Conditions: Clostridium Difficile
Allocation: NA | Intervention model: SINGLE_GROUP
Enrollment: 17
Healthy volunteers: no

Brief summary:
The overarching objective of this study is to address the knowledge gap regarding the short-term and long-term safety of fecal microbiota transplants (FMT). The design will be a prospective, open-label, multi-center longitudinal cohort study to assess the short- and long-term safety of FMT as well as the clinical resolution of diarrhea among 150 patients with 3 or more episodes of clostridium difficile infection (CDI defined as 3 unformed stools over 24 hours for 2 consecutive days and either a positive stool test for CDI or pseudomembranes on colonoscopy/sigmoidoscopy). Subjects will be adult outpatients referred to one of the study centers after at least three recurrent episodes of CDI and previous treatment with at least one 10-day course of oral vancomycin or fidaxomicin. After FMT by colonoscopy/sigmoidoscopy or enema, patients will be followed prospectively and monitored for clinical resolution and adverse events at: 3 days (telephone), 3 weeks (clinical assessment), 8 weeks (telephone), 6 months (telephone), and 12 months (telephone) after FMT. Subjects who recur will be offered a second FMT by colonoscopy with a different donor. Microbiome analysis will be conducted from stool samples at baseline and each of the 5 follow-up intervals.

Detailed description:

Arms:
arm 1: Open label single arm Dosage form: Screened human donor stool, sourced from human-derived microbes generated by healthy, screened donors.

Route of administration: either colonoscopic/sigmoidoscopic FMT or retention enema FMT Dosing Regimen: 250 mL x 1 dose. In the event of a clinical non-response, a repeat single 250 mL dose will occur from a different donor

Interventions: intervention 1: Fecal Microbiota Preparation
Intervention descriptions: intervention 1: Frozen processed human fecal material for treating recurrent Clostridium difficile infections.

Protocol document:
Prot_SAP_000.pdf: 
U.S. Food and Drug Administration (FDA) 
Investigational New Drug Application 
 
 
 
 
 
 
 
 
Sponsor: OpenBiome 
Investigator: Zain Kassam MD, MPH, FRCPC 
196 Boston Avenue 
Suite #2100 
Medford, MA 
02155 
 
Telephone: 857-333-7375 
Fax: 617-575-2201 
 
 
 
 
 
Study Title:  Safety of fecal microbiota Transplantation: OpenBiome Outcomes 
and Longitudinal follow-up (STOOL) for recurrent Clostridium difficile infection 
 
 
 
 
Product:  
Fecal Microbiota Preparations  
 
Proposed Indication:  
Recurrent Clostridium difficile infection  
 
Date Submitted:  
 
October 3, 2014 
 
Edition: 
 
 
1st 
 
ClinicalTrials.gov Identifier: NCT02403622 
 
 
 
 


