVID-19 pandemic, in-person research visits may be restricted. Blood samples for the 
genetic test will only be taken at in-person visits. However, the research coordinator will 
provide the option of giving a blood sample for genetic testing after study completion if the 
participant was unable to provide a blood sample during the study but still would like to 
provide the sample at a later date.  
h) IgG4 against wheat. IgG4 against wheat will be detected using a specific kit (Allergix IgG4 
Food Antibodies - 90 Serum) at baseline. Values over 10 ng/mL will be considered as 
elevated57.  
Details of procedures at each visit are provided in Appendices 2-10. 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 18 of 77 
i) Gluten immunogenic peptides (GIP). Anti-GIP immunochromatographic strips (iVYDAL 
GIP-S kit; Biomedal S.L., Sevilla, Spain) will be used to analyze the gluten content of 
participant stool samples to test for diet compliance. All stool collection details will be 
explained to the patient at V1 and a written explanation will also be provided (Appendix 21).  
j) Changes in systemic immune reactivity will be measured by peripheral blood mononuclear 
cell (PBMC) stimulation. PBMC will be stimulated and levels of pro-inflammatory cytokines 
will be assessed (Appendices 12 and 13). Due to the COVID-19 pandemic, in-person research 
visits may be restricted. Blood samples will only be taken at in-person visits. 
k) Diet composition. This study will include the use of the MealLogger app to take pictures of 
participant meals during the study. Participants will be asked to take pictures of their meals, 
and these pictures will be used by the investigators to categorize meal portion size and relative 
food types (i.e., whether participants have high protein, high fat, high carbohydrate, or high 
vegetable intakes or not).  
l) Symptom duration. This study will evaluate symptom duration after the end of the diet 
challenge. Participants will be asked to complete the IBS-SSS for 7 days after they complete 
the diet challenge to determine how long their symptoms last.  
m) Study results follow-up. At the end of the study when investigators are unblinded, the 
researchers will compile the study results (ex., worsening in IBS symptoms after gluten 
challenge) and provide this information to study participants. A dietary assessment and surveys 
used previously in the study will be sent to study participants immediately prior and one month 
after. 
Early withdrawals 
The following criteria are reasons for early discontinuation (dropouts) during the study: 1-
Patient’s decision; 2-Physician’s decision in case of undue risk with continued participation; 3-
Use of any unauthorized concomitant diets/treatments/medications; 4- Patient contracts 
COVID-19 during the study.  

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 19 of 77 
Medical care will be provided as directed by the study physician and/or the subject’s physician.  
Subjects who withdraw their participation will be requested to return at the next visit for 
evaluation so that their data can be considered for the primary outcome in an Intention to Treat 
analysis.   
If replacement of subjects is not feasible, 20% additional subjects will be recruited to 
compensate for attrition. 
Patient recruitment 
Website 
This clinical study will be advertised on a McMaster webpage (healthsci.mcmaster.ca/diet-in-
ibs/). The website will provide a description of the study, eligibility criteria, and the study 
contact information. The website will be advertised on Google ads (https://ads.google.com/) 
and social media outlets such as Facebook, Twitter, and Instagram. The website will also be 
posted on the Canadian Digestive Health Foundation’s website (http://cdhf.ca/).  
Posters 
Posters will be used to advertise the study. They will be placed at McMaster University 
Medical Centre, in McMaster’s 2F Clinic, and other locations around McMaster University 
campus. The posters will be placed at different hospitals and clinics affiliated with Hamilton 
Health Sciences, such at St. Joseph’s Hospital and Allied Health Professional practices within 
a 15 km radius of McMaster or St. Joseph’s Hospital. Posters will also be posted in pharmacies 
near McMaster University. 
Screening Participants 
The study coordinators will screen potential participants to determine whether they are eligible 
for the study. If the potential participants are registered at Hamilton Health Sciences, the study 
coordinators will ask to contact their physician and request a referral to determine whether they 
are eligible. If participants are not patients registered in Hamilton Health Sciences, they will be 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 20 of 77 
asked for their verbal consent for the study coordinator to contact their medical practitioner to 
provide a referral or further information to assess their eligibility. 
During their appointments, physicians at McMaster’s Gastroenterology Outpatient 2F clinic 
may inform eligible patients about this study. If the patient is interested and consents to be 
contacted about research studies, the physician will inform the study coordinator so that they 
may discuss the study with the patient. 
Data collection 
Case report forms (CRFs) are an integral part of this protocol. All data captured by the 
investigators will directly be recorded on a computer database created using REDCap 
(Research Electronic Data Capture) Software, a secure web application for building and 
managing online surveys and database programs58. Data to be considered as sources in the 
CRFs are: a) Surveys and questionnaires (Appendices 2-10); b) Details of physical 
examination and clinical background; c) Study laboratory sheets; d) Pregnancy urine strips; e) 
Diet assessment: diet initiation, follow-up, and GFD compliance. All other data should be 
checked for accuracy against the patient record. 
To preserve adequate data management, the database will be supervised and monitored by the 
Principal Investigator.  
Videoconferences 
All follow-up visits will be conducted online using either Zoom or telephone, depending on the 
participant’s preference. The participants will be informed about the privacy risks involved in 
video conferencing in the informed consent form. Informed consent will be obtained from 
patients to communicate using Zoom, telephone, email, and/or the secure, encrypted messaging 
app Signal. Patients will be explained the risks related to unauthorized disclosures and/or 
interception of personal health information and steps they can take to help protect their 
information. A link to the Zoom privacy policy (https://zoom.us/privacy) will be included in 
the informed consent form for participants to learn more. 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 21 of 77 
Signal messaging app 
Participants will have the option of communicating with the study coordinators using Signal, a 
secure, encrypted messaging app. As mentioned in HiREB’s Video Conferencing and Consent 
Guidelines (https://hireb.ca/wp-content/uploads/2020/04/Video-Conferencing-and-Consent-
Guidelines.docx), Signal is “an encrypted voice calling or messaging service […] which has 
clear policies about the storing of logs of communication metadata”. Signal does not store 
metadata or use the cloud to back up messages and uses end-to-end encryption by default. A 
link to Signal’s privacy policy (https://signal.org/legal/#privacy-policy) will be provided to 
study participants to learn more. Additionally, the study coordinators will enable the 
‘Disappearing Messages’ feature of Signal such that any text communications to participants 
will be deleted from both devices after 12 hours, as described here: 
https://support.signal.org/hc/en-us/articles/360007320771. Study participants will not be 
required to use Signal but may use it to communicate securely with the study coordinators if 
they prefer.  
Communication with study participants 
Study participants will have the option of communicating with the study coordinators via 
Zoom, phone calls, email, or the secure, end-to-end encrypted messaging platform Signal. 
They will be informed of the privacy risks associated with each method of communication.  
Scripts to contact patients 
If the study coordinators are contacted by a potential participant, then their first contact with 
the participant will include standardized scripts. Scripts for telephone and email contact are 
included in the HiREB application. 
Privacy 
This clinical study will include the use of electronic devices, including phones and computers, 
where study questionnaires and visits will be completed online. The study coordinators will 
use various measures to preserve participant privacy, including: 1) only storing participant 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 22 of 77 
personal information on a secure, encrypted file in the McMaster server; 2) separating personal 
identifiers from study data; 3) communicating with participants on secure platforms, including 
Signal, for messaging or calling; and 4) informing participants of the privacy risks of using 
video conferencing platforms like Zoom.  
Monitoring: 
The Principal Investigator will serve as a monitor during the study. The strategy will involve 
monitoring trial oversight, data, and safety. 
Since no higher risk is expected in any of the study measurements or treatments, a Data Safety 
monitoring board will not be required.  
This clinical trial will be conducted following the principles of ICH (International Conference 
on Harmonization) for Good Clinical Practices and adherence with the applicable regulatory or 
legal requirements. 
COVID-19 Standard Operating Procedures (SOPs) 
Standard operating procedures applicable to clinical and basic research were developed to 
minimize the spread of the COVID-19 coronavirus. They can be found in Appendix 22.  
All research personnel will complete the Ontario COVID-19 Self-Assessment 
(https://www.ontario.ca/page/2019-novel-coronavirus-covid-19-self-assessment) within 24 
hours of going to McMaster University Medical Centre and when they pick up or drop off 
samples for patients. We will follow the guidelines for McMaster University’s Return to 
Research (https://research.mcmaster.ca/phased-return-to-research-activity/), conducting 
research and study visits remotely or in-person depending on the current guidelines.  
Pick-up & drop-off of samples for remote visits 
Due to the COVID-19 pandemic, patients may or may not have in-person research visits at 
McMaster University. For remote visits, study coordinators will mail or drop off the specimen 
collection kit and study intervention to the participant before their study visit and will arrange a 
date and time to pick up the sample from their preferred address (i.e., home or workplace) after 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 23 of 77 
it is collected. Specimens will be transported in a special biohazard container back to 
McMaster University Medical Centre for processing (Appendix 23). Study participants will 
complete the online COVID-19 screening tool (Ontario COVID-19 Self-Assessment) at the 
day of pick-up or drop-off. To coordinate pick-ups and drop-offs, the study coordinators and 
participants may call, email, or use end-to-end encrypted Signal messages 
(https://signal.org/en/), depending on participant preference, once they have been informed of 
the security levels associated with each platform.  
The study coordinators will drive to the participant’s preferred address to pick up the sample 
using a rental car from Enterprise (https://www.enterprise.ca/) and will be covered by 
Enterprise insurance in case of any accident. These samples will be kept in a special biohazard 
refrigerated container approved by Transport Canada to preserve the samples. There will be 
three containers used to ensure safe transportation of study samples: 1) sample collection 
containers with direct contact to participant samples; 2) a secondary container to hold the 
individual samples; and 3) a tertiary cooler with ice or ice packs to hold the secondary 
container. The study coordinator will follow the sample handling protocol in Appendix 24.  
Statistical analysis 
• Sample size calculation 
According to the study design chosen, three independent groups with paired observations were 
considered for sample size calculations. The IBS-SSS is considered to have a minimal 
clinically important difference (MCID) at 50 points52. There is also a within-patient standard 
deviation in IBS-SSS overall score of 68.3 points in IBS-Constipation and 71.4 points in IBS-
Diarrhea59. In order to reject the null hypothesis that there will be no difference in IBS-SSS 
between non-purified gluten and whole wheat flour, purified gluten, and placebo with a power 
of 0.80 and at a significance of a = 0.05, 33 participants are required. 40 participants will be 
recruited to account for 20% attrition.  

