Conditions: Autoimmune Diseases Relapsing Multiple Sclerosis
Keywords: microbiome multiple sclerosis microbiota gut
Allocation: RANDOMIZED | Intervention model: CROSSOVER
Enrollment: 14
Healthy volunteers: yes

Brief summary:
The proposed randomized, open label, with treat as usual control group (standard treatment or any disease modifying drugs), crossover phase II study will be conducted in 40 patients (n=20 per group) with the relapsing forms of multiple sclerosis according to the McDonald 2010 Criteria.

Patients will be randomized into 2 intervention groups. One will receive the FMT from month 1 and for the first 6 months (early intervention group). On the other hand, the other group will be a control group during the first 6 months and will receive the FMT for the last 6 months of the study. Patients will be screened for eligibility based on MS diagnosis and EDSS and if eligible then consented. All qualified patients will not be currently or recently treated with high dose steroids.

Detailed description:
At Visit 1, before FMT(fecal microbial transplantation), patients will be evaluated for their vital signs, medical history and concomitant medications. Also before transplantation, patient's stool will be collected to study their microbial profile, blood collected for analysis to evaluate cytokines levels as well as blood DNA bacteria and finally, urinalysis to assess gut permeability (baseline). Other assessments (prior to the first dose of therapy) include an Expanded Disability Status Scale (EDSS), pregnancy test (if applicable), physical exam and ECG. Blood samples are also taken at month 1 in order to establish a baseline for routine chemistry/hematology. After all these assessments FMT will be performed by a trained nurse via a rectal enema.

FMT for the early intervention group will be at V1, 2, 2.1, 2.2, V3 and V4. FMT randomized to late intervention group will be V4, 5, 6, 6.1.6.2, 6.3 and V7

Both groups, at Visit 1, Visit 4 and Visit 7, patients will be instructed to drink lactulose solution and collect the urine throughout the previous night and first thing in the morning. A proper collecting bottle will be provided and will also undergo a contrast-enhanced brain MRI scan at Robarts Institute London Ontario.

Those randomized to the Early Intervention group, will return to the clinic for visit 2, 1 month after the first FMT(fecal microbial transplantation). Another stool sample to evaluate the microbial before the second FMT will be collected and peripheral blood samples for cytokines and blood bacterial DNA analysis. Both groups this same routine procedure repeated at visits 2.1, 2.2, 3, 4, 5, 6, 6.1.6.2, 6.3 and visit 7. Another safety assessment 2 weeks after FMT is to review any adverse events that may have occurred.

Both groups will have an MRI at M1, M6 and M12.

Arms:
arm 1: Fecal microbiota via enema at Month 1, 2, 3. 4, 5 and month 6 along with stool, urine and blood collection. At months 7, 8, 9, 10, 11 and 12 only stool, urine and blood collection. arm 2: At months 1, 2, 3, 4, 5 and 6, stool, urine and blood collection. Fecal microbiota via enema at month 6, 7, 8, 9, 10, 11and 12 months along with stool, urine and blood collection.

Interventions: intervention 1: Fecal microbiota
Intervention descriptions: intervention 1: fecal microbial transplantation

Protocol document:
Prot_SAP_000.pdf: 
Study Protocol – FMT in MS 
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Ver 3.0  09 OCT 2018 
 
Protocol No. MSFMT -001 
Fecal Microbial Transplantation in Relapsing 
Multiple Sclerosis Patients. 
 
Study Protocol  
 
Protocol Number: MSFMT - 001 
 
 
Test Product: Fecal Microbiota 
 
Phase: II study 
 
 
Sponsor: Dr. Michael Silverman 
 
 
 
Principal Investigator: Michael Silverman, M.D., FRCP, FACP 
 
 +1 416-925-1266 
Protocol Date:  09 OCT2018 
 
 
Amendments - Version: 3.0 
 
 
 


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Protocol No. MSFMT -001 
 
CONFIDENTIALITY STATEMENT 
 
 
THE INFORMATION IN THE FOLLOWING DOCUMENT IS PRIVILEGED AND 
CONFIDENTIAL AND IS PROVIDED TO YOU AS AN INVESTIGATOR, 
POTENTIAL INVESTIGATOR, CONSULTANT OR CONTRACT ORGANIZATION 
FOR REVIEW BY YOU, YOUR DESIGNATE, YOUR STAFF AND APPROPRIATE 
ETHICAL REVIEW COMMITTEE.  BY ACCEPTING THIS DOCUMENT, YOU 
AGREE THAT THE INFORMATION CONTAINED HEREIN WILL NOT BE 
DISCLOSED TO OTHERS WITHOUT WRITTEN AUTHORITY FROM THE 
SPONSOR. EXCEPT TO THE EXTENT NECESSARY TO OBTAIN APPROVAL OF 
THIS PROTOCOL BY AN ETHICAL REVIEW COMMITTEE OR GOVERNMENT 
AGENCY OR IN THE PROCESS OF OBTAINING PATIENT CONSENT TO 
PARTICIPATE IN THIS STUDY. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Protocol No. MSFMT -001 
PROTOCOL/AMENDMENT APPROVAL PAGES 
 
Protocol Version: 3.0  
Date of Version:  10 SEPT 2018 
 
Authored by: 
 
 
_____________________________ 
Signature 
 
_____________________________ 
Date (dd/mm/yy) 
 
 
 
Approved by: 
 
_____________________________ 
 
Signature 
 
 
 
 
 
 
 
 
 
_____________________________ 
 
Date (dd/mm/yy) 
 
 
 
 
 
 
 
 
 


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Protocol No. MSFMT -001 
ABREVIATIONS 
 
MS multiple sclerosis 
CNS central nervous system 
RMS relapsing multiple sclerosis 
MRI magnetic resonance imaging 
FMT fecal microbiota transplantation 
TLR toll-like receptor 
CD cluster designation 
IBD irritable bowels disease 
EAE experimental acute encephalomyelitis 
PSA polysaccharide 
IL interleukin 
SCFA short chain fatty acids 
Th T helper 
KVC kamamicina, colistina and vancomycin 
ROR tyrosine kinase-like orphan receptor 
CDI Clostridium difficile infection 
UC ulcerative colitis 
DNA deoxyribonucleic acid 
RCT randomized clinical trial 
SAE severe adverse event 
EDSS expanded disability status scale 
RN registered nurse 
BMI body mass index 


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Protocol No. MSFMT -001 
HIV human immunodeficiency virus 
HTLV human T lymphotropic virus 
Ig immunoglobulin 
Ag antigen 
Ab antibody 
TTG  transglutaminase 
CMV cytomegalovirus 
EBV Epstein-Bar virus 
MRSA methicillin-resistant Staphylococcus aureus 
VRE vancomycin-resistant Enterococcus 
CRE carbapenemase producing Enterobacteriaciae 
CMRTO College of Medical Radiation Technologists of Ontario 
IUD intrauterine device 
AST aspartate aminotransferase 
ALT alanine transaminase 
GM-CSF granulocyte-macrophage colony stimulating factor 
TNF tumor necrosis factor 
RBC red blood cell 
WBC white blood cell 
INF interferon 
MIP macrophage inflammatory protein 
EDTA Ethylene Diamine Triacetic Acid 
PBS phosphate buffered saline 
RNA ribonucleic acid 
PCR polymerase chain reaction 


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Protocol No. MSFMT -001 
TGF transforming growth factor 
AE adverse events 
CRF clinical research form 
REB research ethics board 
IEC independent ethics committee 
GCP good clinical practice  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
TABLE OF CONTENTS 


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Protocol No. MSFMT -001 
 
1. GENERAL INFORMATION 
1616 
1. Pathophysiology of Multiple Sclerosis and gut bacteria  ................................... 1919 
2. Description of Fecal Microbial Transplantation  ................................................ 1919 
Fecal microbiota transplantation is the process of fecal bacteria transfer from a healthy donor 
into a recipient. The goal is to restore colonic microflora by transplanting healthy bacterial 
flora (e.g. by enema, orogastric tube or by mouth in the form of a capsule containing freeze-
dried material)12. For this study we selected the enema that is a lower risk procedure 
compared to an upper tract endoscopy and more easily accepted for patients than an oral 
capsule of fecal bacteria. An enema will be done to infuse the study treatment into the lower 
bowel through the rectum by a trained nurse.  .................................................................. 1920 
 4.1 Rationale for FMT preparation  .................................................................................. 3130 
4.2 Duration of therapy and study  .................................................................................... 3232 
4.3 Study Termination ...................................................................................................... 3232 
6. TREATMENT OF PATIENTS ....................................................................................... 3434 
6.2.1 Screening, Enrolment and Baseline  .................................................................... 3434 
7.0 ASSESSMENT OF EFFICACY 
3838 
8.0 ASSESSMENT OF SAFETY 3839 
8.1 Head MRI  .................................................................................................................. 3839 
A contrast head MRI will be performed at baseline, 6 months and 12 months for oboth groups 
as a safety measure to evaluate any subclinical activity or any paradoxical effect potentially 
caused by the FMT. .......................................................................................................... 3839 
8.2 Adverse events ........................................................................................................... 3839 
8.2.1 Definition of an Adverse Event ............................................................................. 3839 
8.2.2 Reporting of an Adverse Event ............................................................................. 3939 
8.2.3 Definition of a Serious Adverse Event .................................................................. 4243 


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8.4 Reporting of a serious adverse event ...................................................................... 4243 
8.5 Reporting of a pregnancy during the study ................................................................. 4244 
9. STATISTICAL ANALYSIS 
4344 
9.1 Sample Size Selection ................................................................................................ 4344 
9.2 Demographics ............................................................................................................ 4344 
9.3 Biomarkers ................................................................................................................. 4344 
  
9.4 Safety Analysis ................................................................................................ 4344 
9.5 Criteria for Study Termination ..................................................................................... 4345 
9.6 Missing Values ........................................................................................................... 4345 
9.7 Evaluable Patients ...................................................................................................... 4445 
10.1 Protocol Amendments  ............................................................................................. 4445 
  
10.2 Protocol Deviations ........................................................................................ 4445 
11. ETHICAL CONSIDERATIONS 4445 
11.1 Independent Ethics Committee (IEC)  ....................................................................... 4445 
  
11.2 Ethical Conduct of the Study .......................................................................... 4546 
  
11.3 Patient Information and Consent .................................................................... 4546 
12. DATA HANDLING AND RECORD KEEPING 
4647 
  
12.1 Study Documentation and Record Retention  ................................................. 4647 
12.2 Duties of the Investigational Staff.............................................................................. 4748 


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Protocol No. MSFMT -001 
1. Chris H Polman, Stephen C Reingold, Brenda Banwell, Michel 
Clanet, Jeffrey A Cohen, Massimo Filippi, Kazuo Fujihara, Eva 
Havrdova, Michael Hutchinson, Ludwig Kappos, Fred D 
Lublin, Xavier Montalban, Paul O'Connor, Magnhild Sandberg-
Wollheim, Alan J Thompson, Emmanuelle Waubant, Brian 
Weinshenker, and Jerry S Wolinsky. Diagnostic criteria for 
multiple sclerosis: 2010 Revisions to the McDonald criteria. Ann 
Neurol. 2011 Feb; 69(2): 292–302. 
4849 
 