 
2
2. TABLE OF CONTENTS 
3. INTRODUCTORY STATEMENT AND GENERAL INVESTIGATIONAL PLAN .................... 4 
I. INTRODUCTORY STATEMENT ............................................................................................... 4 
OVERARCHING OBJECTIVE ..................................................................................................... 4 
THE BURDEN OF CLOSTRIDIUM DIFFICILE INFECTION .................................................................... 4 
AN EMERGING TREATMENT: FECAL MICROBIOTA TRANSPLANTATION .......................................... 4 
II. GENERAL INVESTIGATIONAL PLAN ..................................................................................... 5 
III. PRODUCT NAME/CHEMICAL STRUCTURE ........................................................................... 5 
IV. PROPOSED INDICATION(S) ............................................................................................. 6 
V. DOSAGE, ROUTE OF ADMINISTRATION AND DOSING REGIMEN ............................................. 6 
V-A COLONOSCOPIC FMT (FMP250) ................................................................................... 6 
V-B RETENTION ENEMA FMT (FMP250E) ................................................................................ 6 
4. [RESERVED, CFR 312.23] ............................................................................................. 7 
5. INVESTIGATOR’S BROCHURE ..................................................................................... 8 
6. PROTOCOLS ............................................................................................................... 9 
I. STUDY OVERVIEW AND SPECIFIC AIMS ................................................................................ 9 
STUDY TITLE ............................................................................................................................ 9 
SPECIFIC AIMS (SA) ................................................................................................................ 9 
II. SUBJECT INCLUSION/EXCLUSION CRITERIA ....................................................................... 10 
DEFINITIONS ......................................................................................................................... 10 
INCLUSION CRITERIA ............................................................................................................. 10 
EXCLUSION CRITERIA ............................................................................................................. 11 
TARGET ENROLLMENT AND RATIONALE ................................................................................... 11 
III. DESIGN METHODOLOGY ............................................................................................... 12 
IV. THERAPEUTIC ADMINISTRATION: FECAL MICROBIOTA TRANSPLANTATION PROTOCOL ............ 13 
DONOR SELECTION, LABORATORY SCREENING, AND MONITORING ........................................... 13 
SUBJECT PREPARATION …………………………………………………………………………...….…..14 
PREPARATION AND HANDLING OF STOOL FOR FMT INFUSION ................................................... 15 
BASELINE ASSESMENT AND FOLLOW-UP  ……………………………………………………………………     16 
FMT PROCEDURE GUIDELINES ................................................................................................ 16 
IN THE EVENT OF CLINICAL NON-RESPONSE ............................................................................. 17 
WITHDRAWAL ………………………………………………………………………………………………………. 17 
V. STUDY OBSERVATIONS AND MEASUREMENTS .................................................................... 17 
STUDY ENDPOINTS   ………………………………………………………………………………………………… 18 
DATA COLLECTION .............................................................................................................. 18 
DATA ANALYSIS ................................................................................................................... 19 
VI. ADVERSE EVENT MONITORING ...................................................................................... 20 
VI-A. ADVERSE EVENTS REPORTING ....................................................................................... 20 
VI-B. SOLICITED ADVERSE EVENTS ......................................................................................... 22 
VI-C. MONITORING OF ADVERSE EVENTS .............................................................................. 25 
VI-D. ASSESSMENT OF ADVERSE EVENTS ................................................................................ 25 
VI-E. REPORTING SAFETY OBSERVATIONS BY THE INVESTIGATOR TO THE SPONSOR ...................... 26 
VI-F. MONITORING THE STUDY DATABASE AND SUBMITTING SAFETY REPORTS .............................. 27 


 
3
VI-G. HALTING RULES .......................................................................................................... 28 
VII. MINIMIZING RISKS TO HUMAN SUBJECTS ....................................................................... 28 
VII-A. HUMAN SUBJECTS INVOLVEMENT, CHARACTERISTICS AND DESIGN ................................. 28 
VII-B. POTENTIAL RISKS TO SUBJECTS ...................................................................................... 30 
VII-C. POTENTIAL RISKS TO DONORS ..................................................................................... 30 
VII-D. PROTECTION AGAINST RISKS TO SUBJECTS AND DONORS ............................................... 31 
VII-E. SAFETY MONITORING PROTOCOL ................................................................................ 33 
VIII. DATA MONITORING PLAN ........................................................................................... 34 
IX. EDUCATIONAL TRAINING .............................................................................................. 35 
X. POTENTIAL LIMITATIONS ................................................................................................. 35 
XI. INVESTIGATOR CREDENTIALS AND ASSOCIATED BODY CONTACT INFORMATION ................... 35 
XII. CLINICALTRIALS.GOV REQUIREMENTS ………………………………………………………. 36 
7. CHEMISTRY, MANUFACTURING, AND CONTROLS INFORMATION ......................... 36 
8. PHARMACOLOGY AND TOXICOLOGY INFORMATION .......................................... 38 
9. PREVIOUS HUMAN EXPERIENCE WITH THE INVESTIGATIONAL DRUG ..................... 38 
I. INDICATION .................................................................................................................. 38 
II. BACKGROUND OF DISEASE ............................................................................................ 38 
III. SUMMARY OF PREVIOUS HUMAN SAFETY EXPERIENCE ....................................................... 39 
III-A OBSERVATIONAL DATA ................................................................................................. 39 
III-B RANDOMIZED CLINICAL TRIAL DATA ............................................................................... 40 
III-C INVESTIGATOR PRELIMINARY DATA ................................................................................. 40 
III-D OVERALL SUMMARY TABLE  ……………………………………………………………………………….41 
REFERENCES ................................................................................................................. 43 
 