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 24 of 77 
• Statistical analysis 
Statistical testing60 will be performed using the Statistical Package for Social Sciences for 
Windows software (SPSS 20.0; SPSS Inc., Chicago, IL) and R version 3.5.0. The study cohort 
will be characterized descriptively using simple proportions for categorical variables (e.g. 
gender) and means with SDs for parametric continuous variables (e.g. age). Groups will be 
compared using chi-squared tests. Parametric continuous variables will compare means and 
SDs using repeated measures ANOVA. The primary outcome, the IBS-SSS, will be used as a 
dichotomous variable and analyzed using a chi-squared test. Patients will be categorized as 
“worsened their symptoms” if they showed an increase of at least 50 points in overall score, 
and “not worsened” if the score was below that point when dichotomous data is required. The 
IBS-SSS will also be evaluated using repeated measures ANOVA. Baseline and post-treatment 
questionnaires (PROMIS scales, DASS21, etc.) will compare means and SDs using one-way 
ANOVA for between-group comparison or repeated measures ANOVA for within-group 
comparison61. For the secondary outcome (PROMIS scales), separated domain (diarrhea, 
constipation, gas and bloating, or belly pain) scores will also be analyzed using one-way 
ANOVA between groups and repeated measures ANOVA within groups. Secondary outcomes 
will also be adjusted for multiple comparisons using Tukey’s post-hoc test and an overall p-
value of 0.05 for significance.  
A p-value of <0.05 will be considered statically significant. Biochemical measures (AGA and 
blood tests) will be reported as means and SDs if parametric or medians and IQRs if non-
parametric. Correlation between changes in IBS-SSS and PROMIS scales and changes in 
intestinal microbiota will be tested using a Pearson's correlation coefficient if parametric, or a 
Spearman’s correlation if non-parametric. Subgroup analysis of primary and secondary 
outcomes will be performed considering presence/absence of AGA, whether the patient has a 
celiac-compatible genotype (HLA DQ2/8 positive), and whether the participants are compliant 
vs non-compliant to the study protocol. 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 25 of 77 
 
• COVID Analyses 
Participants in our study will be asked whether they were tested for COVID-19 during the 
pandemic and whether they tested positive in a short questionnaire at each visit. Participants 
reporting a positive test will drop from the study and an early termination visit will be 
performed.  
 
 
• Biological Sample Processing 
Stool 
Stool samples will be kept cold until further processing. On the day of collection, samples will 
be aliquoted into 6 different 2 mL cryovials in an anaerobic chamber. Aliquots will be stored at 
-80℃ until the analysis.  
Urine 
Urine samples will be kept cold from collection until further processing, using an insulating 
bag with ice packs. The research coordinator will pick up the urine sample at the patient’s 
home within the first 30-45 minutes after collection. On the day of collection, urine will be 
aliquoted into 0.5 mL sterile microtubes and stored at -80℃.  
Blood 
Blood samples will be collected in green-topped heparin-containing tubes, red-topped tubes 
with coagulant, and lavender-topped tubes with EDTA. Serum and plasma will then be 
separated by centrifugation within 1 hour after collection to prevent hemolysis. Plasma will be 
aliquoted into 1.5 mL tubes and stored at -80℃.  
 
• Analysis of the Microbiota: Molecular approach  
 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 26 of 77 
To understand the relationship between IBS states and the gut microbiota, we propose to 
analyze the microbial communities at two levels: (i) microbial diversity (16S rRNA gene 
analyses) and (ii) metabolic capabilities (metagenomics, or “bulk”, DNA sequencing). Illumina 
sequencing of 16S rRNA genes will help us to comprehensively monitor the variation in 
microbial taxonomic representation of IBS patients over time. On the other hand, deep 
sequencing of bulk DNA will provide a picture of the genetic composition of samples, which 
will indicate the types of functions available to such microbial communities.  
For 16S rRNA gene sequence analyses, we will use the QIIME software platform. This 
platform enables the following steps: aligning sequences using secondary-structure 
information, clustering sequences into operational taxonomic units (OTUs) by percent 
similarity, assigning taxonomic information to unique clusters, generating averaged 
accumulation (rarefaction) curves for each sample, and establishing estimates of total diversity. 
Ethical aspects 
The study protocol will be submitted by one of the investigators for approval by the Hamilton 
Integrated Research Ethics Board (HiREB). Commencement of the clinical trial is not 
permitted without written approval by the ethics committee. The HiREB must be notified of all 
subsequent additions or changes to the study protocol. Notification of the HiREB is also 
required in the event of a serious adverse event (SAE) during the clinical trial. This trial will be 
conducted according to the principles and rules laid down in the Declaration of Helsinki 
(Appendix 25) and its subsequent amendments. 
Protection of the subject’s confidentiality 
Confidentiality of all study participants will be maintained; codes for subject identification 
(ID) will be utilized (i.e. participant, P, and number of enrolment; P001) and documents 
relating their identity to their ID will be kept separate from their study records. 
Informed consent 
When the investigator has determined that the subject is an appropriate candidate for the study, 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 27 of 77 
the study will be described and explained by a researcher during an interview either in person, 
via phone, via end-to-end encrypted Signal calls, or on the online videoconferencing tool 
Zoom. Due to the COVID-19 pandemic, online visits were introduced using the online video 
conferencing platform Zoom. Subjects will be informed of the privacy risks of conducting 
online visits on Zoom before they consent to participating in this study. The investigator will 
fully answer all participant questions. A copy of the information letter and informed consent 
form will be provided to the subject. Informed consent will be obtained from each subject by 
the investigator prior to V1 and enrolment in the study, either in written format or as an e-
consent. If consent is obtained in-person, the consent form will be signed and dated by the 
subject and the investigator. The consent form will be completed in two copies: the first copy 
is kept in the investigator’s file and is checked by the monitor while the second is given to the 
patient. Participants will also have the option of having their informed consent form mailed to 
them to complete at home. Once the consent form is signed and dated, participants will mail 
the consent forms back using a pre-paid, pre-addressed envelope. If the consent is obtained 
online, participants will complete e-consent forms using either the REDCap E-Consent 
Framework or the DocuSign eSignature platform (https://www.docusign.ca/). The consent 
form will be electronically signed and dated by the participant and investigator. The 
Farncombe Institute’s REDCap is hosted on a secure McMaster server and the e-consent forms 
would be collected and stored in a different REDCap project from the study CRFs to ensure 
that the participant ID is kept separate from any identifying information. Only REDCap users 
with ‘full data set’ privileges will be able to download archived e-consent forms. DocuSign is a 
secure eSignature platform that is commonly used at Hamilton Health Sciences and McMaster 
University Faculty of Health Sciences. As our lab already uses REDCap for our data 
collection, we will use REDCap in the interim but plan to collect consents using DocuSign if 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 28 of 77 
we get a department, faculty, or institute-wide account. 
No subject will undergo study-related procedures before completion of the informed consent.  
Handling adverse events (AEs) 
 
An AE is defined as any untoward occurrence in a patient or clinical investigation subject who 
has been administered an investigational product. It does not necessarily require a causal 
relationship with the treatment. AEs include abnormal laboratory findings, illnesses, signs, 
and/or symptoms that occur or worsen during the course of the study. They also include 
occasions when subjects contact the investigator or their private physician and are examined or 
given medical direction. They may or may not lead to the withdrawal of the subject from the 
study. AEs are generally classified as serious or non-serious. All AEs occurring during the 
study will be reported and recorded whether or not they are considered to be serious (SAEs), 
non-serious, and/or related to the treatment. The following information will be required in each 
case: subject ID and date, description of event, duration, frequency, intensity, seriousness, 
action taken, outcome and sequel, relationship to test product, and definitions. Documentation 
of a SAE that requires a separate form will be completed by the investigators in each case 
(Appendix 26). In case of any adverse event occurrence, adequate care will be provided by the 
research physicians. 
Impact of the study 
 
Traditional IBS management approaches often rely on a combination of dietary changes, 
antispasmodics, antidiarrheal, laxatives, antidepressants, and analgesics62. However, the 
efficacy, safety, and tolerability of these treatments have not been examined in well-controlled 
RCTs. A systematic review of the efficacy and safety of traditional therapies found that none 
of these agents demonstrated level I evidence to support their use in the treatment of IBS63. 
Furthermore, the adverse effects of some IBS treatments may outweigh the potential benefits. 
It is not surprising that IBS patients are often dissatisfied with the efficacy and tolerability of 
traditional treatment options. These treatments only address isolated symptoms rather than the 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 29 of 77 
cause, thereby leading to repeated doctor visits, multiple medications, and frequent 
prescription changes. This is costly, contributing to a tremendous economic burden on both 
society and the health care system64. Additionally, symptoms experienced by IBS patients 
significantly impact their quality of life65–67.  
Adopting a GFD is becoming increasingly popular with IBS patients, as well as the general 
American population, despite the fact that it is 2-3 times costlier68. Several IBS patients either 
self-start or are recommended to start a GFD by their physicians in order to alleviate their 
symptoms, despite the fact there is little evidence gluten is a trigger. Thus, it is crucial to have 
a better understanding of the real effect of diet in IBS patients and that a rational approach to 
the prescription of a GFD is undertaken. The results of this study will contribute to greater 
knowledge of the potential mechanisms and effects of gluten and ATIs, leading to more 
effective future dietary treatments for the global IBS epidemic. 