 
 
 
 
 
 
CLINICAL STUDY SYNOPSIS 
 
Funding:  
Chapman Chair in MS Clinical 
Research - LHSC and LHSF 
Investigator:  
Marcelo Kremenchutzky +1 4Marcelo 
Kremenchutzky16-925-1266 
 
Name of test product:  Fecal microbiota 
 
 
Title of the study:  Fecal Microbial Transplantation in Relapsing Multiple 
Sclerosis Patients 
 


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Protocol No. MSFMT -001 
Principal Investigator and study center:   
Michael Silverman, M.D., FRCP, FACP  
St. Joseph’s Health Care London 
268 Grosvenor St.  
London Ontario 
N6A 4V2 
 
Clinical phase: II 
 
Primary objectives:  
o To evaluate effects of fecal microbial transplant (FMT) from healthy donors on 
biological biomarkers as peripheral blood cytokines levels of relapsing multiple 
sclerosis [RMS] patients from baseline to up to 12 months  
Secondary objective: 
o To evaluate the safety of  FMT in MS patients  
o To evaluate tolerability of FMT in MS patients 
 


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Protocol No. MSFMT -001 
Trial Design: 
The proposed randomized, open label, controlled [treatment as usual control group], 
crossover study will be conducted in 40 patients (n=20 per group) diagnosed with the 
relapsing forms of multiple sclerosis according to the McDonald 2010 Criteria1.   
Based on the diagnostic criteria patients will be further screened to confirm 
eligibility and if eligible then consented.  
Patients will be randomized 1:1 into two groups. One group will receive FMT, done 
by rectal enema administered by a RN, at baseline, month 1, month 2, month 3, 
month 4, and month 5 (early intervention group). The other group will receive 
treatment as per standard of care (treatment as usual control group) in the beginning 
of the study. For the last 6 months they will also receive FMT at month 6, month 7, 
month 8, month 9, month 10, and month 11 (late intervention group).  
Clinical visits for both groups will be performed at baseline, and months 1 to 12 
inclusive. During these visits a physical exam with vital signs, including a 
standardized neurological assessment Expanded Disability Status Scale – EDSS) will 
be done.  
Patient’s stools will be collected before each FMT procedure in both groups to 
evaluate gut microbial composition. 
Urine will be collected to evaluate gut permeability at baseline, 6 and 12 months for 
both groups. 
Peripheral blood samples also will be collected to evaluate circulating  cytokine 
levels ( IL-1β, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-13, IL- 15, IL-17A, 
IL-17E/IL-25, IL-17F, IL-21, IL-22, IL-23, IL-27, IL-28A, IL-31, IL-33, GM-CSF, 
IFNγ, MIP-3α, TNFα and TNFβ)  at baseline, months 1 to 12 inclusive for  both 
groups. 
 
Peripheral blood samples will also be collected to evaluate blood DNA bacteria at 
baseline, and months 1 to 12 inclusive for both groups. 
 
Brain MRI with gadolinium [3T MRI machine at Robarts Research Institute] will be 
performed at baseline, 6 and 12 months for both groups as a safety biomarker [to rule 
out paradoxical effects; i.e. significant increase in MS activity as defined by Cutter’s 
rule: 5 or more newly enhancing white matter lesions in the brain compared to the 
previous scan].   
In the event of early termination, a visit will be performed whenever possible, 
including a final blood sample and all end of treatment assessments that should be 
completed as soon as possible. All patients with at least 1 week of treatment will be 
 


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Protocol No. MSFMT -001 
eligible for analysis.   
Number of Patients:   
A maximum of 40 patients will be enrolled in intervention part of this study and 
randomized 1:1 into one of the two groups [20 patients per group] using an internet 
based randomizer software [https://www.randomizer.org/]. We may screen up to an 
extra 10 patients to compensate for possible drop outs, withdraw consent or those 
who discontinued for any other reason. 
We will also recruit up to 10 healthy volunteers to provide a blood sample [1 tube, 5 
ml] to serve as a reference arm for the cytokine analysis. This reference group will 
be seen only once and will not participate in any  other study activities. 
Consequently, the total number of participants in this study will be n=50. 


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Protocol No. MSFMT -001 
Inclusion criteria for patients:  
1. Have a confirmed diagnosis of relapsing MS defined by the 2010 Revised 
McDonald Criteria for the Diagnosis of Multiple Sclerosis1   
2.  Have a baseline Expanded Disability Status Scale (EDSS) ≤ 7.0  
3. Age equal or older than 18 years of age 
4. Be able to attend all clinic appointments without interruption 
5. Be proficient in English  
6. Be willing and able to give written informed consent 
 
7. Negative blood pregnancy test at screening 
 
Exclusion criteria for patients  
 
1. Not meeting all of the above inclusion criteria 
2. Pregnancy [current or planned for the near future] or breastfeeding 
3. All qualified patients will not be currently or recently [90 days] treated with 
immunosuppressive therapy [i.e. chemotherapy] or high dose corticosteroids [i.e. 
1g of prednisone or greater or equivalent]. Immune-Modulatory therapies are 
allowed as per standard of care. 
4. Ongoing use of antibiotics 
5. Presence of a chronic intestinal disease e.g. celiac, mal-absorption, colonic 
tumor 
6. Concomitant inflammatory diseases 
7. Standard of care exclusions for MRI scans as per LHSC standards 
 
Healthy controls Selection: 
1. Age equal or older than 18 
2. Written informed consent 
3. Self reportedly “healthy”, in particular not having a history of any disease or 


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Protocol No. MSFMT -001 
disorder, or any treatment ever for such [ie lupus, cancer, etc], that in the 
opinion of the PI could be possibly related to changes in the immune system 
that would alter the results of a cytokine in blood analysis. 


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Protocol No. MSFMT -001 
Test product, dose and mode of administration, batch number to be provided by Dr 
Silverman's Microbiology lab at St. Joseph’s Health Care, London, ON. For this 
study, fecal samples from healthy donors will be screened as per Health Canada 
guidelines. About 50-70g of the fecal sample is blended in 220ml saline in a 
Stomacher and filtered and transferred to an enema bag for transplantation. From 
here on this is known as “FMT” or “study medication”. 
Lot no. XXXXXX 
 
Duration of treatment/study:  
FMT rectal enema will be administered as scheduled per group, for up to 6 months. 
Criteria for evaluation: 
o Circulating cytokines levels (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-
12p70, IL-13, IL-15, IL-17A, IL-17E/IL-25, IL-17F, IL-21, IL-22, IL-23, IL-27, 
IL-28A, IL-31, IL-33, GM-CSF, IFNγ, MIP-3α, TNFα and TNFβ). 
o Gut microbiome through feces analysis 
o Blood DNA bacteria 
o Gut permeability through urine analysis  
o All adverse events occurring during this study will be recorded.  MS clinical 
functional status will be monitored using EDSS to assess any changes during the 
treatment period, as well as head MRI as a safety surrogate. 
Statistical methods: 
Adverse events will be coded using MedDRA dictionary. Incidence of adverse 
events by the severity, relationship to treatment, and outcome will be provided. 
Narratives of all serious adverse events and events causing death will be included. 
Laboratory parameters, vital signs and other safety parameters will be documented 
with the use of descriptive statistics. 
 
 
 
 


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Protocol No. MSFMT -001 
1. GENERAL INFORMATION 
 
Protocol Number and Title of the Study 
Fecal Microbial Transplantation in Relapsing Multiple Sclerosis Patients. 
 
Sponsor 
Dr. Michael Silverman 
Signature Authorization 
 
 
Clinical Study Sites 
London Health Sciences Centre 
University Hospital 
339 Windermere Road 
London, Ontario, N6A 5A5 
 
Robarts Research Institute 
Western University 
1151 Richmond St. N 
London, Ontario, N6A 5B7 
 
St. Joseph’s Health Care London 
268 Grosvenor St. 
London, Ontario, N6A 4V2 
Dean Cumming School of Medicine 
University of Calgary 


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Protocol No. MSFMT -001 
 2500 University Dr NW,  
Calgary, Alberta,  T2N 1N4 
 
Clinical Trial Leader 
Michael Silverman, M.D., FRCP, FACP 
Investigator 
Michael Silverman, M.D., FRCP, FACPTel.: +1 519-646-6311 
Email: Michael.silverman@sjhc.london.on.ca 
 
Clinical Assessment  
Sarah Morrow, M.D., FRCPC 
Lynn McEwan, MS NP 
Clinical Neurological Science Department 
University of Western Ontario 
London, ON, Canada 
 
Cytokines Analytical Laboratory 
Steven M Kerfoot, PhD 
Assistant Professor 
 
Kate Parham, PhD 
Postdoctoral Associate 
 
Department of Microbiology and Immunology 
University of Western Ontario 
London, Ontario, Canada 
 
  
Microbiome Analysis 
 
Jeremy P. Burton, BSc, MSc, PhD (Otago), dBA 
Division of Urology, Department of Surgery/ Department of Microbiology & Immunology,  
Lawson Health Research Institute and Canadian Centre for Human Microbiome and Probiotics 


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Protocol No. MSFMT -001 
University of Western Ontario 
London, Ontario, Canada 
 
Urine Analytical Laboratory 
 
 
Dr Jon Meddings, MD FRCPC 
Dean Cumming School of Medicine 
University of Calgary, Calgary, Alberta, Canada 
 
 
Stool Analytical Laboratory 
 
Michael S. Silverman, MD, FRCP 
 
Seema Nair Parvathy, PhD 
 
Department of Medicine, Division of Infectious Diseases 
University of Western Ontario 
London, Ontario, Canada 
 
Biostatistics and Data Management 
Prof Yves Bureau, PhD 
Assistant Professor of Medical Biophysics Department 
University of Western Ontario 
Lawson Health Research Institute 
London, Ontario, Canada 
 
 
 
 
2. BACKGROUND INFORMATION 
Multiple sclerosis (MS) is an autoimmune, inflammatory, demyelinating disease of the 
central nervous system (CNS) influenced by genetic susceptibility and some environmental 
factors not yet fully understood3. Relapsing forms of MS (RMS) are generally characterized by 
clearly defined periods of worsening neurological symptoms [i.e. relapse] followed by full or 
partial recovery [i.e. with sequelae and residual deficits upon recovery].   Relapsing forms of MS 