 
Appendix 1: OpenBiome Drug Master File Authorization Letter  
 
Appendix 2: OpenBiome Donor Risk Factor Questionnaire  
 
Appendix 3: OpenBiome Donor Prospective Health Status Questionnaire  
 
Appendix 4: STOOL Pre-enrollment Check List and Case Report Forms 
 
Appendix 5: STOOL Subject Contact Schedule 
 
Appendix 6: Informed Consent  
 
Appendix 7: Principal Investigator CV 
 
Appendix 8: 21 CFR 312.8 Cost Recovery – Support for Qualification  
 
 


3. INTRODUCTORY STATEMENT AND GENERAL INVESTIGATIONAL PLAN  
I. Introductory Statement 
Overarching Objective 
There is emerging concern regarding suboptimal efficacy of standard antibiotic 
treatment for recurrent Clostridium difficile infection, leading to expanded interest in 
fecal microbiota transplantation, a highly promising therapy. However, little is known 
about the safety, particularly long-term, of fecal microbiota transplants as current 
retrospective data has had sporadic, inconsistent follow-up. The overarching objective 
of this study is to address this knowledge gap by collecting safety data from a 
multicenter cohort in a prospective, longitudinal manner. 
The burden of Clostridium difficile infection  
Clostridium difficile infection (CDI) has recently been reported as the most common 
healthcare-associated infection in the United States, and the emergence of 
community-acquired CDI is particularly concerning.1 2 CDI is associated with a spectrum 
of symptoms ranging from diarrhea to abdominal pain, and severe infections may lead 
to toxic megacolon and death. The clinical burden of CDI is significant. In the United 
States, the incidence of CDI per 10,000 inpatient hospitalizations doubled between 2000 
and 2005 (5.5 versus 11.2 cases).3 Moreover, CDI presents substantial economic 
ramifications. Nosocomial CDI increases the cost of an otherwise matched 
hospitalization by four-fold, translating to a reported cost of up to $4.8 billion/year in the 
United States.4–6  
 
The suboptimal efficacy of standard antibiotic treatment for CDI has prompted growing 
concerns. Since 2000, failure rates of metronidazole, the recommended first-line 
treatment for mild to moderate CDI, have increased from 2.5% to more than 18%.7 
Other reports from Canada, the United Kingdom, and Spain have also documented 
resistance to metronidazole and reduced susceptibility to vancomycin in CDI.8–10 What 
is most concerning about the landscape of CDI is the incidence of recurrent CDI 
following antibiotic treatment with metronidazole, vancomycin or fidaxomicin – which 
continues to increase and has become a major challenge.11–14 After 2 or more episodes 
of recurrence, the risk of subsequent recurrence has been reported to exceed 60% with 
antibiotic treatment.12 15 16 Evidence-based treatment options are limited for patients 
experiencing multiple recurrent CDI episodes as highlighted in the most recent US 
clinical guidelines.17 
An emerging treatment: Fecal Microbiota Transplantation  
Given the shortcomings of standard antibiotic therapy, particularly in patients with 
recurrent CDI, alternative approaches have been explored. Although its mechanism of 
action is not fully elucidated, fecal microbiota transplantation (FMT) is a unique therapy 
that aims to address the underlying mechanism of recurrent CDI. Specifically, FMT aims 
to restore ‘colonization resistance’, re-establish diversity, and facilitate microbial 
homeostasis in order to protect against toxigenic CDI.18–20 Systematic review/meta-
analysis of observational data has described clinical cure in approximately 90% of 
patients, where a placebo response seems unlikely in a patient population with 


 
5
prolonged and severe symptoms in the context of previous standard antibiotics.21 
Additionally, small randomized controlled trials have reported similarly impressive 
efficacy in a difficult to treat patient population.22 23  
 
Although the efficacy of FMT appears convincing, there is a paucity of robust safety 
data for FMT. The current safety profile has been collected predominantly from 
retrospective reviews or systematic reviews of case series data, approaches that have 
methodological limitations in assessing the safety of FMT.21 24 25 Broadly, the literature 
contains inconsistent long-term follow-up of adverse events, which have been 
spontaneously reported rather than actively sought, in turn increasing the risk of 
underreporting. 21 Randomized controlled trials have also had brief follow-up and one 
trial was stopped early for benefit at interim analysis, limiting the ability to meaningfully 
assess the long-term safety profile of FMT.22 23 Although the current body of literature 
does not signal any significant safety risk for FMT, a high-quality prospective longitudinal 
study is needed to confirm the safety profile of FMT among recurrent CDI patients. 
 