June 2022- Version 6 
                                                                                                     Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
 
Page 30 of 77 
References 
 
1.  
Boivin M. Socioeconomic impact of irritable bowel syndrome in Canada. Can J 
Gastroenterol. 
2001;15 
Suppl 
B:8B-11B. 
papers://5aecfcca-9729-4def-92fe-
c46e5cd7cc81/Paper/p53689 
2.  
Sanger GJ, Alpers DH. Development of drugs for gastrointestinal motor disorders: 
Translating science to clinical need. Neurogastroenterol Motil. 2008;20(3):177-184. 
doi:10.1111/j.1365-2982.2008.01084.x 
3.  
Gibson PR, Shepherd SJ. Evidence-based dietary management of functional 
gastrointestinal symptoms: The FODMAP approach. J Gastroenterol Hepatol. 
2010;25(2):252-258. doi:10.1111/j.1440-1746.2009.06149.x 
4.  
McKenzie YA, Alder A, Anderson W, et al. British Dietetic Association evidence-based 
guidelines for the dietary management of irritable bowel syndrome in adults. J Hum Nutr 
Diet. 2012;25(3):260-274. doi:10.1111/j.1365-277X.2012.01242.x 
5.  
Gaesser GA, Angadi SS. Gluten-free diet: Imprudent dietary advice for the general 
population? J Acad Nutr Diet. 2012;112(9):1330-1333. doi:10.1016/j.jand.2012.06.009 
6.  
El-Chammas K, Danner E. Gluten-Free Diet in Nonceliac Disease. Nutr Clin Pract. 
2011;26(3):294-299. doi:10.1177/0884533611405538 
7.  
van Overbeek F, Uil-Dieterman I, Mol I, Köhler-Brands L, Heymans H, Mulder C. The 
daily gluten intake in relatives of patients with coeliac disease compared with that of the 
general Dutch population. Eur J Gastroenterol Hepatol. 1997;9(11):1097-1099. 
8.  
Catassi C, Fabiani E, Iacono G, et al. A prospective, double-blind, placebo-controlled trial 
to establish a safe gluten threshold for patients with celiac disease. Am J Clin Nutr. 
2007;85(1):160-166. doi:85/1/160 [pii] 
9.  
Lebwohl B, Granath F, Ekbom A, et al. Mucosal healing and mortality in coeliac disease. 
Aliment Pharmacol Ther. 2013;37(3):332-339. doi:10.1111/apt.12164 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 31 of 77 
10.  
Ferch CC, Chey WD. Irritable bowel syndrome and gluten sensitivity without celiac 
disease: Separating the wheat from the chaff. Gastroenterology. 2012;142(3):664-666. 
doi:10.1053/j.gastro.2012.01.020 
11.  
Verdu EF. Editorial: Can gluten contribute to irritable bowel syndrome? Am J 
Gastroenterol. 2011;106(3):516-518. doi:10.1038/ajg.2010.490 
12.  
Eswaran S, Tack J, Chey WD. Food: the forgotten factor in the irritable bowel syndrome. 
Gastroenterol Clin North Am. 2011;40(1):141-162. doi:10.1016/j.gtc.2010.12.012 
13.  
Carroccio A, Mansueto P, Iacono G, et al. Non-celiac wheat sensitivity diagnosed by 
double-blind placebo-controlled challenge: exploring a new clinical entity. Am J 
Gastroenterol. 2012;107(12):1898-1906; quiz 1907. doi:10.1038/ajg.2012.236 
14.  
Verdu EF, Huang X, Natividad J, et al. Gliadin-dependent neuromuscular and epithelial 
secretory responses in gluten-sensitive HLA-DQ8 transgenic mice. Am J Physiol 
Gastrointest Liver Physiol. 2008;294(1):217-225. doi:10.1152/ajpgi.00225.2007 
15.  
Biesiekierski JR, Newnham ED, Irving PM, et al. Gluten causes gastrointestinal 
symptoms in subjects without celiac disease: a double-blind randomized placebo-
controlled 
trial. 
Am 
J 
Gastroenterol. 
2011;106(3):508-514; 
quiz 
515. 
doi:10.1038/ajg.2010.487 
16.  
Wahnschaffe U, Schulzke JD, Zeitz M, Ullrich R. Predictors of clinical response to 
gluten-free diet in patients diagnosed with diarrhea-predominant irritable bowel 
syndrome. 
Clin 
Gastroenterol 
Hepatol. 
2007;5(7):844-850; 
quiz 
769. 
doi:10.1016/j.cgh.2007.03.021 
17.  
Schuppan D, Pickert G, Ashfaq-Khan M, Zevallos V. Non-celiac wheat sensitivity: 
differential diagnosis, triggers and implications. Best Pract Res Clin Gastroenterol. 
2015;29(3):469-476. doi:10.1016/j.bpg.2015.04.002 
18.  
Zevallos VF, Raker V, Tenzer S, et al. Nutritional Wheat Amylase-Trypsin Inhibitors 
Promote Intestinal Inflammation via Activation of Myeloid Cells. Gastroenterology. 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 32 of 77 
2017;152(5):1100-1113.e12. doi:10.1053/j.gastro.2016.12.006 
19.  
Tatham AS, Shewry PR. Allergens to wheat and related cereals. Clin Exp Allergy. 
2008;38(11):1712-1726. doi:10.1111/j.1365-2222.2008.03101.x 
20.  
Junker Y, Zeissig S, Kim S-J, et al. Wheat amylase trypsin inhibitors drive intestinal 
inflammation via activation of toll-like receptor 4. J Exp Med. 2012;209(13):2395-2408. 
doi:10.1084/jem.20102660 
21.  
Catassi C, Elli L, Bonaz B, et al. Diagnosis of non-celiac gluten sensitivity (NCGS): The 
salerno experts’ criteria. Nutrients. 2015;7(6):4966-4977. doi:10.3390/nu7064966 
22.  
El-Salhy M, Ostgaard H, Gundersen D, Hatlebakk JG, Hausken T. The role of diet in the 
pathogenesis and management of irritable bowel syndrome (Review). Int J Mol Med. 
2012;29(5):723-731. doi:10.3892/ijmm.2012.926 
23.  
National Center for Health Statistics. Ambulatory Health Care Data. U.S. Department of 
Health & Human Services. Published 2017. Accessed September 17, 2017. 
https://www.cdc.gov/nchs/ahcd/index.htm 
24.  
Mitchell CM, Drossman DA. Survey of the AGA membership relating to patients with 
functional gastrointestinal disorders. Gastroenterology. 1987;92(5 Pt 1):1282-1284. 
doi:10.1016/S0016-5085(87)91099-7 
25.  
Pietzak M. Celiac disease, wheat allergy, and gluten sensitivity: when gluten free is not a 
fad. 
JPEN 
J 
Parenter 
Enteral 
Nutr. 
2012;36(1 
Suppl):68S-75S. 
doi:10.1177/0148607111426276 
26.  
Marcason W. Is there evidence to support the claim that a gluten-free diet should be used 
for weight loss? J Am Diet Assoc. 2011;111(11):1786. doi:10.1016/j.jada.2011.09.030 
27.  
Terazono E. Healthy appetites drive jump in sales of gluten-free foods. Financial Times. 
Published 2017. Accessed September 10, 2017. https://www.ft.com/content/4ec0f2f2-
2c0a-11e7-9ec8-168383da43b7 
28.  
Higgins JA. Whole grains, legumes, and the subsequent meal effect: implications for 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 33 of 77 
blood glucose control and the role of fermentation. J Nutr Metab. 2012;2012:829238. 
doi:10.1155/2012/829238 
29.  
Rosén LAH, Östman EM, Shewry PR, et al. Postprandial glycemia, insulinemia, and 
satiety responses in healthy subjects after whole grain rye bread made from different rye 
varieties. 1. J Agric Food Chem. 2011;59(22):12139-12148. doi:10.1021/jf2019825 
30.  
Russo F, Riezzo G, Chiloiro M, et al. Metabolic effects of a diet with inulin-enriched 
pasta in healthy young volunteers. Curr Pharm Des. 2010;16(7):825-831. 
doi:10.2174/138161210790883570 
31.  
Harris KA, Kris-Etherton PM. Effects of whole grains on coronary heart disease risk. 
Curr Atheroscler Rep. 2010;12(6):368-376. doi:10.1007/s11883-010-0136-1 
32.  
Kelly G. Inulin-type prebiotics: a review. (Part 2). Altern Med Rev. 2009;14(1):36-55. 
http://www.ncbi.nlm.nih.gov/pubmed/19364192 
33.  
Abrams SA, Griffin IJ, Hawthorne KM, et al. A combination of prebiotic short- and long-
chain inulin-type fructans enhances calcium absorption and bone mineralization in young 
adolescents. Am J Clin Nutr. 2005;82(2):471-476. doi:82/2/471 [pii] 
34.  
Reddy BR. Noncaloric Benefits of Carbohydrates. Nestle Nutr Inst Workshop Ser. 
2015;82:27-37. doi:10.1159/000381999 
35.  
Tarini J, Wolever TMS. The fermentable fibre inulin increases postprandial serum short-
chain fatty acids and reduces free-fatty acids and ghrelin in healthy subjects. Appl Physiol 
Nutr Metab. 2010;35(1):9-16. doi:10.1139/H09-119 
36.  