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Protocol No. MSFMT -001 
include relapsing remitting or secondary progressive.  In RMS, there is no clinically obvious 
disease progression during the periods between disease relapses4.  
Relapses are typically treated with a high dose of glucocorticoids over a short period of time 
[3-5 days]. Certain immunomodulatory agents, including interferon beta preparations, glatiramer 
acetate, natalizumab, fingolimod, dimethyl-fumarate, alemtuzumab and teriflunomide, have 
shown beneficial effects for patients with RMS (decreased relapse rate and a slower 
accumulation of brain lesions on MRI).  However, these treatments may be associated with 
significant toxicity and limited efficacy over time4.  
2.1.Pathophysiology of Multiple Sclerosis and gut bacteria  
The gut microbiome has recently emerged as a potential contributor to MS 
pathogenesis5,6. Previously reviewed data from murine model of autoimmune demyelinating 
disease7,8 suggest that gut microbiota can powerfully modulate immune-mediated demyelination. 
Moreover, studies involving MS patients have shown that their microbial composition is 
different compared to healthy controls9,10,11 and this could be related to disease development and 
activity12. Fecal microbial transplantation (FMT) from healthy donors is already used to restore 
normal fecal floral and treat colitis caused by Clostridium difficile. Therefore, we proposed that 
fecal microbial transplantation from a healthy donor could change the fecal flora composition of 
RMS patients, have a measurable effect on biomarkers of inflammation and possibly influence 
clinical or MRI disease activity. 
We conducted a review about gut microbiome and MS accepted for publication (Mult 
Scler 2016 Dec 12. Breaking down the gut microbiome composition in multiple sclerosis).  
2.2.Description of Fecal Microbial Transplantation  
Fecal microbiota transplantation is the process of fecal bacteria transfer from a healthy 
donor into a recipient. The goal is to restore colonic microflora by transplanting healthy 
bacterial flora (e.g. by enema, orogastric tube or by mouth in the form of a capsule 
containing freeze-dried material)12. For this study we selected the enema that is a lower risk 
procedure compared to an upper tract endoscopy and more easily accepted for patients than an 
oral capsule of fecal bacteria. An enema will be done to infuse the study treatment into the lower 
bowel through the rectum by a trained nurse.  
 
 
2.3. Summary of literature on MS and Gut Microbiome  
There is a growing body of evidence that gut microbiota goes beyond classical physiological 
functions such as vitamin production, digestion of nutrients, or protection against pathogens. 
Under normal physiological conditions, the human gut microbiota is a homeostatic ecosystem.  


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Protocol No. MSFMT -001 
Disruption of this equilibrium can result in what is known as “dysbiosis” and has been associated 
with several conditions, such as gastrointestinal diseases, obesity, diabetes, chronic periodontitis, 
vaginosis, atopic diseases, non-alcoholic steatohepatitis, and Alzheimer’s disease13.  
Some studies have shown that MS patients have an alteration in their microbiota composition 
compared to healthy individuals. In a pilot study of the gut microbial in MS, Cantarel et al. found 
decreased Faecalibacterium among seven RMS patients compared to eight controls14. Miyake et 
al. compared 20 Japanese RMS patients to 40 controls and similarly found that that there was a 
depletion of species belonging to butyrate-producing Clostridium clusters XIVa and IV, 
including Faecalibacterium prausnitzii and Eubacterium rectale14, among MS patients. Chen et 
al. found depletion of the butyrate-producing family Erysipelotrichaceae, which belongs to 
Firmicutes, among 31 patients with RMS compared to 36 controls15. Interestingly, however, the 
authors also noted an increased abundance of Blautia and Dorea genera, both belonging to 
Clostridium cluster XIVa, among their RMS patients15. Tremlett et al. also noted a depletion of 
butyrate-producing Faecalibacterium, Lachnospiraceae, Ruminococcaceae and Butyricimonas 
among 18 cases of early pediatric MS compared to 17 controls16. Two observational studies, the 
first by Gandhi et al. and the second by Jangi et al., reproducibly identified a reduction in the 
Butryicimonas genus among MS patients, which belongs to the Bacteroidetes phylum but is 
capable of producing butyrate17,18. In another pediatric study, Tremlett et al. followed 17 RRMS 
patients over a mean of 19.8 months and found that depletion of Fusobacterium genera, 
belonging to the Fusobacteria phylum, was associated with increased relapse risk19. Although 
presence of Fusobacteria has been associated with autoimmunity, including juvenile idiopathic 
arthritis and ulcerative colitis, Fusobacterium is also a butyrate producer20-22. The depletion of 
butyrate-producing bacteria in MS suggests that this pro-inflammatory alteration of the gut 
microbiota may be linked to disease pathogenesis. 
Rumah et al. identified the first RMS patient with epsilon toxin (ETX)-producing C. 
perfringens Type B in her GI tract23. ETX is produced by Clostridium perfringens types B and 
D35. It disrupts the blood-brain barrier and binds myelin, leading to its consideration as a 
potential MS trigger35. The authors found reduced non-toxigenic Clostridium perfringens Type A 
in the gut microbial of MS patients, and suggest this may create an ecological niche for ETX-
producing Clostridium perfringens24. Among their MS patients, 10% had antibodies specific to 
ETX in CSF and plasma compared to only 1% of controls, indicating increased immune system 
exposure to ETX among MS patients24.  
Furthermore, Miyake et al. found reductions of Bacteroidetes, including several Bacteroides 
species and Prevotella copri, among their RMS patients16. Similarly, Baum et al. compared 54 
patients with MS to healthy controls and found reduced Bacteroidaceae, a family belonging to 
Bacteroidetes26. Tremlett et al. also found decreased Bacteroidaceae among their patients with 
early pediatric MS27. The relevance of these findings may relate to Lipid 654, a bacterially-
derived lipodipeptide produced by a number of Bacteroidetes that was found to have lower 
serum levels among MS patients compared to healthy controls and Alzheimer’s patients15, 27. 


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Protocol No. MSFMT -001 
Lipid 654 binds toll-like receptor 2 (TLR2)28; although TLRs are critical to innate immune 
system activation, low-level tonic TLR2 stimulation by Lipid 654 may be immunoregulatory via 
maintenance of immune system tolerization28,29. Decreased Lipid 654-producing Bacteroidetes 
may therefore be pro-inflammatory, again plausibly linking the gut microbial to MS 
immunopathogenesis.  
Cekanaviciute et al. identified decreased Parabacteroides distasonis, which belongs to 
Bacteroidetes, among 51 MS patients compared to 55 controls29. Similarly, Chen et al. identified 
decreased Parabacteroides as well as Prevotella among their patients with RRMS compared to 
controls30. Oral administration of Parabacteroides distasonis antigens attenuates experimental 
murine colitis, with evidence of an immunomodulatory effect on cytokine production and 
increased mesenteric lymph node CD4+ T-regulatory cells19. Cekanaviciute et al. found 
Parabacteroides distasonis also augmented the CD4+ T-regulatory cell phenotype, suggesting 
that it may be linked to MS immunopathogenesis via its influence on CD4+ T-regulatory cell 
production31.  
Two studies, the first by Gandhi et al. comparing 53 patients with MS to 44 healthy controls, 
and the second by Jangi et al. comparing 60 patients with MS to 43 healthy controls, 
reproducibly found increased Methanobrevibacter in the gut microbial of MS patients18,19. Jangi 
et al. also identified higher breath methane among a cohort of MS patients compared to 
controls19, which may be due to increased gut Methanobrevibacter. However, the association 
between increased methane production and constipation is a potential confounder that should be 
controlled for in future studies, given the high prevalence of constipation in MS32, 33. MS patients 
with antibody reactivity to Methanobrevibacter-derived lipids had increased T-cell expression of 
pro-inflammatory cytokines, and increased Methanobrevibacter was associated with reactivity to 
a myelin sheath component19. These findings suggest a link between Methanobrevibacter and T-
cell auto reactivity causing inflammation and demyelination in MS.  
Several other differences in the gut microbiota have been identified in MS. Cekanaviciute et 
al. found that MS patients had increased Acinetobacter calcoaceticus, which belongs to 
Proteobacteria31. Molecular database analysis identified similarity between Acinetobacter 
calcoaceticus and an encephalitogenic myelin peptide34,35,. Elevated anti-Acinetobacter 
antibodies have also been found in MS patients, implicating molecular mimicry as a potential 
immunopathogenic link between Acinetobacter and MS35,36. Cekanaviciute et al. identified a 
reduction in CD4+ T-regulatory cells after T-cell stimulation with Acinetobacter calcoaceticus, 
suggesting it may also promote autoimmunity by impairing T-regulatory cell function31.  
Chen et al. found increased abundance of a number of Proteobacteria among their patients 
with RMS, including Pseudomonas and Mycoplana27. Proteobacteria have been associated with 
IBD by driving inflammation and promoting gut dysbiosis, which may contribute to the 
development of other autoimmune diseases such as MS37.  


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Protocol No. MSFMT -001 
Jangi et al. found increased Akkermansia among MS patients, which belongs to the phylum 
Verrucomicrobia19. Akkermansia muciniphila has been found to facilitate infection and 
inflammation by Salmonella Typhimurium in gnotobiotic mice38. In contrast, however, reduced 
Akkermansia muciniphila has been observed in IBD and metabolic syndrome39. The implications 
of increased Akkermansia in MS are therefore unclear; there may be a direct immunopathogenic 
relationship, Akkermansia may indirectly facilitate the growth of other pro-inflammatory 
bacteria, or other mechanisms may contribute. Jangi et al. also found that increased Akkermansia 
and Methanobrevibacter positively correlated with expression of genes among T-cells and 
monocytes implicated in MS pathogenesis; in contrast, butyrate-producing Butyricimonas was 
inversely correlated with these genes, suggesting that gut microflora may contribute to MS 
pathogenesis by polarizing to an inflammatory rather than immunomodulatory T-cell and 
monocyte phenotype19. 
Baum et al. noted increased Bifidobacterium among MS patients, which belong to 
Actinobacteria31. Tremlett et al. similarly found an increase in Bifidobacterium among patients 
with early pediatric MS compared to controls20. Bifidobacterium, commonly administered as 
probiotic therapy, has been linked to IBD40. However, Bifidobacterium animalis and a probiotic 
mixture including Bifidobacterium bifidum significantly ameliorated EAE activity, with evidence 
of inhibited Th1/Th17 polarization and regulatory T-cell induction41,42. The reason for this 
conflicting data regarding Bifidobacterium in demyelinating disease is unclear; further study into 
the potential role of Bifidobacterium and other probiotic therapy in MS is needed.  
Finally, fecal microbial transplantation is used to treat patients with recurrent Clostridium 
difficile infection (CDI), an antibiotic-associated bacterial colitis that may be life-threatening43,44. 
Antimicrobial therapy disrupts normal intestinal microflora and facilitates CDI; transplantation 
of healthy donor stool is believed to restore fecal microbial diversity in the recipient and prevent 
CDI recurrence43,44. As evidence of gut dysbiosis in MS mounts, FMT from donors free of 
autoimmune disease may be a consideration to restore microbial balance in those patients. 
Borody et al. reported neurologic benefit in three MS patients who underwent FMT for 
constipation45. The neurologic improvement was dramatic, including recovery of bowel and 
bladder function and regained ability to walk. Nevertheless, enthusiasm for these findings is 
tempered by the absence of controls, the diagnosis of ‘atypical’ MS in two patients, and the 
unclear biologic plausibility regarding how FMT would reverse established MS disease burden. 
On this ground, we propose that fecal microbial transplantation from healthy donors to MS 
patients could restore microbial gut balance and ultimately influence disease activity.  
 