OpenBiome is a 501(c)(3) non-profit organization that is the first public FMT stool bank in 
the United States for CDI non-responsive to standard therapy. Under the FDA’s current 
regulatory framework of enforcement discretion, OpenBiome has partnered with more 
than 100 health care institutions in over 30 states to expand safe, affordable access to 
FMT. Accordingly, our institute is uniquely positioned to meaningfully assess the safety 
profile of FMT, as our group and others have identified an absence of high-quality, 
prospective studies interrogating safety.21  
II. General Investigational Plan 
 
The design will be a prospective, open-label, multi-center longitudinal cohort study to 
assess the short- and long-term safety of FMT as well as the clinical resolution of diarrhea 
among 150 patients with 3 or more episodes of CDI (defined as 3 unformed stools over 
24 hours for 2 consecutive days and either a positive stool test for CDI or 
pseudomembranes on colonscopy/sigmoidoscopy).26 Subjects will be adult outpatients 
referred to one of the study centers after at least three recurrent episodes of CDI and 
previous treatment with at least one 10-day course of oral vancomycin or fidaxomicin. 
After FMT by colonoscopy/sigmoidoscopy or enema, patients will be followed 
prospectively and monitored for clinical resolution and adverse events at:  
3 days (telephone), 6 weeks (clinical assessment), 6 months (telephone or clinical 
assessment), and 12 months (telephone or clinical assessment) after FMT. Subjects who 
recur will be offered a second FMT by colonoscopy with a different donor. Microbiome 
analysis will be conducted from stool samples at baseline and each of the 4 follow-up 
intervals. 
III. Product Name/Chemical Structure 
 
Fecal Microbiota Preparations 


 
6
IV. Proposed Indication(s) 
 
Recurrent Clostridium difficile infection 
V. Dosage, Route of Administration and Dosing Regimen  
V-A Colonoscopic/sigmoidoscopic FMT (FMP250) 
 
Dosage Form: Screened human donor stool, 
 
 
 homogenized, filtered 
 and aliquoted to sterile 250mL 
vessels. 
 
Route of Administration: Infusion will occur in the most proximal region of the colon that 
is technically feasible. The material will be infused through the working channel of the 
endoscope. 
 
Dosing Regimen: 250 mL x 1 dose. In the event of a clinical non-response (defined in 
‘Protocol’ Section), a repeat single 250 mL dose will occur from a different donor. 
V-B Retention Enema FMT (FMP250E) 
 
Dosage Form: Screened human donor stool, 
 
 
 homogenized, filtered 
 and aliquoted to sterile 250mL 
vessels. 
 
Route of Administration: Lubricated enema nozzle will be inserted into rectum and 
contents expelled into the distal colon with the subject requested to retain material for 
at minimum 30 minutes.  
 
Dosing Regimen: 250mL x 1 dose. In the event of clinical non-response (defined in 
‘Protocol’ Section), a repeat single 250 mL dose via colonoscopy will occur from a 
different donor. 
 