Neyrinck AM, Delzenne NM. Potential interest of gut microbial changes induced by non-
digestible carbohydrates of wheat in the management of obesity and related disorders. 
Curr 
Opin 
Clin 
Nutr 
Metab 
Care. 
2010;13(6):722-728. 
doi:10.1097/MCO.0b013e32833ec3fb 
37.  
Lee A, Newman JM. Celiac diet: its impact on quality of life. J Am Diet Assoc. 
2003;103(11):1533-1535. doi:10.1016/S0002 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 34 of 77 
38.  
Lee AR, Ng DL, Zivin J, Green PHR. Economic burden of a gluten-free diet. J Hum Nutr 
Diet. 2007;20(5):423-430. doi:10.1111/j.1365-277X.2007.00763.x 
39.  
Verdu EF, Armstrong D, Murray JA. Between celiac disease and irritable bowel 
syndrome: the “no man’s land” of gluten sensitivity. Am J Gastroenterol. 
2009;104(6):1587-1594. doi:10.1038/ajg.2009.188 
40.  
Mills EJ, Chan A-W, Wu P, Vail A, Guyatt GH, Altman DG. Design, analysis, and 
presentation of crossover trials. Trials. 2009;10:27. doi:10.1186/1745-6215-10-27 
41.  
Piantadosi S. Clinical Trials: A Methodologic Perspective: Second Edition. 2nd ed. 
(Shewhart WA, Wilks SS, eds.). John Wiley & Sons, Inc.; 2005. doi:10.1002/0471740136 
42.  
Friedman LM, Furberg CD, DeMets DL, Reboussin DM, Granger CB. Fundamentals of 
Clinical Trials. 5th ed. Springer International Publishing; 2015. doi:10.1007/978-3-319-
18539-2 
43.  
Canadian Celiac Association and Dietitians of Canada. Gluten-Free Eating. Published 
2011. 
Accessed 
September 
17, 
2017. 
http://www.celiac.ca/b/wp-
content/uploads/2013/07/Gluten_Free_Eating_Apr_2011.pdf 
44.  
Hodge A, Patterson AJ, Brown WJ, Ireland P, Giles G. The Anti Cancer Council of 
Victoria FFQ: relative validity of nutrient intakes compared with weighed food records in 
young to middle-aged women in a study of iron supplementation. Aust N Z J Public 
Health. 2000;24(6):576-583. doi:10.1111/j.1467-842X.2000.tb00520.x 
45.  
Merchant AT, Dehghan M. Food composition database development for between country 
comparisons. Nutr J. 2006;5:2. doi:10.1186/1475-2891-5-2 
46.  
Leffler DA, Dennis M, Edwards George JB, et al. A simple validated gluten-free diet 
adherence survey for adults with celiac disease. Clin Gastroenterol Hepatol. 
2009;7(5):530-536, 536.e1-2. doi:10.1016/j.cgh.2008.12.032 
47.  
Skodje GI, Sarna VK, Minelle IH, et al. Fructan, Rather Than Gluten, Induces Symptoms 
in Patients With Self-Reported Non-Celiac Gluten Sensitivity. Gastroenterology. 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 35 of 77 
2018;154(3):529-539.e2. doi:10.1053/j.gastro.2017.10.040 
48.  
Elli L, Tomba C, Branchi F, et al. Evidence for the Presence of Non-Celiac Gluten 
Sensitivity in Patients with Functional Gastrointestinal Symptoms: Results from a 
Multicenter Randomized Double-Blind Placebo-Controlled Gluten Challenge. Nutrients. 
2016;8(2):84. doi:10.3390/nu8020084 
49.  
Zanini B, Baschè R, Ferraresi A, et al. Randomised clinical study: gluten challenge 
induces symptom recurrence in only a minority of patients who meet clinical criteria for 
non-coeliac gluten sensitivity. Aliment Pharmacol Ther. 2015;42(8):968-976. 
doi:10.1111/apt.13372 
50.  
Biesiekierski JR, Peters SL, Newnham ED, Rosella O, Muir JG, Gibson PR. No effects 
of gluten in patients with self-reported non-celiac gluten sensitivity after dietary reduction 
of fermentable, poorly absorbed, short-chain carbohydrates. Gastroenterology. 
2013;145(2):320-8.e1-3. doi:10.1053/j.gastro.2013.04.051 
51.  
Hill 
P. 
Low 
FODMAP 
Muesli 
Bars. 
Accessed 
March 
24, 
2018. 
https://www.monashfodmap.com/blog/low-fodmap-muesli-bars/ 
52.  
Francis CY, Morris J, Whorwell PJ. The irritable bowel severity scoring system: a simple 
method of monitoring irritable bowel syndrome and its progress. Aliment Pharmacol 
Ther. 1997;11(2):395-402. Accessed April 2, 2018. https://journals-scholarsportal-
info.libaccess.lib.mcmaster.ca/pdf/02692813/v11i0002/395_tibsssibsaip.xml 
53.  
Spiegel BMR, Hays RD, Bolus R, et al. Development of the NIH Patient-Reported 
Outcomes Measurement Information System (PROMIS) gastrointestinal symptom scales. 
Am J Gastroenterol. 2014;109(11):1804-1814. doi:10.1038/ajg.2014.237 
54.  
Kim ER, Rhee P-L. How to interpret a functional or motility test - colon transit study. J 
Neurogastroenterol Motil. 2012;18(1):94-99. doi:10.5056/jnm.2012.18.1.94 
55.  
Evans RC, Kamm MA, Hinton JM, Lennard-Jones JE. The normal range and a simple 
diagram for recording whole gut transit time. Int J Colorectal Dis. 1992;7(1):15-17. 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 36 of 77 
doi:10.1007/BF01647654 
56.  
Megiorni F, Mora B, Bonamico M, et al. A rapid and sensitive method to detect specific 
human lymphocyte antigen (HLA) class II alleles associated with celiac disease. Clin 
Chem Lab Med. 2008;46(2):193-196. doi:10.1515/CCLM.2008.049 
57.  
Zar S, Benson MJ, Kumar D. Food-specific serum IgG4 and IgE titers to common food 
antigens in irritable bowel syndrome. Am J Gastroenterol. 2005;100(7):1550-1557. 
doi:10.1111/j.1572-0241.2005.41348.x 
58.  
Harris PA, Taylor R, Thielke R, Payne J, Gonzalez N, Conde JG. Research electronic data 
capture (REDCap)--a metadata-driven methodology and workflow process for providing 
translational research informatics support. J Biomed Inform. 2009;42(2):377-381. 
doi:10.1016/j.jbi.2008.08.010 
59.  
Colucci R, Gambaccini D, Ghisu N, et al. Influence of the serotonin transporter 5HTTLPR 
polymorphism on symptom severity in irritable bowel syndrome. PLoS One. 
2013;8(2):e54831. doi:10.1371/journal.pone.0054831 
60.  
Dallal GE. The Computer-Aided Analysis of Crossover Studies. Gerard E. Dallal. 
Published 
2012. 
Accessed 
September 
17, 
2017. 
http://www.jerrydallal.com/lhsp/crossovr.htm 
61.  
Roalfe AK, Roberts LM, Wilson S. Evaluation of the Birmingham IBS symptom 
questionnaire. BMC Gastroenterol. 2008;8:30. doi:10.1186/1471-230X-8-30 
62.  
Adam B, Liebregts T, Holtmann G. [Irritable bowel syndrome]. Dtsch Med Wochenschr. 
2005;130(8):399-401. doi:10.1055/s-2005-863064 
63.  
Fennerty MB. Traditional therapies for irritable bowel syndrome: an evidence-based 
appraisal. 
Rev 
Gastroenterol 
Disord. 
2003;3 
Suppl 
2:S18-24. 
http://www.ncbi.nlm.nih.gov/pubmed/12775999 
64.  
Johanson JF. Options for patients with irritable bowel syndrome: contrasting traditional 
and novel serotonergic therapies. Neurogastroenterol Motil. 2004;16(6):701-711. 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 37 of 77 
doi:10.1111/j.1365-2982.2004.00550.x 
65.  
Svedlund J, Sjödin I, Dotevall G. GSRS--a clinical rating scale for gastrointestinal 
symptoms in patients with irritable bowel syndrome and peptic ulcer disease. Dig Dis Sci. 
1988;33(2):129-134. http://www.ncbi.nlm.nih.gov/pubmed/3123181 
66.  
Patrick DL, Drossman DA, Frederick IO, DiCesare J, Puder KL. Quality of life in persons 
with irritable bowel syndrome: development and validation of a new measure. Dig Dis 
Sci. 1998;43(2):400-411. http://www.ncbi.nlm.nih.gov/pubmed/9512138 
67.  
Drossman DA, Patrick DL, Whitehead WE, et al. Further validation of the IBS-QOL: a 
disease-specific quality-of-life questionnaire. Am J Gastroenterol. 2000;95(4):999-1007. 
doi:10.1111/j.1572-0241.2000.01941.x 
68.  
Missbach B, Schwingshackl L, Billmann A, et al. Gluten-free food database: the 
nutritional quality and cost of packaged gluten-free foods. PeerJ. 2015;3:e1337. 
doi:10.7717/peerj.1337 
69.  
Sapone A, Bai JC, Ciacci C, et al. Spectrum of gluten-related disorders: consensus on new 
nomenclature and classification. BMC Med. 2012;10:13. doi:10.1186/1741-7015-10-13 