 
2.4 Pre-clinical studies  
Previously reviewed animal research has examined the role of the gut microbiota in 
experimental autoimmune encephalomyelitis (EAE), a murine model of autoimmune 


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Protocol No. MSFMT -001 
demyelinating disease46. It was shown that germ-free mice devoid of gut microbiota are resistant 
to EAE, but generally immunodeficient47,8.  
 The zwitterionic capsular polysaccharide A (PSA) produced by Bacteroides fragilis was 
shown to be protective against EAE, with evidence of a PSA-dependent decrease in the pro-
inflammatory cytokine IL-17 and enhanced conversion of IL-10-producing CD4+ T-regulatory 
cells16. PSA-mediated induction as well enhanced immunosuppressive function of human T-
regulatory cells has also been shown in vitro30,49. PSA uniquely exemplifies a single gut 
microbial-derived product that has reproducibly demonstrated anti-inflammatory properties and 
seems protective against immune-mediated demyelination, suggesting therapeutic potential in 
MS30,49. Furthermore, Firmicutes producing the SCFA butyrate are increased in patients assigned 
to a plant-based rather than animal-based diet, and such SCFAs may have a beneficial anti-
inflammatory effect in EAE and MS50,51.  
Some probiotic bacteria have been shown to improve EAE, with less pro-inflammatory  
and increased CD4+ T-regulatory cell responses41,42,52-55.,  Bifidobacterium animalis and a 
probiotic mixture including Bifidobacterium bifidum significantly ameliorated EAE activity, with 
evidence of inhibited Th1/Th17 polarization and regulatory T-cell induction41,42.Whether there is 
any benefit to dietary probiotic supplementation in MS, however, is unknown, and caution may 
even be warranted given increased Bifidobacterium was  identified in some MS gut 
microbiomestudies for example.26,31 
The use of antibiotics to reduce the gut bacterial load is protective against EAE, with 
reduced pro-inflammatory cytokine production and enhanced CD4+ T-regulatory cell 
proliferation30,48. Yokote56 et al. studied the possible effects of altering gut flora by antibiotic 
treatment on EAE. First, mice treated with kamamicina, colistina and vancomycin (KCV) had a 
change in their gut flora compared to the control group proved by DNA microarray analysis after 
KCV. The experimental mice group was treated starting one week before immunization. After 
the EAE induction, the experimental group had less clinical manifestations, less mononuclear 
cells infiltrations on lumbar spinal cord and reduced demyelination compared to the control 
group. Moreover, they also studied the cells response in vitro with antigen stimulation. 
Interestingly, the proliferation response of the mononuclear cells were not affected in the 
experimental group but they produced less inflammatory cytokines (IL-6 and 17) and showed a 
reduced expression of ROR, an important transcription factor for Th17, together with enhance 
production of IL-10.  
In summary, animal models suggest that the gut microbiota can powerfully modulate 
immune-mediated demyelination. Moreover, that inflammatory cytokines can be influenced by 
microbiota changes in a measurable fashion.     
 
2.5 Rationale for the use of FMT for the treatment of multiple sclerosis 
patients  


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Protocol No. MSFMT -001 
Gastrointestinal disorders have more data published in the literature on this matter. A 
recent systematic review by Chapman et al. of FMT for CDI included 29 studies, 2 of them 
randomized control trials, 4 prospective and 23 retrospective. The authors concluded that FMT is 
a highly effective therapy for refractory or recurrent CDI with a success rate of 83-100% with an 
acceptable safety profile57. Another systematic review and meta-analysis by Sun et al. of FMT 
for ulcerative colitis (UC) identified 2 RCTs, 1 open-label case-control study, and 8 non-control 
cohort studies about the efficacy and safety of FMT in UC patients. They concluded that FMT 
may be an efficacious and safe alternative therapy for UC, at least when the standard therapy has 
failed or is unacceptable58. 
FMT has also been studied in metabolic disorders by Vrieze et al. This was a double-
blind, randomized controlled study where half of the 18 male patients with metabolic syndrome 
were transplanted their own feces (control group), while the other half were transplanted feces 
from thin donors (experimental group). The experimental group showed significant increase in 
insulin sensitivity after six weeks from FMT59.  
Fecal matter transplantation in patients has been tested in a few medical conditions. Most 
reports consist in open label or non-controlled studies with a small number of subjects. 
Moreover, the fecal matter transplant may vary the way the transplant is prepared as the route of 
administration which makes comparisons even harder. Even though more robust evidence about 
the efficacy and application of this procedure is lacking, one can conclude that FMT is feasible 
and safe for a phase II clinical study. 
 The rationale for treatment of MS patients with FMT in a phase II study is to restore a gut 
microbiome composition to one similar to a healthy person, and measure its possible effects in 
biomarkers as pro and anti-inflammatory cytokines related to MS pathophysiology. We plan to 
further study gut bacteria interaction in MS by DNA blood bacteria and gut permeability 
analysis.  
2.6 Patient population and study rationale  
This is essentially an immunological study, and therefore patients with RMS, regardless of 
the disease duration, will be enrolled in this study. They will be in a standard of care treatment 
for their MS and may or may not be on treatment with disease modifying therapy as per their 
own individual case. On the other hand, patients must be off of systemic high dose 
corticosteroids for at least 90 days.  
The primary aim of this study is to determinate if FMT can affect the immunological 
cytokine profile of MS patients. We also aim to evaluate the safety and tolerability of this 
treatment in this population. To our knowledge, a study like this has not yet been published 
anywhere else in the world.  
 
2.7 Potential risks for FMT and study procedures 


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Protocol No. MSFMT -001 
Two randomized control Trials (RCTs) reported mild adverse events attributed to FMT, 
including diarrhea, cramping, belching, nausea, abdominal pain, bloating, transient fever, and 
dizziness 41. Serious adverse events were also described, including a case of Fournier gangrene61, 
but none were attributed to the FMT. 
Eight case-series studies (reporting on 70 patients) did not explicitly mention harms; all 
others specifically commented on harms (or lack thereof). Possible procedure-related harms, 
including micro perforation with colonoscopy62  and gastrointestinal bleeding63,peritonitis64, and 
pneumonia64  with use of the upper gastrointestinal tract route, were rarely reported. One case-
series study reported on FMT use in 80 immuno compromised patients65. Although serious 
adverse events (2 deaths and 10 hospitalizations) occurred in 12 patients (15%); 4 were 
considered related to FMT, and 5 were possibly related.  
Because rare adverse events may be first reported as a case report, we examined case 
reports for such events. One report66 described a patient with abdominal pain and hypotension 3 
days after FMT by means of a gastro jejunostomy tube placed through an indwelling gastric tube. 
The patient had pneumoperitoneum, toxic megacolon, and polymicrobial bacteremia and 
subsequently died. Long-term data on harms were not reported. 
According to published articles, transient adverse responses after FMT have been reported, 
including mild fever, abdominal pain, diarrhea, exhaust, flatulence, and fatigue. However, these 
adverse effects are self-limiting. De Leon et al66 reported a UC patient quiescent for more than 
20 years who developed a flare of UC after FMT. This case gives us cautionary information 
concerning FMT being used to treat CDI with UC. Moreover, a recent paper reported a UC 
patient who had a cytomegalovirus infection after performing home FMT without donor 
screening67. As extracts of feces are mediators between the donor and recipient, FMT has the 
potential for transmitting occult infections even when strict donor screening is performed. 
More recently Wang et al published a systematic review on safety of FMT. A total of 50 
studies including original full-text articles, letters to the editor, abstracts of scientific 
conferences, case reports and case series which were published between 1913 and 2015 were 
reviewed. Totally 78 kinds of AEs were revealed with a total incidence rate of 28.5%. Among 
the 42 publications, 5 kinds were definitely and 38 kinds were probably related to FMT. The 
commonest FMT attributable AE was abdominal discomfort, which was reported in 19 
publications. For upper gastrointestinal routes of FMT, 43.6% (89/204) patients were 
compromised by FMT-attributable AE, while the incidence dropped to 17.7% (76/430) for lower 
gastrointestinal routes. In contrast, the incidences of serious adverse events (SAEs) were 2.0% 
(4/196) and 6.1% (40/659) for upper and lower gastrointestinal routes, respectively. A total of 44 
kinds of SAEs occurred in 9.2% patients, including death (3.5%, 38/1089), infection (2.5%, 
27/1089), relapse of inflammatory bowel diseases (0.6%, 7/1089) and Clostridium difficile 
infection (0.9%, 10/1089). 


Study Protocol – FMT in MS 
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Ver 3.0  09 OCT 2018 
 
Protocol No. MSFMT -001 
The paucity of RCTs is notable. As a result, there is a dearth of high-quality evidence to 
guide clinicians and policymakers on how to apply this promising but largely untested therapy. 
The overall low quality of the available evidence evaluating FMT is important and indicates that 
additional research is needed. However, the information available suggests that FMT can be 
safely done.    
3. STUDY OBJECTIVES AND PURPOSE  
This is a prospective, cross-over, open label, randomized 1:1 early vs delayed 
groups, interventional [FMT] vs treatment as usual controlled trial to explore the effects of 
FMT from a healthy donor to RMS patients and investigate whether this can influence 
disease activity based on a panel of biological markers.    
 
 3.1 Primary outcome 
To evaluate if FMT from healthy donors can significantly change cytokine profile in 
peripheral blood samples of RMS patients.  
3.2 Secondary outcomes 
We are also aiming to evaluate if FMT can possibly alter gut permeability through urinalysis 
and peripheral blood DNA bacteria. We also aim to evaluate treatment safety using the 
Expanded Disability Status Scale (EDSS) and MRI as surrogates for any paradoxical worsening 
in MS disease activity. We are monitoring adverse events to assess tolerability. 
4. STUDY DESIGN  
The proposed randomized, open label, with treat as usual control group, crossover phase II 
study will be conducted in 40 patients (n=20 per group) with the relapsing forms of multiple 
sclerosis according to the McDonald 2010 Criteria (Pollman 2011).  
Patients will be randomized into 2 intervention groups. One will receive the FMT from 
baseline and for the first 6 months (early intervention group). On the other hand, the other group 
will be a control group during the first 6 months and will receive the FMT for the last 6 months 
of the study (fFigure 1). The schedule of study procedures and evaluations for both groups is 
given in Tables 1 and 2. Patients will be screened for eligibility based on MS diagnosis and 
EDSS and if eligible then consented. All qualified patients will not be currently or recently 
treated with high dose steroids.  
 