June 2022- Version 6 
                                                                                                     Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
 
Page 38 of 77 
APPENDIX 1: Graphic of Dietary Trends in the US69 
From:  
BMC Med. 2012; 10: 13.  
Published online 2012 February 7. doi: 10.1186/1741-7015-10-13 
 
 
Figure 3 
 
Click on image to zoom 
 
Trend of three different diets (low carbohydrate diet, fat-free diet, and gluten-free diet), 
in the USA during the period 2004 to 2011. For the American general population, 
adopting a gluten-free diet is becoming an increasingly popular option. The market for 
gluten-free food and beverage products grew at a compound annual growth rate of 28% 
from 2004 to 2011, eclipsing the low carbohydrate diet and the fat-free diet in 2008, to 
finish with almost $1.6 billion in retail sales in 2010. By 2012 the market is expected to 
reach about $2.6 billion in sales. The fact that approximately three million Americans 
suffer from celiac disease and only a fraction of these patients have been diagnosed 
implies that patients suffering other forms of proven gluten reaction, including gluten 
sensitivity and wheat allergy, contribute to this market growth. The rest of the market is 
filled either by people who undertake the diet as occasional consumers (no medical 
necessity) or by individuals affected by maladies that have been claimed to be affected 
by gluten exposure, including autism spectrum disorder, attention deficit hyperactivity 
disorder, multiple sclerosis and irritable bowel syndrome, but for which there is no 
evidence of the effectiveness of the diet.

June 2022- Version 6 
                                                                                                     Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
 
Page 39 of 77 
APPENDIX 2: Irritable Bowel Syndrome Symptom Severity Score (IBS-SSS) 
 
 
 
 
 
 
Diagnosis and Management of IBS in Adults
IBS Severity Score
Figure 2.
Scoring system to assess the severity of irritable bowel syndrome (IBS) symptoms.
Adapted with permission from Francis CY, Morris J, Whorwell PJ. The irritable bowel severity scoring
system: a simple method of monitoring irritable bowel syndrome and its progress. Aliment Pharmacol Ther.
1997;11(2):401.
Copyright © 2012 by the American Academy of Family Physicians.
This content is owned by the AAFP. A person viewing it online may make one printout of the material and may
use that printout only for his or her personal, non-commercial reference. This material may not otherwise be
downloaded, copied, printed, stored, transmitted or reproduced in any medium, whether now known or later
invented, except as authorized in writing by the AAFP. Contact afpserv@aafp.org for copyright questions and/or
permission requests.

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 40 of 77 
APPENDIX 3: PROMIS Scale: Bloating & Gas 
 
 
 
PROMIS Scale v1.0 – Gastrointestinal Gas and Bloating 13a  
 
 
Gastrointestinal Gas and Bloating    
 
 
Please respond to each question or statement by marking one box. 
 
In the past 7 days… 
 
 
 
 
 
 
 
 
 
 
1 
GISX94 
 Did you have swelling in your belly? 
 
 
B 
No →  If No, go to #5 
 
 
A 
Yes  
2 
GISX95 
 How bad did the swelling in your belly get? 
 
 
1 
Not bad at all 
 
 
2 
A little bad 
 
 
3 
Somewhat bad 
 
 
4 
Quite bad 
 
 
5 
Very bad 
3 
GISX96 
 How much did the swelling in your belly interfere with your day-to-day activities? 
 
 
1 
Not at all 
 
 
2 
A little bit 
 
 
3 
Somewhat 
 
 
4 
Quite a bit 
 
 
5 
Very much 
Last Updated: 2 September 2016 
© 2010-2016 PROMIS Health Organization and PROMIS Cooperative Group           Page 1 of 5 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 41 of 77 
 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 42 of 77 
 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 43 of 77 
 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 44 of 77 
 
 
 
 
 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 45 of 77 
APPENDIX 4: PROMIS Scale: Belly Pain 
 
 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 46 of 77 
 
 
 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 47 of 77 
APPENDIX 5: PROMIS Scale: Diarrhea 
 
 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 48 of 77 
 
 
 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 49 of 77 
APPENDIX 6: PROMIS Scale: Constipation 
 
 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 50 of 77 
 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 51 of 77 
 
 
 
 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 52 of 77 
APPENDIX 7: Stool consistency (Bristol Stool Form Scale): (all patients) 
Please indicate the most common pattern of stool consistency that you had over the last seven 
days according to this scale: 
 
 
APPENDIX 8: Stool frequency:  
Please indicate the average number of bowel movements (i.e., number of times you have 
opened your bowels) per day in the last 7 days:    
 
Average bowel movements per day:  __ 
 
 
 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 53 of 77 
APPENDIX 9: DASS-21 Questionnaire 
 
 
DASS21  
Name: 
 
 
 
 
 
Date: 
 
 
Please read each statement and circle a number 0, 1, 2 or 3 which indicates how much the statement 
applied to you over the past week. There are no right or wrong answers. Do not spend too much 
time on any statement. 
 
The rating scale is as follows: 
 
0 
Did not apply to me at all 
1 
Applied to me to some degree, or some of the time 
2 
Applied to me to a considerable degree or a good part of time 
3 
Applied to me very much or most of the time 
 
1 (s) 
I found it hard to wind down 
 
0 
1 
2 
3 
2 (a) 
I was aware of dryness of my mouth 
 
0 
1 
2 
3 
3 (d) 
I couldn’t seem to experience any positive feeling at all 
 
0 
1 
2 
3 
4 (a) 
I experienced breathing difficulty (e.g. excessively rapid breathing, 
breathlessness in the absence of physical exertion) 
 
0 
1 
2 
3 
5 (d) 
I found it difficult to work up the initiative to do things 
 
0 
1 
2 
3 
6 (s) 
I tended to over-react to situations 
 
0 
1 
2 
3 
7 (a) 
I experienced trembling (e.g. in the hands) 
 
0 
1 
2 
3 
8 (s) 
I felt that I was using a lot of nervous energy 
 
0 
1 
2 
3 
9 (a) 
I was worried about situations in which I might panic and make a fool 
of myself 
 
0 
1 
2 
3 
10 (d) 
I felt that I had nothing to look forward to 
 
0 
1 
2 
3 
11 (s) 
I found myself getting agitated 
 
0 
1 
2 
3 
12 (s) 
I found it difficult to relax 
 
0 
1 
2 
3 
13 (d) 
I felt down-hearted and blue 
 
0 
1 
2 
3 
14 (s) 
I was intolerant of anything that kept me from getting on with what I 
was doing 
 
0 
1 
2 
3 
15 (a) 
I felt I was close to panic 
 
0 
1 
2 
3 
16 (d) 
I was unable to become enthusiastic about anything 
 
0 
1 
2 
3 
17 (d) 
I felt I wasn’t worth much as a person 
 
0 
1 
2 
3 
18 (s) 
I felt that I was rather touchy  
 
0 
1 
2 
3 
19 (a) 
I was aware of the action of my heart in the absence of physical 
exertion (e.g. sense of heart rate increase, heart missing a beat) 
 
0 
1 
2 
3 
20 (a) 
I felt scared without any good reason 
 
0 
1 
2 
3 
21 (d) 
I felt that life was meaningless 
 
0 
1 
2 
3 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 54 of 77 
 
 
 
 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 55 of 77 
APPENDIX 10: Somatic Symptoms (PHQ-15) 
 
 
 
 
 
PHYSICAL SYMPTOMS   
(PHQ-15) 
 
 
During the past 4 weeks, how much have you been bothered by any of the following problems? 
 
 
Not 
bothered 
at all 
(0) 
Bothered  
a  
little 
(1) 
Bothered 
a 
lot  
(2) 
a.   Stomach pain 
F 
F 
F 
b.   Back pain 
F 
F 
F 
c.   Pain in your arms, legs, or joints (knees, hips, etc.) 
F 
F 
F 
d.   Menstrual cramps or other problems with your periods   
WOMEN ONLY 
F 
F 
F 
e.   Headaches 
F 
F 
F 
f.    Chest pain 
F 
F 
F 
g.   Dizziness 
F 
F 
F 
h.   Fainting spells 
F 
F 
F 
i.    Feeling your heart pound or race 
F 
F 
F 
j.    Shortness of breath 
F 
F 
F 
k.   Pain or problems during sexual intercourse 
F 
F 
F 
l.   Constipation, loose bowels, or diarrhea 
F 
F 
F 
m. Nausea, gas, or indigestion 
F 
F 
F 
n.  Feeling tired or having low energy 
F 
F 
F 
o.  Trouble sleeping 
F 
F 
F 
                                                      
                                                   (For office coding: Total Score T_____  =  _____   +   _____ ) 
 
Developed by Drs. Robert L. Spitzer, Janet B.W. Williams, Kurt Kroenke and colleagues, with an educational grant 
from Pfizer Inc.  No permission required to reproduce, translate, display or distribute. 
 
 
 
 
           

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 56 of 77 
APPENDIX 11: SHAPE Method – Procedure   
  
SITZMARKS diagnostic test helps physicians select the best therapeutic option, based on 
colon transit time, for adult patients with severe constipation who have otherwise negative GI 
evaluations. 
• 
Convenient gelatin capsules 
• 
Efficient pre-cut radiopaque rings 
• 
Time saving and cost effective 
• 
CPT code 99070 
• 
10 capsules per package; each capsule contains 24 Radiopaque polyvinyl chloride 
markers of 1 mm X 4.5 mm 
SUGGESTED DIRECTIONS TO THE PHYSICIAN: 
 
 
(1 capsule: each capsule contains 24 markers) 
 
Step 1: 
 
1. On day 0, direct patient to take SITZMARKS capsule by mouth with water, preferably 
with confirmation by office observation. Instruct patient to abstain from using 
laxatives, enemas or suppositories of any kind for 2 days. 
2. Arrange a flat-plate abdominal X-ray on day 2 to determine the location and the extent 
of elimination of the radiopaque markers. 
3. Patients who expel 4-20 (16.7-83.3%) markers have grossly normal colonic transit. 
4. Patients who retain 6 or more markers may have follow-up abdominal X-rays within 
several days. 
5. For patients whose markers accumulate in the rectosigmoid or when markers are 
retained diffusely, Step 2 may be warranted 
 
 
Reading the Results: 
 
If 80% of the markers are passed by day 5, colonic transit is grossly normal. If remaining markers 
are scattered about the colon, the condition is most likely hypomotilty or colonic inertia. If 
remaining markers are accumulated in the rectum or rectosigmoid, the condition is most likely 
functional outlet delay, e.g., internal rectal prolapse, anismus. 
 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 57 of 77 
 
A. If 5-20 markers remain, 
patient has grossly normal 
colonic transit. 
B. Most rings are scattered 
about the colon. Patient most 
likely has hypomotility or 
colonic inertia. 
C. Most rings are gathered in 
the rectosigmoid. Patient has 
functional outlet obstruction. 
 
 
 
 
 
 
 
 
 
 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 58 of 77 
APPENDIX 12: Isolation and Cryopreservation of Human PBMC from Whole Blood 
 
(ValentinLEDUCQ, June 2014) 
1. Principle. 
 
Peripheral blood mononuclear cells (PBMC) are isolated from whole blood collected in heparin 
or citrate vacutainer tubes, by use of a density gradient centrifugation procedure. Then, PBMC 
can be frozen and stored into a cryoprotective media containing 10% DMSO and FBS, in order 
to have an acceptable yield and viability post-storage.  
 
2. Materials and equipment. 
a. PBMC isolation. 
• Histopaque (Histopaque-1077, #10771 Sigma) 
• 50 ml conical sterile centrifuge tubes 
• 15 ml conical sterile centrifuge tubes 
• PBS without Ca/Mg (PAA) 
• RPMI-1640 complete (10%FBS, 10mM Penicillin Streptomycin, L-glutamine) 
• Trypan blue 0.4% 
b. PBMC cryopreservation. 
• Sterile Cryovials 2 ml 
• RPMI-1640 complete (10%FBS, 10mM Penicillin Streptomycin, L-glutamine) 
• 2X freezing medium (20% DMSO in FBS) 
• DMSO Anhydrous (LifeTechnologies #15250-061) 
• PBS 1X without Ca/Mg (PAA) 
• Hemocytometer  
• Mr. Frosty freezing container 
• -80°C freezer 
• Liquid nitrogen freezer 
 
3. Procedure. 
a. PBMC isolation. 
• After carefully checking the sample identification on the blood tubes, remove the whole 
blood from the heparin tubes and transfer to its corresponding 50ml centrifuge tubes.  
• Mix the Histopaque-1077 prior to opening the bottle. 
• Gently layer the blood onto an equal volume of Histopaque-1077, in a 15 ml conical 
centrifuge tube. Split the blood of one sample in as many tubes you need to use all the 
blood. 
• Centrifuge at room temperature for 30 minutes at 400g, without brake. 
• Remove the plasma and white cell interface and transfer to the new appropriately 
labeled 50ml conical tube. Try to retrieve all the cells from the interface without 
disturbing the red pellet at the bottom of the ficoll layer. 
• Add RPMI w/o FBS to the new tube up to 50mls.  Be sure to add at least an equal 
volume of RPMI. If this is not possible in a single tube, split into two 50 ml tubes. 
• Centrifuge for 10 minutes at room temperature at 400g. Remove the supernatant from 
the pellet carefully and discard in an appropriate manner. 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 59 of 77 
• Resuspend each pellet in a small volume of RPMI, and then fill each tube to 50ml with 
RPMI w/o FBS. 
• Centrifuge for 10 minutes at room temperature at 400g. Remove the supernatant from 
the pellet carefully and discard in an appropriate manner. 
• Resuspend cells for counting in 1ml complete RPMI at room temperature.  
• Dilute 10µl of the cell suspension into 490µl PBS 1X + 500µl Trypan blue 0,4% (100X 
dilution factor).  
• With a cover-slip in place, transfer 10µl into the chamber of the hemocytometer. Count 
cells in the 100 squares : 
 
!"## $#
⁄
 =  ("##) × 10! 
 
• Freeze the cells according to the Cryopreservation protocol, freezing about 10.106 cells 
per vial. Freeze at least 3 vials per sample. Thus, if there are less than 30.106 cells in a 
sample, split evenly into at least 2 vials. 
b. PBMC cryopreservation. 
• Label cryovials to freeze the sample (sample identification, number of cells). 
• Take an appropriate volume of the sample to harvest 30.106 cells, and complete with 
cRPMI to a final volume of 1,5 ml. If there are less than 30.106 cells total, just 
complete the sample with cRPMI to a final volume of 1,5 ml. 
• Add 1,5 ml 2X freezing medium at room temperature to the 1,5 ml cell suspension. 
Gently swirl the tube when adding the freezing medium and dispense 1ml per cryovial.  
• Place the cryovials in a room temperature Mr. Frosty freezing container.  
• Place the freezing container as soon as possible into the -80°C freezer.  
• Transfer the cryovials to liquid nitrogen tank after 1 to 14 days for long-time 
conservation.  
 
 
 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 60 of 77 
APPENDIX 13: PBMC isolation and preparation 
 
Peripheral blood mononuclear cells (PBMC) are isolated from whole blood collected in 
heparin or citrate vacutainer tubes, by use of a density gradient centrifugation procedure 
(Ficoll). 
 
PBMC are re-suspended at 106 cells/ml into complete RPMI and plated in a 96 wells culture plate 
(180µl of the cell suspension, 0.18x106 cells per well). The cells then rest for 12 hours at 37°C, 
5% CO2 before any stimulation procedure. 
 
PBMC stimulation. 
 
PBMC are then stimulated (in duplicate) using the following stimuli for 24 hours at 37°C, 5% 
CO2. 
 
Stimuli 
Final 
Concentratio
n 
MDP (invivogen, tlrl-mdp) 
10 µg/ml 
Curdlan (invivogen, tlrl-cura) 
10 µg/ml 
Heat Killed C. albicans 
106 CFU/ml 
Heat Killed E. Coli 
106 CFU/ml 
LPS e.coli (sigma, #12630) 
10 ng/µl 
PMA (invivogen, tlrl-pma) 
500 ng/ml 
Ionomycin (sigma, #I0634) 
1 µg/ml 
PBS 1X 
/ 
 
After 24 hours, the supernatant is collected and stored at -80°C for further analysis (using 
multiplex beads array). 
 
 
 
 
 
 
 
 
 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 61 of 77 
 
APPENDIX 14: Study Design 
 
 
 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 62 of 77 
APPENDIX 15: GFD Instructions 
 
 
 
 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 63 of 77 
APPENDIX 16: Food Frequency Questionnaire (SPOR Study) 
 
 
 
 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 64 of 77 
APPENDIX 17: Expert Dietitian Evaluation of GFD Adherence 
 
In general, how compliant do you believe the participant is with the GFD (include accidental 
and/or intentional ingestion of gluten in analysis)? 
 