 
 
 
20 MS 
20 MS 
Late intervention 
Control 
Control 


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Ver 3.0  09 OCT 2018 
 
Protocol No. MSFMT -001 
 
 
 
 
 
 
 
 
 
 
 
At Visit 1, before FMT, patients will be evaluated for their vital signs, medical history and 
concomitant medications. Also before transplantation, patient’s stool will be collected to study 
their microbial profile, blood analysis to evaluate cytokines levels as well as blood DNA bacteria 
and finally, urinalysis to assess gut permeability (baseline).  
Stool samples for all microbiome analysis will be collected in the form of a used toilet paper 
packed in a black pouch and transferred in ambient conditions to the laboratory at the Lawson 
Health Research Institute at St. Joseph’s Hospital and stored at -80 °C until the time of DNA 
extraction.  
Peripheral blood will also be collected by standard technique venipuncture by a venipuncture 
certified and experienced RN. Cytokines levels will be assessed by Luminex in the Department 
of Microbiology and Immunology at the University of Western Ontario and blood DNA bacteria 
will be evaluated in St. Joseph Microbiology Laboratory. 
For gut permeability patients will be instructed to drink lactulose solution and collect the 
urine throughout the night and first thing in the morning. A proper collecting bottle will be 
provided.  Once the urine sample bottle reaches the laboratory at St. Joseph’s Hosptial, London, 
Ontario the total volume will be measured and an aliquot of 30ml total, 10mL in each sterile 
urine container (no other additives) will be separated and stored at -20C and sent on dry ice to 
Dr. Meddings laboratory at Calgary, Alberta. All biological material will be transported 
according to biosafety regulations. 
 Patients will also undergo a contrast-enhanced brain MRI scan at Robarts Institute in 
London Ontario. Other baseline assessments (prior to the first dose of therapy) include an EDSS, 
pregnancy test (if applicable), and physical exam.  Blood samples are also taken at this visit in 
order to establish a baseline for routine chemistry/hematology.  After all this assessments FMT 
will be performed by a trained nurse via a rectal enema 
Patients will return to the clinic for visit 4 weeks after the first FMT.  Another stool sample 
to evaluate the microbial before the second FMT will be collected. Also peripheral blood 
samples for cytokines and blood bacterial DNA analysis. This same routine procedure will be 
Figure 1. Study Design 


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Ver 3.0  09 OCT 2018 
 
Protocol No. MSFMT -001 
repeated at month 2, 3, 4 and 5 for the early intervention group and at month 6, 7, 8, 9, 10 and 11 
for the late intervention group. Both groups will come to clinic at baseline andmonth 1 to 12 
inclusive, even if they are not receiving FMT. During all visits, a stool sample, a cytokine sample 
and a blood DNA bacteria sample will be collected. It is worth mentioning that at 6 and 12 
months another urine analysis will be done to evaluate gut permeability and MRI study at 
Robarts Institute, London, Ontario for safety assessment will be performed for both groups. 
Another safety assessment in each visit after FMT is to review of any adverse events that 
may have occurred. All patients receiving at least 1 week of treatment will be eligible for 
analysis.   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


Study Protocol – FMT in MS 
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Protocol No. MSFMT -001 
 
Table 1. Early Intervention Group Schedule 
 
Study Months 
Pre-
Visit 
Baselin
e 
Month 1 
Month 2 
Month 3 
Month 4 
Month 5 
Mont
h 6 
Mont
h 7 
Mont
h 8 
Mont
h 9 
Mont
h 10 
Mont
h 11 
Mont
h 12 
Visit Duration 
1.5hour
s 
1.5hours 
1.5hour
s 
1.5hour
s 
1.5hour
s 
1.5hour
s 
1.5hour
s 
45min 
45min 
45min 
45min 
45min 
45min 
45min 
Informed 
Consent Form 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Fecal 
Transplantatio
n 
 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
 
 
Height  
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Weight, Vital 
Signs 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Stool Sample 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Urine: Gut 
Permeability 
 
X 
 
 
 
 
 
X 
 
 
 
 
 
X 
Circulating 
Cytokine 
Levels and 
Blood DNA 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
MRI 
 
X 
 
 
 
 
 
X 
 
 
 
 
 
X 
Routine 
Hematology 
and Urinalysis 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Physical Exam/ 
EDSS 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
 


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Protocol No. MSFMT -001 
 
 
Table 2. Late Intervention Group Schedule 
Study Months 
Pre-
Visit 
Baselin
e 
Month 
1 
Month 
2 
Month 
3 
Month 
4 
Month 
5 
Month 
6 
Month 
7 
Month 
8 
Month 
9 
Month 
10 
Month 
11 
Month 
12 
Visit Duration 
1.5 
hour
s 
45mins 
45min
s 
45min
s 
45min
s 
45min
s 
45min
s 
1.5hour
s 
1.5hour
s 
1.5hour
s 
1.5hour
s 
1.5hour
s 
1.5hour
s 
1.5hour
s 
Informed 
Consent Form 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Fecal 
Transplantatio
n 
 
 
 
 
 
 
 
X 
X 
X 
X 
X 
X 
 
Height  
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Weight, Vital 
Signs 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Stool Sample 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Urine: Gut 
Permeability 
 
X 
 
 
 
 
 
X 
 
 
 
 
 
X 
Circulating 
Cytokine 
Levels and 
Blood DNA 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
MRI 
 
X 
 
 
 
 
 
X 
 
 
 
 
 
X 
Routine 
Hematology 
and Urinalysis 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Physical Exam 
/ EDSS 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 


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Protocol No. MSFMT -001 
 
 
 
 4.1 Rationale for FMT preparation  
The processing of stool samples for FMT via enema is shown in the chart below. Sample 
processing shall be done in sterile conditions in Biosafety cabinet level67.   
 
 
 
FMT will be administered via rectal enema.  Microflora will be studied from stool 
samples collected prior to administration of the FMT and will be stored at -80C. Before and 
after procedure the patients will be tested as per the chart given above. Data than shall be 
recorded periodically and analyzed for statistical significance. 
 
 
Frozen fecal sample* from 
standard donor (~50-70g) is mixed 
with 220ml sterile saline. 
Mixed in a stomacher for homogenizing and Slurry filtered 
(pore size 0.5mm) and transferred to an enema bag and 
delivered to patient. 
Patient is requested to lie on his lateral 
side to retain slurry as long as able 
(~30min) 


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Protocol No. MSFMT -001 
 
 
 
4.2 Duration of therapy and study  
Patients will be screened prior to starting treatment with FMT and randomized between 2 
groups. One, early intervention group, will receive FMT for the first 6 months and the other will 
be a control group treated as standard only. After 6 months, the early intervention will not 
receive the transplantation anymore and the control group will receive FMT for the remaining 6 
months. Study drug will be administered for a period of 6 months for each group total.   
Patients may withdraw consent at any time.  Study drug may be discontinued at any time by 
the investigator if doing so is deemed to be in the best interest of the patient.  However, the 
patient should be encouraged to continue in the study for safety follow-up. 
4.3 Study Termination 
The Investigator reserves the right to terminate this study at any time.  In terminating the 
study, the Investigator will ensure that adequate consideration is given to the protection of the 
patients’ safety and interests.  
 
5. SELECTION AND WITHDRAWAL OF PATIENTS 
5.1 Donor Screening Criteria 
Donors will be selected according to a standard protocol (appendix A). This is included 
in this protocol for information only. Donors will have been already selected and material will be 
already processed by Dr. Silverman’s Microbiology lab at St Joseph’s Hospital, London, 
Ontario, as per their standard of care. 
5.2 Patient selection 
5.2.1 Patient Inclusion criteria 
o Have a confirmed diagnosis of relapsing (relapsing-remitting or secondary 
progressive) MS defined by the 2010 Revised McDonald Criteria for the Diagnosis of 
Multiple Sclerosis1.Any disease duration will be accepted. 
o Have a baseline EDSS of = or <7.0 
o Older than 18 years of age. 
o Be able to attend all clinic appointments without interruption 
o Patients must be able to understand English sufficiently well to understand and 
comply with the clinic and medication schedules and procedures. 
o Be willing and able to give written informed consent 


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Protocol No. MSFMT -001 
o Negative blood pregnancy test at screening 
 
5.2.2 Patient Exclusion criteria 
o Not meeting all of the above inclusion criteria 
o Pregnancy or breastfeeding 
o Current or recent [in the last 90 days] exposure to high dose corticosteroids 
o Ongoing use of antibiotics  
o Presence of a chronic intestinal disease e.g. Celiac, malabsorption, Colonic tumor 
o Inability to provide informed written consent. 
o Immunosuppression from transplantation, HIV, Cancer chemotherapy or ongoing use 
of any immunosuppressive agents.  
o Concomitant inflammatory diseases 
o Any contra-indications for MRI. Participants are to be screened by a CMRTO 
certified MRI Technologist in order to determine the MRI compatibility or exclusion 
of implantable/external devices according to the manufacturer’s safety guidelines. 
The devises include cerebral aneurysm clips, neuro-stimulator, mechanical heart 
valves, cardiac stents, IUDs, vena cava filters, shunts, embolization coils, cochlear 
implants, non-removable prosthesis/artificial limbs. Contraindications are pacemaker 
of defibrillator, shrapnel/metallic fragments, previous brain surgery, seizure, severe 
claustrophobia, weight or body index that will prevent a successful MRI study.  
 
 5.3 Recruitment of trial patients.  
Successful recruitment will be 3-6 patients per month on average. The goal is a total 
recruitment of 20 patients/12 months given the high numbers of competing studies, missed 
patients, consent failure rates and the refusal of an FMT strategy. We expect to monitor success 
with recruitment throughout the study and make revisions to recruitment procedures and 
eligibility criteria as necessary.  
 
5.3.1 Recruitment of Healthy Volunteers. 
Advertising will be posted throughout campus and hospitals in London announcing this study 
and calling for self-reported healthy individuals 18 y/o and older to participate in this study as a 
“control group”. We will recruit up to 10 [ten] volunteers. Participation will entail a single visit 
to the clinic for proper written informed consent prior to obtaining 1 tube, 5 ml of blood via 
standard of care venipuncture in one arm administered by a qualified registered nurse or lab 
technician. The tube with blood will be labeled with a study ID and sent to Dr Kerfoot’s 
Immunology lab for analysis in a completely de-identified fashion. This “control group” will not 
have any further participation in this study.   
  
 
 


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Protocol No. MSFMT -001 
5.4 Adherence to protocol.  
Successful adherence will be defined as =90% of patients progressing to FMT and all follow-
up. Preliminary estimates of non-administration of the trial intervention are needed, along with 
strategies that maximize exposure to the intervention.   
 