1. Excellent 
● Participant eats gluten fewer than 3 times per year 
● Uses only celiac-friendly restaurants or asks thorough questions when dining out 
● Follows “when in doubt, leave it out” motto 
● Review of diet reveals no obvious gluten sources 
● Checks medications, supplements, and body care products that enter nose, eyes, and mouth 
● Reads labels well 
● Has eliminated cross-contamination potential in the kitchen 
 
2. Good 
● Participant eats gluten once per month 
● Asks useful questions in restaurants 
● Shows a high level of confidence in following the diet 
● Review of diet may show minimal hidden gluten exposure 
● Checks medications, supplements, and body care products 
● Reads labels well 
● Does not eat contaminated oats 
● Has eliminated most, if not all, cross-contamination from kitchen 
 
3. Fair 
● Participant eats gluten 2 to 3 times per month 
● Does not ask questions in restaurants or when dining out—guesses or takes chances 
● Checks some but not all medications, supplements, and body care products 
● Shows some confusion over label reading 
● Review of diet shows some obvious gluten exposure 
● Consumes gluten on occasion—intentionally or unintentionally—per diet/lifestyle recall 
● May rely on partner/family member for some caregiving regarding diet 
● Has not eliminated cross-contamination potential from kitchen 
● May have mental or behavioral issues that make following the diet more difficult 
● May have language skills that make dietary adherence more difficult 
 
4. Poor 
● Participant eats gluten 1 to 2 times per week 
● Does not ask questions in restaurants or when dining out 
● Does not check medications, supplements, or body care products 
● Consumption of gluten containing foods for religious reasons (e.g., Communion/Passover) 
● Requires repeated diet education on hidden gluten, and so forth 
● Cheats regularly (1/wk) and intentionally 
● May rely often/completely on partner/family member to answer my questions 
● Has contaminated kitchen area 
● May have mental or behavioral issues that make following the diet more difficult 
● May have language skills that make dietary adherence more difficult 
 
5. Very poor 
● Participant eats gluten more than 2 times per week 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 65 of 77 
● Does not ask questions in restaurants or when dining out 
● Does not check medications, supplements, or body care products 
● Takes Communion or Passover regularly 
● Requires repeated diet education on hidden gluten, and so forth 
● Cheats regularly (2/wk or more) and intentionally 
● May rely often/completely on partner/family member to answer my questions 
● Has contaminated kitchen area 
● May have mental or behavioral issues that make following the diet more difficult 
● May have language skills that make dietary adherence more difficult 
● May show poor adherence/motivation as a result of denial, anger, or budgetary, family, or 
social concerns 
 
6. No GFD 
Participant does not follow the GFD 
 
Food Label Quiz 
Circle ingredients that participant incorrectly classifies. 
(1) Dehydrated potatoes 
(2) Oat gum 
(3) Sugar 
(4) Corn oil 
(5) Partially hydrogenated corn oil 
(6) Sea salt 
(7) Soy lecithin 
(8) Wheat flour 
(9) Leavening (sodium bicarbonate) 
(10) Natural flavors 
(11) Sucrose 
(12) Molasses 
(13) Spices 
(14) Wheat starch 
(15) Tomato paste 
(16) Dextrose 
(17) Malt extract 
(18) Maltodextrin 
(19) Extracts of paprika 
(20) Citric acid 
(21) Beef fat 
(22) Soy flour 
(23) Corn syrup solids 
(24) Barley malt flour 
(25) Lactic acid 
(26) Egg yolk 
(27) Casein 
(28) Peanut oil 
(This ingredient list is a modified ingredient list for potato 
chips from a major manufacturer.) 
 
Record Review/Recall 
Review of 3-day food record or 24-hour diet recall 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 66 of 77 
APPENDIX 18: Gluten Purification Protocol 
 
1. Make sure the temperature of the room is 4-8 degrees Celsius. 
2. Wash gluten with 5 mL/g of a solution of 50 mM ammonium bicarbonate and 1 M sodium 
chloride by soaking it in a bowl for 2 hours. 
3. After 2 hours, strain the gluten in a strainer or cloth. 
4. Wash the gluten with 5 mL/g of a solution of 50 mM ammonium bicarbonate and 1 M sodium 
chloride by soaking it in a bowl for 2 hours. 
5. After the 2 hours, again strain the gluten in a strainer or cloth. 
6. Wash the gluten with 5 mL/g of a solution of 50 mM ammonium bicarbonate and 1 M sodium 
chloride by soaking it in a bowl for 2 hours. 
7. After the 2 hours, again strain the gluten in a strainer or cloth. 
8. Wash the gluten with 5 mL/g of a solution of 50 mM ammonium bicarbonate and 1 M sodium 
chloride by soaking it in a bowl for 10 minutes. 
9. After the 10 minutes, again strain the gluten in a strainer or cloth. 
10. Wash the gluten with 5 mL/g of a solution of 50 mM ammonium bicarbonate and 1 M sodium 
chloride by soaking it in a bowl for 10 minutes. 
11. After the 10 minutes, again strain the gluten in a strainer or cloth. This should be thorough, 
should de-enrich by 80-90%.  
12. Lyophilize. 
13. Store in container – any type (glass, plastic, etc).  
 
 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 67 of 77 
APPENDIX 19: Granola Bars Recipe (Modified) 
 
Makes 14 servings x 16 batches = 224 granola bars 
Ingredients 
375 g (4.5 cups) rolled oats x 16 batches = 6000 g of oats 
140 g of gluten flour x 16 batches = 2240 g of gluten total 
140 g of whole wheat flour x 16 batches = 2240 g of flour total 
4.6 g (1 tsp) baking soda x 16 batches = 73.6 g of baking soda 
4.33 g (1 tsp) vanilla extract x 16 batches = 73.6 g or 80 mL 
157.8 g (2/3 cup) olive oil x 16 batches = 2667 mL olive oil 
350 g (1 cup) rice malt syrup x 16 batches = 4000 mL 
77.5 g (1/3 cup) packed brown sugar x 16 batches = 1240 g of brown sugar 
177 g semisweet chocolate chips x 12 batches = 2142 g of chocolate chips 
25 g pumpkin seeds (or, if chocolate-free, then 145 g pumpkin seeds) 
 
Directions 
1. Preheat oven to 325 degrees F (165 degrees C). Lightly grease one 9x13 inch pan. 
2. In a large mixing bowl combine the oats, flour, baking soda, vanilla, oil, rice malt syrup 
and brown sugar. Stir in the chocolate chips and pumpkin seeds. 
3. Lightly press mixture into the prepared pan. Bake at 325 degrees F (165 degrees C) for 
18 to 22 minutes or until golden brown. Let cool for 10 minutes then cut into bars. Let 
bars cool completely in pan before removing or serving. 
 
 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 68 of 77 
APPENDIX 20: Information Sheet for Urine Collection at Home 
 
Dear study participant, 
For the current study, we kindly ask you to collect your urine sample on the days that were 
communicated to you by the study staff. They will provide you with all necessary materials for 
collection purposes (plastic 50 mL container with orange top).  
 
Collect your urine (Date: __/____/____) 
Collect the first morning urine (mid-stream portion) directly into a plastic bottle provided by 
the investigators.  
Write the time and date of urination on the urine container.  
 
Preserve the Sample 
You must put the bottle in the fridge (temperature 4-8C) in an upright position until you 
transfer it to the study staff.  
 
Transfer to the study staff 
Transport the urine sample in an upright position in a plastic bag (together with stool sample) 
as soon as possible.  
 
 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 69 of 77 
APPENDIX 21: Information sheet for stool collection at home 
 
Dear Sir or Madame, 
 
Please read the following the instructions carefully.  
 
For the current study, we kindly ask you to collect a stool sample before each visit. The study 
staff provided you with all necessary material for collection purposes (plastic plate, sample 
container, aluminum bag, AnaeroGen sachet, isotherm bag, ice block). You should put the ice 
block in the freezer as soon as possible. 
Please collect your stool sample within 24 hours of your study visit by following the procedure 
below: 
 
Collection of stool  
Ø Defecate directly onto the plastic plate (or the cardboard plate, if provided) 
Ø Transfer approximately 5 teaspoons (25 mL or just under 2 tbsp) of stool sample into 
the plastic container (white top container, 50 mL) using the provided spatula 
Ø Close the lid but pay attention to leave it a bit open to allow the air to exchange 
Ø Note the date and time of defecation on the container, as well as study ID or name 
 
Preserve the stools 
Ø Open the Oxoid sachet by tearing at the level of the notch 
Ø Take the paper sachet that is inside and immediately place it into the smaller plastic bag 
Ø Transfer the sample pot in the upright position in the smaller plastic bag (the pot lid 
must remain slightly opened to allow air exchange) 
Ø Immediately seal the bag 
Ø Place the smaller bag into the larger plastic bag and seal the larger bag 
Ø After 30 minutes, put the bag in the FREEZER in the upright position until you transfer 
it to the study staff 
 
 
Transfer to the STUDY Staff 
Ø Transport the samples in an upright position in the isotherm bag after having added the 
ice block that was placed in advance in the freezer 
 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 70 of 77 
APPENDIX 22: General rules for research coordinators and study participants 
 
1. Prior to On-Site Arrival 
• 
Ensure that research coordinator and study participant have completed self-screen and 
have passed all screening criteria as required by MDCL and HHS sites 
• 
Ensure having a clean mask as required per current masking regulations for McMaster 
University/HHS site 
• 
Once on-site, a sign-in sheet will be available to document the research associate’s 
name and arrival/departure time 
 
2. Follow proper hygiene: 
• 
Wash hands thoroughly and regularly, following proper hand washing hygiene 
guidelines -- be sure to wash hands when entering and before leaving lab 3N59 
• 
Practice hand hygiene directly after contact with high touch areas 
• 
In areas with no proximity to a sink, use alcohol-based hand sanitizer (ensure hand 
sanitizer is available in the work area) 
• 
Avoid touching eyes, face, and mouth 
 