5.5 Safety 
We will monitor patients closely for potential adverse events. We plan to administer the FMT 
through a standard rectal enema done by a trained Registered Nurse. Full screening of the donor 
to rule out transmissible blood-borne, or fecal pathogens, as well as history and physical 
examination to rule out any underlying health conditions [as performed in previous studies70,71  
and as recommended in the recent Guidance document from Health Canada], will assure the 
safety of the procedure. It is important to note that FMT has been found to be extremely safe in 
multiple previous studies, with no documented complications ever reported when performed in 
medically stable patients.  
6. TREATMENT OF PATIENTS 
6.1 Study Drug 
Fecal microbiota. 
 
6.2 Study Evaluations 
All summary of study procedures and evaluations at each visit were provided in Tables 1 
and 2 (previous chapter).  
 
6.2.1 Screening, Enrolment and Baseline  
An informed consent form must be signed prior to initiating any procedures or 
evaluations for the purpose of the study (Appendix B).  After the informed consent form has 
been signed patients will be randomized to receive FMT for the first 6 months of the study or to 
be a standard per treatment control group that will received treatment at the last 6 months of the 
study.  
The following evaluations at baseline visit and prior to FMT will be performed in both 
groups: 
o Medical history; 
o Vital signs (including pulse, respiration rate, blood pressure and temperature); 
o Physical Exam (including weight in kg); 
o Concomitant medications; 
o Dietary history; 


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Protocol No. MSFMT -001 
o Expanded Disability Status Score (EDSS); 
o Blood samples will be taken for haematology (including haemoglobin, red blood cell 
count, white blood cell count, differential and platelets); 
o Blood samples will be taken for serum chemistry (sodium, potassium, chloride, uric 
acid, creatinine, blood urea nitrogen, total protein, calcium, albumin, total bilirubin, 
AST, ALT, alkaline phosphatase, glucose); 
o Blood sample for multiplex cytokine profiling (includes pro-inflammatory (IL-6, IL-
15, IL-17, GM-CSF, TNFα) and anti-inflammatory (IL-4, IL-10 and IL-14) 
cytokines).  
o Clean catch, mid-stream urine for urinalysis and micro analysis (including pH, 
protein, glucose, ketones, nitrites, leukocyte esterase, RBCs, WBCs) and gut 
permeability; 
o Blood sample for blood bacterial DNA 
o Head MRI as baseline 
 
After evaluation, the FMT via rectal enema will be performed by a RN in each visit.  
 
 
6.2.2 Subsequent visits  
On all subsequent visits, prior to FMT the following evaluations will be performed:  
o Physical examination  
o Rapid (urine) pregnancy test for women of childbearing potential; 
o Vital signs (including pulse, respiration rate, blood pressure and temperature); 
o Concomitant medications; 
o EDSS; 
o Blood samples will be taken for haematology (including haemoglobin, red blood cell 
count, white blood cell count, differential and platelets); 
o Blood samples will be taken for serum chemistry (including: sodium, potassium, 
chloride, uric acid, creatinine, blood urea nitrogen, total protein, calcium, albumin, total 
bilirubin,, AST, ALT, alkaline phosphatase, glucose); 
o Clean catch, mid-stream urine for urinalysis and micro analysis (including pH, protein, 
glucose, ketones, nitrites, leukocyte esterase, RBCs, WBCs,); 
o Blood sample for blood bacterial DNA 
o Blood sample for multiplex cytokine profiling (includes pro-inflammatory (IL-6, IL-15, 
IL-17, GM-CSF, TNFα) and anti-inflammatory (IL-4, IL-10 and IL-14) cytokines).  


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Protocol No. MSFMT -001 
After evaluation, the FMT via rectal enema will be performed by a RN in each visit.  
At baseline, 6 and 12 months a head MRI will be performed. At these time points urine will 
be also collected to perform gut permeability analysis.  
 
At any time during the study, if a patient has experienced an adverse event then they may 
be asked to return to the clinic for an unscheduled visit.  Patients with adverse events will be 
followed to resolution.  
 
6.2.3 Peripheral blood cytokines levels  
Cytokine levels will be measures as previously described72. Peripheral blood will be 
collected into EDTA blood collection tubes ensuring complete mixing with anticoagulant to 
avoid clotting. Within 60 minutes of blood collection samples will be spun for 10 minutes at 
4500xg. The plasma will be collected and aliquoted into cryovials. All samples will be collected 
and stored at -80C before the plasma is measured for 25 immunological cytokines using a 
luminex-based mulitiplexed immunoassay. The Human Th17 Magnetic bead panel 
(HTH17MAG-14K, Millipore) will be used in this study to detect changes in Interleukin 1β (IL-
1β), IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-13, IL-15, IL-17A, IL-17E/IL-25, IL-17F, 
IL-21, IL-22, IL-23, IL-27, IL-28A, IL-31, IL-33, granulocyte macrophage colony stimulating 
factor (GM-CSF), interferon γ (IFNγ), macrophage inflammatory protein 3α (MIP-3α), tumour 
necrosis factor α (TNFα) and TNFβ. Plasma samples will be run in duplicate and assays will be 
standardized as per product specifications. Plasma cytokine levels in healthy controls will be 
compared to levels in MS patients at all time points. Moreover, cytokine levels of MS patients at 
baseline (D0) will be compared to levels at 12 months to determine whether intervention (FMT) 
had an effect on pro- and anti-inflammatory cytokine levels. 
 
6.2.4 Blood DNA bacteria  
Up to 10mL blood will be collected in a 3mL K2EDTA Whole Blood Collection Tube 
manufactured by BD (BD Product number: 367856). Isolation of bacterial DNA from blood is 
obtained by adding 1.5 ml of whole blood to 6 ml of 0.17 M ammonium chloride. The sample 
are incubated for 20 minutes at 37° C and centrifuged for 10 minutes at 10 000 rpm. The 
supernatant is removed and the precipitate is suspended in 100μl of solution of lysozyme (2 
mg/ml) and lysostaphin (0.2 mg/ml) in PBS buffer. Sample are then transferred to Eppendorf 
tubes with glass beads (700-1100 μm) and subjected to mechanical disruption for 20 seconds at a 
speed of 4.0 m/s. Again, sample are incubated for 30 minutes at 37° C and centrifuged for 10 
minutes at 12 000 rpm. 
 
6.2.5 Stool collection 


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Protocol No. MSFMT -001 
Fecal samples will be collected from patients at baseline, and months 1 to 12 inclusive 
prior to receiving their scheduled FMTs. To collect the stool samples the participants will collect 
a piece of visibly soiled toilet paper and place it into a DNA-free bag. The bag will be 
transported to the Lawson Health Research Institute at St. Joseph’s Hospital and stored at -80 °C 
until the time of DNA extraction when all samples are collected. DNA will be extracted using 
the MO BIO Powersoil 96-well soil isolation kits, as adapted for the Human Microbiome Project. 
After DNA extraction, the V4 region of the 16S rRNA gene will be amplified using validated 
bar-coded PCR primer sets.  After amplification, the samples will be sent for Illumina MiSeq 
sequencing (London Regional Genomics Centre). The reads generated from sequencing will be 
demultiplexed, taxonomically assigned by BLAST, and analyzed using the program R. 
Microbiome analysis will follow the protocol as described in Bisanz et al.4 A principal 
component analysis will be conducted and ALDEx2 tests will be performed to determine what 
the main differences in bacterial composition are between samples. The microbial composition at 
baseline will be compared to the other time points to determine how long it takes for the 
recipient to revert to their original microbiota. The donor microbiota will also be compared to 
each time point to determine how similar the patient’s bacterial composition is to the donors after 
transplant. 
 
6.2.6 Urine analysis for gut permeability  
Patients will be instructed not to consume alcohol on the day of the test or 5 days 
previously. Also, to eat supper before 6:00 pm, and then no food or drink is to be consumed after 
this time. At bedtime, patients will be advised to empty bladder and discard urine. 
A packet containing sugar (lactulose) and mannitol will be provided. Patients will be 
instructed to empty the packet into a glass of water according to the table below.  
 
Weight 
Lactulose Mannitol  Sucrose 
Tap water 
45kg-up 
5 grams 
2.0 grams 100 grams 450ml 
 
 
Throughout the night and first thing in the morning, all urine must be collected and 
brought to St Joseph’s Health Care.  
 
 
6.3 Sample Size analysis 
It was assumed that the acceptable Type I error will be 0.05 and power will be 0.8. 
Moderate effect size effect is expected. This is a study of a kind that has never been done before 
in this population. From similar studies in other populations or other interventions on a similar 
population, a 40% change in cytokine levels (ie TGFbeta1) analysis is expected, as reported on 
other cytokine study with no pharmacological intervention, such as vitamin D supplementation73.  


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Protocol No. MSFMT -001 
 
6.4 Study duration 
 
One year.  
7.0 ASSESSMENT OF EFFICACY 
As this study is an immunological study, the following efficacy outcomes will be 
evaluated:  
o Changes in peripheral blood cytokines within RMS patients between baseline and months 1 
to 12 inclusive, for both groups. Luminex kit will be used.   
o Changes on microbiome after FMT by analyzing the gut bacterial composition through 
stool analysis and blood bacteria DNA at baseline, and months 1 to 12 inclusive for both 
groups.  
o Gut permeability through urine analysis at baseline, 6 and 12 months for both groups.  
 
 
8.0 ASSESSMENT OF SAFETY 
 
8.1 Head MRI  
A contrast head MRI will be performed at baseline, 6 months and 12 months for both 
groups as a safety measure to evaluate any subclinical activity or any paradoxical effect 
potentially caused by the FMT. 
8.2 Adverse events 
8.2.1 Definition of an Adverse Event 
An adverse event (AE) is defined as any undesirable physical, psychological, or behavioural 
event experienced by a subject in conjunction with their participation in the clinical trial, 
regardless of whether the event is procedure- or study product-related.  This includes any 
condition (including a pre-existing condition) that was: 
▪ Not present prior to study treatment initiation but appeared or reappeared following initiation 
of study treatment. 
▪ Present prior to study treatment, but worsened during study treatment.  (This could include 
any condition resulting from concurrent illness, reactions to concomitant medications, or 
progression of disease states).   
The occurrence of an AE will be based on changes in the subject’s physical examination, 
laboratory results, and/or signs and symptoms. 
 


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Protocol No. MSFMT -001 
8.2.2 Reporting of an Adverse Event 
All AEs regardless of causal relationship are to be recorded in the case report form (CRF) 
and other source documentation.   
The Investigator must determine the intensity of any AEs according to a three-point 
scale: mild, moderate, severe, and reported on specific AE pages of the CRF. If the severity of an 
AE worsens during study drug administration, only the worst intensity should be reported on the 
AE page. If the AE lessens in intensity, no change in the severity is required.  
 
Mild 
Event may be noticeable to subject; does not influence daily activities; the AE resolves 
spontaneously or may require minimal therapeutic intervention;  
 
 
Moderate 
Event may make subject uncomfortable; performance of daily activities may be 
influenced; intervention may be needed; the AE produces no sequelae. 
 
Severe 
Event may cause noticeable discomfort; usually interferes with daily activities; subject 
may not be able to continue in the study; the AE produces sequelae, which require 
prolonged therapeutic intervention. 
 
A mild, moderate or severe AE may or may not be serious. These terms are used to 
describe the intensity of a specific event (as in mild, moderate, or severe myocardial infarction). 
However, a severe event may be of relatively minor medical significance (such as severe 
headache) and is not necessarily serious. Seriousness rather than severity serves as a guide for 
defining regulatory reporting obligations. 
The Investigator must also determine the causal relationship using the guidelines listed in 
Table 8.1 and Section 8.1.1.  Life threatening AEs will be recorded as serious adverse events 
(SAEs) in the CRF and will be reported to the medical monitor. 
All AEs will be followed until resolution.  Resolution is defined as the disappearance of 
all signs or symptoms related to the adverse event or the stabilization of the adverse event with 
residual signs or symptoms that are not expected to improve. 


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Protocol No. MSFMT -001 
 
Criteria for Determining Category of Relationship of Clinical Adverse Events to 
Treatment 
 
 
Not 
related 
 
This category applies to those adverse events which, after careful 
consideration, are clearly and incontrovertibly due to extraneous 
causes (disease, environment, etc.)  
 
 
Unlikely 
(must 
have 
two) 
 
In general, this category can be considered applicable to those adverse 
events which, after careful medical consideration at the time they are 
evaluated, are judged to be unrelated to the test drug.  An adverse 
event may be considered unlikely if or when: 
· 
It does not follow a reasonable temporal sequence from 
administration of the test drug.  
· 
It could readily have been produced by the subject’s clinical 
state, environmental or toxic factors, or other modes of therapy 
administered to the subject. 
· 
It does not follow a known pattern of response to the test drug. 
· 
It does not reappear or worsen when the drug is re-administered. 
 
 
Possibly 
(must 
have 
two) 
 
This category applies to those adverse events for which, after careful 
medical consideration at the time they are evaluated, a connection with 
the test drug administration appears unlikely but cannot be ruled out 
with certainty.  An adverse event may be considered possibly related if 
or when:  
· 
It follows a reasonable temporal sequence from administration 
of the test drug. 
· 
It could not readily have been produced by the subject’s 
clinical state, environmental or toxic factors, or other modes of 
therapy administered to the subject. 
· 
It follows a known pattern of response to the test drug. 


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Probably 
(must 
have 
three) 
This category applies to those adverse events for which, after careful 
medical consideration at the time they are evaluated, are felt with a 
high degree of certainty to be related to the test drug.  An adverse 
event may be considered probably related if or when: 
· 
It follows a reasonable temporal sequence from administration 
of the test drug. 
· 
It could not be reasonably explained by the known 
characteristics of the subject’s clinical state, environmental or 
toxic factors, or any other modes of therapy administered to the 
subject. 
· 
It disappears or decreases on cessation or reduction in dose.  
There are important exceptions when an adverse event does not 
disappear upon discontinuation of the drug, yet drug-
relatedness clearly exists (e.g. bone marrow depression, fixed 
drug eruptions, tardive dyskinesia).  
· 
It follows a known pattern of response to the test drug. 
 
 
Definitely 
(must 
have 
all) 
 
This category applies to those adverse events which, the Investigator 
feels are incontrovertibly related to test drug.  An adverse event may 
be assigned an attribution of definitely related if or when: 
· 
It follows a reasonable temporal sequence from administration 
of the test drug. 
· 
It could not be reasonably explained by the known 
characteristics of the subject’s clinical state, environmental or 
toxic factors, or other modes of therapy administered to the 
subject. 
· 
It disappears or decreases on cessation or reduction in dose 
with re exposure to drug.  (Note:  this is not to be construed as 
requiring re-exposure of the subject; however, a category of 
definitely related can only be used when a recurrence is 
observed.)  
· 
It follows a known pattern of response to the test drug. 
 
 
 
 
 


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Protocol No. MSFMT -001 
8.2.3 Definition of a Serious Adverse Event 
Adverse events that are to be considered serious include the following: 
 
o Death, regardless of cause, which occurs within 30 days of the last dose of study drug 
or if after 30 days is a result of delayed toxicity due to administration of the study 
drug. 
o Immediately life-threatening AE  
o Permanently disabling event. 
o Hospitalization or prolonged hospitalization excluding those for protocol-specific 
procedures. 
o Congenital anomaly. 
“Life threatening” means that the study participant was, in the opinion of the Investigator, 
at immediate risk of death from the reaction as it occurred.  Any other event thought by the 
Investigator to be serious should also be reported. 
The term “severe” is used to grade intensity and is not synonymous with the term 
“serious”. 
 
8.4 Reporting of a serious adverse event 
In the event of a SAE, regardless of any opinions on relationship to the study drug, the 
Medical Monitor is to be notified.  In the event that the Medical Monitor cannot be reached, 
other personnel will be available to receive the report.   
All SAEs that are unexpected (i.e., not identified in nature, severity, or frequency in the 
current Investigator’s Brochure), whether considered to be drug related or not, require a SAE 
Report Form be completed.  All SUSARS (suspected unexpected serious adverse events) must be 
reported in writing to the Concerned Research Ethics Board (REB).  
A death occurring during the study or which comes to the attention of the Investigator 
within 30 days after discontinuation of study drug, whether considered treatment related or not, 
must be reported. 
Preliminary reports of SAEs will be followed by detailed descriptions which will include 
copies of the hospital case reports, autopsy reports, and other documents as requested and 
applicable.   
 
8.5 Reporting of a pregnancy during the study 
In principle, pregnancy and the lactation period are exclusion criteria for clinical studies 
involving investigational drugs, which are not directly related to the respective conditions. In the 
event of a pregnancy occurring during the course of this particular study, the subject should be 


Study Protocol – FMT in MS 
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Ver 3.0  09 OCT 2018 
 
Protocol No. MSFMT -001 
withdrawn from study, but closely followed-up during the entire course of the pregnancy and 
postpartum period. All recommendations described in the investigational drug brochure during 
pregnancy and lactation have to be carefully considered. 
 
Parental and neonatal outcomes must be recorded even if they are completely normal and 
without Adverse Events. Off-spring should be followed up for at least 8 weeks after delivery. 
Longer observation periods may be considered if an adverse outcome of the pregnancy was 
observed. 
 
9. STATISTICAL ANALYSIS 
9.1 Sample Size Selection 
This is an exploratory proof of concept immunological study.  As such the number of 
patients in each treatment group was arbitrarily chosen in lieu of a convenience sampling for this 
type of study.  
9.2 Demographics 
Demographic variables such as age, race, etc. and baseline status (medical history, physical 
exam, etc.) will be documented by dose level and/or overall, as applicable. 
9.3 Biomarkers 
We will analyze mean differences between two independent groups whilst subjecting 
participants to repeated measures overtime to assess variability between- and within-subjects 
variable. Therefore, we will use the mixed-design ANOVA model. 
  
9.4 Safety Analysis 
Adverse events will be coded using MedDRA dictionary. Incidence of adverse events by 
the severity, relationship to treatment, and outcome will be provided. Narratives of all serious 
adverse events and events causing death will be included. Laboratory parameters, vital signs and 
other safety parameters will be documented with the use of descriptive statistics.   
9.5 Criteria for Study Termination 
The study continues until all 40 patients have been treated.  
9.6 Missing Values 
No imputation for missing values will be utilized. 


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Protocol No. MSFMT -001 
9.7 Evaluable Patients 
All patients who received 1 week of study treatment will be included in all analyses and 
listings. 
10. QUALITY CONTROL AND QUALITY ASSURANCE PROCEDURE ANALYSIS 
 
 10.1 Protocol Amendments   
In the event that a protocol change is proposed for all patients, the following procedure for 
a protocol amendment will be followed. Each investigator(s) must sign and date the amendment 
prior to implementation. All major protocol amendments must be submitted to and approved by 
health authorities. In addition, the investigator(s) must report all protocol amendments to, and 
receive all required approvals from, Independent Ethics Committee (IEC) prior to the 
implementation of any protocol amendment at the study center. All protocol amendments will be 
listed in the amended protocol.   
Administrative changes to the protocol which do not impact the design or conduct of the 
study will be submitted to health authorities and IEC as notifications. 
  
10.2 Protocol Deviations 
No deviations from this protocol will be permitted without the prior written approval of the 
investigators.  Any such changes which may affect a patient’s treatment or informed consent, 
especially those increasing potential risks, must receive prior approval by the IEC.  Other 
administrative revisions which may impact the clinical portion of a study will be duly reported to 
the IEC by the Investigator   
 
11. ETHICAL CONSIDERATIONS 
11.1 Independent Ethics Committee (IEC)  
Good Clinical Practice (GCP) requires that the Investigator’s Brochure, clinical protocol, 
any protocol amendments, the informed consent and all other forms of patient information 
related to the study (e.g., advertisements used to recruit patients) and any other necessary 
documents for each participating site be reviewed by an IEC.  Any amendments to the protocol 
will require IEC approval prior to implementation of any changes made to the study design.  All 
expedite Serious Adverse Events should be reported to the IEC.  During the conduct of the study, 
the investigator should promptly provide written reports to the IEC on any changes that affect the 
conduct of the study and/or increase the risk to patients.  


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Protocol No. MSFMT -001 
  
11.2 Ethical Conduct of the Study 
The study will be conducted in accordance with the protocol, the International Conference 
on Harmonization Harmonized Tripartite Guidelines E6 for Good Clinical Practice, ethical 
principles that have their origin in the Declaration of Helsinki, 2013 and all applicable local 
regulations.  
  
11.3 Patient Information and Consent 
 
Patients attending a clinic specializing in the care of patients with MS will be approached 
by their Physician or nurse regarding their interest in participation. If they express interest and 
consent they will then meet with the investigator and his representative. At this time, the patient 
will be questioned regarding inclusion and exclusion criteria, and if eligible will request 
informed consent for participation. The consent will contain all the essential elements of 
informed consent set forth in ICH E6. The investigator or his/her representative will explain the 
nature of the study to the patient, and answer all questions regarding this study. The informed 
consent must be reviewed, signed and dated by the patient and the person administering the 
consent prior to screening the patient for the study.  A copy of the informed consent form will be 
given to the patient and the original will be placed in the patient’s medical record if in 
accordance with the institution’s standard operating procedures. An entry must also be made in 
the patient’s chart to confirm that informed consent was obtained prior to any study-related 
procedures and that the patient received a signed copy.  To protect the confidentiality of the 
patient, all reports and communications relating to the study will identify patients by assigned 
patient identifier only. Potential donors will be recruited from student groups or staff volunteers 
in Western University. 
 
11.4 Benefits of the study 
 
 
11.4.1 Participant’s benefit 
 
 MS patients may or may not be benefitted by the outcome of the study. We hope to 
demonstrate that human gut microbiome may impact MS cytokines profile and that FMT is 
feasible. We aim to further increase the understanding about MS pathology.  
 