3.  Etiquette 
• 
Cover coughs and sneezes (cough/sneeze into upper sleeve, elbow, or tissue) 
• 
Turn away from others when coughing or sneezing 
• 
Respect and maintain 2 meters physical distancing 
 
4.  Masking 
• 
A mask must be worn in all public or shared spaces and at the time of contact with a 
study participant 
• 
When taking a new mask, ensure to wash your hands or apply hand sanitizer prior to 
reaching for fresh stock 
 
5.  Cleaning and Disinfection 
• 
At the start of work and prior to leaving, wipe down everything you have used with 
70% ethanol (desktop, computer keyboard, telephone, computer screen, benchtops, 
fridge, freezer and lab equipment) 
• 
Use spray bottles of 70% ethanol and paper towels to disinfect common touchpoints 
(door handles, light switches, desk, etc.) 
• 
When traveling between rooms, please use paper towels to handle the doorknob and 
avoid contaminating your hands. Dispose of towel when you reach your destination. 
• 
Avoid sharing of work tools and equipment where possible 
 
6.  Shared Infrastructure 
• 
Common areas within the laboratory and office must adhere to physical distancing 
rules and be sanitized after use   
• 
Mechanisms will be in place to ensure common shared areas will not be congested 
 
7.  Physical Distancing 
• 
The research coordinator and study participant must maintain a distance of 2 meters 
from each other 
• 
You may chat with co-workers at break, but physical distancing rules must apply 
 
8.  Departure 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 71 of 77 
• 
Please leave campus/HHS site promptly once essential tasks are completed 
 
RESEARCH PLAN FOR LAB SPACE (3N59) 
 
1. Research activities will be performed involving the work of Research Associates to 
support time-sensitive wet lab research which cannot occur from home – i.e. collection 
and processing of samples. These activities will be performed in room 3N59. 
2. All staff are fully trained for the task they will execute and have adopted a “shift work” 
schedule to ensure that physical distancing is maintained. One (1) Research Associate 
from the team will be permitted on site at a time. Shifts will be tracked using a Google 
Calendar. 
3. Self-monitoring of symptoms will continue to be vital. Anyone who feels unwell or 
may have been in contact with a possible COVID-19 case must not come to campus 
and should follow the advice on Ontario’s COVID-19 self-screening website. The 
number of personnel in each room will be limited to ensure sufficient space to allow for 
appropriate physical distancing (2 meters). 
 
CLEANING THE LAB AND OFFICE SPACE 
 
1. Each day, research staff are responsible to regularly wipe down common 
surfaces/equipment using 70% ETOH. In general, every time a piece of equipment is 
used it should be wiped down BEFORE AND AFTER use with 70% ETOH. These 
areas include, but are not limited to: fridge/freezer door handles, centrifuge(s), door 
handles, drawer pulls, benchtops, faucets/taps, Lab Specimen Binder, computer, 
keyboard, desktop, printers, etc. 
2. Housekeeping will increase the frequency of cleaning of highly touched surfaces, but 
you should avoid touching door handles wherever possible. 
3. Keep all benches clean and tidy. All lab benches are to be wiped down with 70% 
ETOH before and after each use. 
4. All shared equipment is to be wiped down with 70% ETOH before and after all 
processing. 
 
ENTERING AND EXITING MCMASTER UNIVERSITY HSC/MUMC SITE 
 
1. Entrance and exit procedures for the Health Sciences Centre is determined by the 
Faculty of Health Sciences. Please refer to the most recent notice in regard to entry 
requirements. 
2. Consider the use of stairs to move between floors 
3. Do not enter an elevator if 2 meters distancing is not possible 
4. Everyone entering research workplaces must then proceed to wipe down both the 
external and internal door handles with 70% ETOH before engaging in any work. This 
ensures a clean door handle for the next person. 
5. Wash hands or use hand sanitizer after touching high-touch surfaces 
6. Upon exiting research workplaces ensure that all high touch areas including common 
lab benches, desks, keyboards, drawer handles, freezer/fridge handles door handles, 
light switches have been wiped down with 70% ETOH. 
 
CHECKLIST FOR ENGAGING IN RESEARCH WORK IN OFFICE ROOMS  
 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 72 of 77 
1. Enter room and immediately proceed to wipe down external and internal door handles 
with 70% ETOH with paper towels 
2. Contain your personal belongings in a drawer at your desk.  Use of high touch items 
such as a cell phone should be minimized 
3. Ensure PPE is used as per guidelines 
4. Wipe down your desk area, drawer handles, keyboard, mouse and all benches and lab 
equipment with 70% ETOH that are needed or used to complete your work 
5. Complete your work in a timely fashion, do not hang around the office/lab 
unnecessarily 
6. Physical distancing must be maintained during breaks. Remove all lab PPE before 
exiting the lab 
7. When you have finished your lab work, wipe down all benches and equipment with 
70% ETOH 
8. Gather your things and do a final wipe down of your desk, drawer handles, and interior 
door handles with 70% ETOH. Leave the premises promptly after completing your 
daily tasks. 
 
 
 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 73 of 77 
APPENDIX 23: Biohazard Container for Study Samples 
 
 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 74 of 77 
APPENDIX 24: COVID-19 Measures for Patient Sample Handling 
 
1. Preparing sample collection kits: Once the study coordinators thoroughly wash and 
sanitize their hands, they will prepare the stool and urine collection kits. The collection 
kit components are stored in a separated, low-traffic research staff room. 
2. Mailing/drop off collection kits: Stool and urine collection kits will be placed in a 
package and will not be opened until delivered to the study participant’s address. 
3. Instructions for proper stool/urine collection: 
a. Participants will be instructed to wash their hands and wear gloves while 
collecting the samples. 
b. After collecting the sample, participants will clean the collection tube and place 
it in a double hermetic bag. 
c. The participants will then dispose of gloves and other used materials. 
4. Instructions for pick up and transport: After the participant collects the sample, they 
will complete the online COVID-19 screening tool (Ontario COVID-19 Self-
Assessment). If they pass the screening, they will coordinate a contactless pickup with 
the study coordinator where the sample will be placed in a specialized hermetic hazard 
container approved by Transport Canada. 
5. Instructions for stool sample processing (aliquoting) and storage: 
a. Once samples are placed in the anaerobic chamber, the study coordinator will 
sanitize or wash their hands and put on gloves. 
b. Clean surfaces with 70% alcohol and make sure that other samples, cultures, 
and objects are far enough to prevent contamination if there are spills or 
splashes during sample aliquoting. 
c. Aliquot stool samples slowly from primary tube to smaller tubes placed in a 
clean plastic sample box. 
d. Once aliquoting is done, clean chamber surfaces and the closed sample box with 
alcohol 70%. 
e. Dispose of all used materials in a double biohazard bag. Remove the sample 
box and biohazard bags from the anaerobic chamber. 
f. Samples will be stored in a -80 freezer. 
 
 
 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 75 of 77 
APPENDIX 25: Declaration of Helsinki 
 
 
 
 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 76 of 77 
APPENDIX 26: Serious Adverse Events Form 
 
 
Serious Adverse Event (SAE)  
Report Form Version 3.0 
1 of 2 
13Jan2014 
 
Serious Adverse Event (SAE)  
Report Form 
STUDY NAME 
Protocol Number: 
 
Site Name: 
 
Pt ID: 
 
Date Participant Reported: 
            /                   /                        . 
 d d 
m m m 
y y 
y 
y 
 
1. 
SAE onset date: 
___ ___ / ___ ___ ___ / ___ ___ ___ ___ 
 
d 
d 
m m m 
y 
y 
y 
y 
2. 
SAE stop date:  
___ ___ / ___ ___ ___ / ___ ___ ___ ___ 
 
d 
d 
m m m 
y 
y 
y 
y 
3. 
Location of SAE: 
 
4. 
Was this an unexpected adverse event? 
 Yes 
 No 
5. 
Brief description of participants with no personal identifiers: 
Sex:  
 F 
 M 
Age:  
 
Diagnosis for study participation: 
 
 
 
6. 
Brief description of the nature of the SAE (attach description if more space is needed):  
 
 
 
 
7. 
Category of the SAE: 
 Date of death 
___/_____/_______ 
 
 
(dd/mmm/yyyy) 
 Life threatening 
 Hospitalization – initial or prolonged 
 Disability/incapacity 
 Congenital anomaly/birth defect 
 Required intervention to prevent permanent 
impairment 
 Other: 
 
8. 
Intervention type: 
 Medication or nutritional supplement (specify): 
 
 Device (specify): 
 
 Surgery (specify): 
 
 Behavioral/lifestyle (specify): 
 

June 2022- Version 6  
 
 
 
                                                              Diet in IBS 
Study No 09.25.NRC 
CONFIDENTIAL 
 
Page 77 of 77 
 
 
Serious Adverse Event (SAE)  
Report Form Version 3.0 
2 of 2 
13Jan2014 
9. 
Relationship of event to intervention: 
 Unrelated (clearly not related to the intervention) 
 Possible (may be related to intervention) 
 Definite (clearly related to intervention) 
10. Was study intervention discontinued due to event? 
 Yes 
 No 
11. What medications or other steps were taken to treat the SAE? 
 
 
12. List any relevant tests, laboratory data, and history, including preexisting medical conditions: 
 
 
13. Type of report: 
 Initial 
 Follow-up 
 Final 
Signature of principal investigator:  _______________________________  Date:  _________________