 
11.4.2 Benefits to the society:  
 
The goal of this project is to establish a comprehensive research team consisting of 
Neurologists, Neuroradiologists, Immunologists and Microbiome scientists to develop local 
expertise which would enable the present as well as future studies. Potential future interventions 
would be planned based on the study. In particular we will look at how long the transplanted 
microbiome persists and whether certain organisms are critical in the success of the intervention 
and thus could be selected for in a subsequent more focused microbial supplementation 
interventions (probiotics) or with selective antibiotics to remove harmful bacteria.  


Study Protocol – FMT in MS 
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Protocol No. MSFMT -001 
 
11.5 Potential risks/harms/inconveniences 
 
 We will monitor closely for potential adverse events. Full screening of the donor to rule 
out transmissible blood, or fecal pathogens, as well as history and physical examination to rule 
out any underlying health conditions, will assure the safety of the procedure. It is important to 
note that FMT has been found to be extremely safe in multiple previous studies.  All details of 
the study shall be discussed with the patient’s and donor and included in the informed consent 
forms. There is the potential of a previously unrecognized illness in the donor, to be identified 
through screening. Potential donors will be counseled regarding this and will receive pre and 
posttest counseling for HIV and other screened diseases. Any potential donors who are identified 
to have a significant health issue during screening will be referred for appropriate medical care.  
 
12. DATA HANDLING AND RECORD KEEPING 
  
12.1 Study Documentation and Record Retention  
For this study all data will be collected in a case report form (CRF).  CRFs will be 
maintained for each patient. The Investigator will be responsible for the recording of all data on 
the CRFs provided to them, as certified by the Investigator’s signature and date.  Should any 
value be significantly different from normal, the Investigator will comment in the appropriate 
sections provided in the CRFs.  
All CRF corrections are to be made by the investigator or his/her study site designee and 
need to be documented by date. 
Data recorded in each patient’s CRF must have first been documented in the patient’s 
medical record or other source documentation (e.g., laboratory reports, progress notes, etc.) 
CRFs will be reviewed and compared with corresponding source documents by the Clinical 
Monitor who will determine the CRFs acceptability. Copies of completed CRFs will be reviewed 
by the Clinical Monitor.  
The Investigator Site File (ISF) must be maintained in an organized fashion with no co-
mingling of any other non-study-related materials. The study file must contain all study-related 
correspondence (e.g., Ethics Committee correspondence, correspondence to/from the Sponsor 
and/or its designee, patients’ signed informed consents, copy of the protocol and any/all 
amendments, copy of signed regulatory agency forms. etc.)  In addition, the study file must 
contain contact information (i.e., name, address, telephone) for each patient who participates in 
the study. The patient contact information is for emergency or medical follow-up purposes 
related to this study. It is not submitted to the Sponsor, unless specifically required for the 
regulatory purposes. 


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Protocol No. MSFMT -001 
All case report forms and all source documents (e.g., informed consent forms; adverse 
event reports; drug dispensing/disposition records; medical record information and reports as 
described above) must be retained in the ISF by the investigator for the maximum period of time 
defined as: 
1. Twenty-five years or  
2.For a minimum of two(2) years following last approval of a marketing application 
in an ICH region and until there are no pending or contemplated marketing applications in 
an ICH region or  
3. At least 2 years after the discontinuation of clinical development of this product  
 
12.2 Duties of the Investigational Staff 
In addition to the specifications of this protocol, the investigator and all individuals 
responsible to the investigator who assist in the conduct in the study are obliged to conduct this 
study in accordance with currently applicable regulations. 
 
The investigator will provide copies of the protocol and all pertinent information as 
necessary to the study staff, and will discuss the material with them to ensure they are fully 
informed regarding the drug and the conduct of the study. 
 
12.3 Use of Information and Publication 
 
Publication of the interim analysis and final study report are planned. The Sponsor-
Investigator (Dr. Kremenchutzky) will determine authorship for each manuscript based on 
contributions to the study design, study execution, and manuscript completion. No author will be 
included without prior authorization but the intention is to be broadly inclusive of all study 
investigators who make active contributions as outlined by The ICJME criteria for authorship 
(Authorship credit should be based on 1) substantial contributions to conception and design, 
acquisition of data, or analysis and interpretation of data; 2) drafting the article or revising it 
critically for important intellectual content; and 3) final approval of the version to be published. 
Authors should meet conditions 1, 2, and 3). 
 
 
 
 
 
 
 
 
 


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Protocol No. MSFMT -001 
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29. Cekanaviciute E, Runia T, Debelius J, et al. The influence of microbiota on the adaptive 
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30. Aminov RI. Role of archaea in human disease. Front Cell Infect Microbiol. 2013;3:42. 
 
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32. Ebringer A, Hughes L, Rashid T, Wilson C. Acinetobacter immune responses in multiple 
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35. Mukhopadhya I, Hansen R, El-Omar EM, Hold GL. IBD—what role do Proteobacteria 
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36. Ganesh BP, Klopfleisch R, Loh G, Blaut M. Commensal Akkermansia muciniphila 
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37. Png CW, Lindén SK, Gilshenan KS, et al. Mucolytic bacteria with increased prevalence 
in IBD mucosa augment in vitro utilization of mucin by other bacteria. Am J 
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38. Wang W, Chen L, Zhou R, et al. Increased proportions of Bifidobacterium and the 
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39. Kwon H-K, Kim G-C, Kim Y, et al. Amelioration of experimental autoimmune 
encephalomyelitis by probiotic mixture is mediated by a shift in T helper cell immune 
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40. Ezendam J, de Klerk A, Gremmer ER, van Loveren H. Effects of Bifidobacterium 
animalis administered during lactation on allergic and autoimmune responses in rodents. 
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41. van Nood E, Vrieze A, Nieuwdorp M, et al. Duodenal infusion of donor feces for 
recurrent Clostridium difficile. N Engl J Med. 2013;368(5):407-415. 
 
42. Shahinas D, Silverman M, Sittler T, et al. Toward an understanding of changes in 
diversity associated with fecal microbiome transplantation based on 16S rRNA gene deep 
sequencing. MBio. 2012;3(5). 
 
43. Borody T, Leis S, Campbell J, Torres M, Nowak A. Fecal Microbiota Transplantation 
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44. Mielcarz DW, Kasper LH. The Gut Microbiome in Multiple Sclerosis. Curr Treat 
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45. Berer K, Mues M, Koutrolos M, et al. Commensal microbiota and myelin autoantigen 
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46. Hooper L V, Littman DR, Macpherson AJ. Interactions between the microbiota and the 
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47. Telesford KM, Yan W, Ochoa-Reparaz J, et al. A commensal symbiotic factor derived 
from Bacteroides fragilis promotes human CD39 + Foxp3 + T cells and T reg function. Gut 
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48. David LA, Maurice CF, Carmody RN, et al. Diet rapidly and reproducibly alters the 
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50. Lavasani S, Dzhambazov B, Nouri M, et al. A novel probiotic mixture exerts a 
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51. Takata K, Kinoshita M, Okuno T, et al. The lactic acid bacterium Pediococcus 
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52. Maassen CBM, Claassen E. Strain-dependent effects of probiotic lactobacilli on EAE 
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54. Hiroaki Yokote,Sachiko Miyake, J. Ludovic Croxford, Shinji Oki, Hidehiro Mizusawa, 
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55. Chapman BC, Moore HB, Overbey DM, Morton AP, Harnke B, Gerich ME, Vogel JD. 
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60. MacConnachie AA, Fox R, Kennedy DR, Seaton RA. Faecal transplant for recurrent 
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67. Hamilton MJ, Weingarden AR, Sadowsky MJ, Khoruts A. Standardized frozen 
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APPENDIX A 
Donor Screening Criteria 
Donor inclusion criteria: 
A healthy donor who has a normal body mass index (BMI of 18.5 – 24.9) and who 
satisfies the following criteria will be selected for the study and screened for all known potential 
transmissible agents. 
 
Donor exclusion criteria: 
o Any underlying metabolic disease including; hypertension, hyperlipidemia, diabetes, 
insulin insensitivity, atherosclerosis  
o A history of any gastrointestinal or liver disorders or cancers.  Including but not limited 
to; gastroesophageal reflux, peptic ulcer disease, celiac disease, inflammatory bowel 
disease (Crohn’s disease or ulcerative colitis), microscopic colitis, motility disorders 
(including gastropariesis and irritable bowel syndrome) and diverticular disease. 
o Previous surgery to the intestine, liver or gallbladder (except remote appendectomy). 
o History of any malignancy 
o Use within 3 months of any antibiotics or probiotics. 
o Hospitalization within 3 months. 
o Recent travel to a developing country (within 3 months) 
o New Sexual Partner (within 3 months). 
o Street drug use 
o Family history of diabetes, early onset coronary disease or gastrointestinal or liver 
disease, colon cancer, familial malignancy 
o Psychiatric history (major affective disorder, psychotic illness, ongoing use of any 
psychiatric medications) 
o Any positive laboratory results for a transmissible pathogen 
o Alcohol intake with a cut off value of <10g/d in women and <20g/d in men. 
 


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Donor laboratory screening: Summary of Tests and Procedures for Donor 
Screening is given below. For information purposes only. 


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Protocol No. MSFMT -001 
Visit 
 
 
Screening/ Visit 1 
30min-1hr 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Tests and Procedures  
(as applicable based on the protocol) 
 
Assess body mass index (BMI of 18.5–24.9) 
Physical examination 
Use within 3months of any antibiotics or 
probiotics 
Hospitalization within 3 months  
Collect past medical history, family history 
of diabetes or coronary disease, psychiatric 
history 
Recent travel to a developing country (within 
3 months) 
New Sexual Partner (within 3 months). 
Street drug use 
Alcohol intake with a cut off value of <10g/d 
in women &<20g/d in men. 
Stool (1g): Microbiology/Immunology tests: 
Bacterial culture, Ova and Parasites and C. 
difficile. If donor has travelled to areas where 
other infections occur we will screen for 
those as well.  
Stool 
for 
Transplantation 
- 
~50 
- 
70g/transplantation  
Urine (5ml): Chlamydia and Gonorrhea 
Blood screening (5-10ml): Biochemical 
assays:, liver function tests 
Microbiology/Immunology tests: Screen for 
HIV 1/2, HTLV 1/2, Hepatitis A IgM, 
Hepatitis BsAg, Hepatitis BcAb, Hepatitis C, 
H. pylori antibody and Anti TTG antibody. 
Serology 
for 
Syphilis, 
Strongyloides, 
Schistosomiasis, Amebiasis, CMV and EBV. 
Throat and Rectal swab: for Chlamydia and 
Gonorrhea 
Nasal swab and rectal swab: for drug 
resistant organisms including MRSA, VRE 


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Visit 2  
10min 
 
 
 
 
and 
CRE 
(carbapenemase 
producing 
Enterobacteriaciae) 
 
 
 
To drop off Stool samples collected fresh or 
within 3days of the transplantation. 
 


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