 
 
 
 
 
 
 
 
 
 
 
 


 
7
4. [RESERVED, CFR 312.23]  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


 
8
5. INVESTIGATOR’S BROCHURE 
 
[Follows] 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


 
9
6. PROTOCOL 
I. Study Overview and Specific Aims 
 
The proposed multi-center, open-label, longitudinal cohort study aims to capture the 
safety profile of FMT in recurrent CDI, as well as to contribute additional efficacy data to 
the existing body of evidence. The proposed study includes collaboration with 
Investigators who are able to define the microbial landscape of patients pre- and post-
FMT, and further elucidate if there is a microbial signature that predisposes patients to 
respond, fail treatment, or have adverse events from FMT.  
Study Title 
Safety of fecal microbiota Transplantation: OpenBiome Outcomes and Longitudinal 
follow-up (STOOL) for recurrent Clostridium difficile infection 
Specific aims (SA)  
 
SA1. Determine the short-term (<6 weeks) safety of FMT for the prevention of 
further CDI recurrence. 
Hypothesis: Careful donor screening for both infectious and non-infectious yet 
potentially microbiome-associated conditions minimize the risk of donor to 
recipient transmission of disease. Therefore, FMT will not be associated with 
meaningful number of related serious adverse events, recognizing that the 
recurrent CDI patient population is conventionally elderly with multiple 
comorbidities and may be at risk for FMT procedure-related complications 
compared to otherwise young and healthy individuals based on conventional 
literature.  
 
SA2. Determine the long-term (6-12 months) safety of FMT for the prevention of 
further CDI recurrence. 
Hypothesis: Careful donor screening for both infectious and non-infectious yet 
potentially microbiome-associated conditions minimize the risk of donor to 
recipient transmission of disease. Therefore, FMT will not be associated with 
meaningful number of related serious adverse events, recognizing that the 
recurrent CDI patient population is conventionally elderly with multiple 
comorbidities and may be at risk for developing new conditions, new 
medications or adverse events including death, but unrelated to FMT material.  
 
SA3. Determine the longitudinal robustness of FMT clinical cure rate for the 
prevention of further CDI recurrence. 
Hypothesis:  We predict a clinical cure rate in keeping with the current literature 
(80-90%) at the12 month follow-up time interval. This predicted longitudinal 
response rate would exclude patients who require further antibiotics that put 
them at risk for CDI reinfection, not true recurrence.  
 
 


 
10
SA4. Collect pre- and post-FMT stool samples from patients with recurrent CDI as 
well as donor stools for microbiome analysis. 
Hypothesis: Prior to FMT, recurrent CDI patients will have deficiencies of certain 
normal colonic flora.22 After FMT, these microbial populations will be restored, 
resulting in a durable transition to a diverse, stable community that resembles the 
composition of the healthy stool donor. We expect that this transformation will be 
stable in the absence of challenge with antimicrobial therapy.  
II. Subject Inclusion/Exclusion Criteria 
Definitions 
 
Clostridium difficile infection: At least 3 unformed stools over 24 hours for 2 
consecutive days and either positive stool test (ELISA or PCR) for 
Clostridium difficile toxins or pseudomembranes on colonoscopy (SHEA-
IDSA guidelines).26 
 
Clinical Cure: Clinical resolution of diarrhea (<3 unformed stools for 2 
consecutive days) with maintenance of resolution at 6-week follow-up 
period, at minimum, and no further need for antibiotic therapy for CDI. In 
keeping with SHEA-IDSA guidelines and an absence of ‘test of cure,’ 
subjects who meet aforementioned definition will be considered a clinical 
cure regardless of any incidental follow-up stool testing employed.26 
 
Clinical Non-Response: Ongoing or development of diarrhea and the 
requirement for antibiotic therapy for CDI.  
Inclusion criteria 
 
Adult (age 18-75 years old) 
 
Outpatient  
 
Third or further documented CDI episode and 
 
Unable to maintain CDI cure after standard therapy with oral vancomycin 
or fidaxomicin 
o Previous treatment with at least one course of tapered/pulse 
vancomycin or 
o Inability to taper or stop vancomycin or fidaxomicin without 
developing diarrhea requiring antibiotic therapy. 
 
Improvement of CDI symptoms on vancomycin or fidaxomicin   
Exclusion criteria 
 
Unable to comply with study follow-up procedures at discretion of MD 
 
Unable to provide informed consent at discretion of MD 
 
Participating in another clinical trial 
 
Pregnant or nursing currently or planned pregnancy in next 1 year 
 
Evidence of toxic megacolon or gastrointestinal perforation 
 
Peripheral white blood cell count >30 x 109/L and/or temperature >38 
degrees Celsius  
 
Admission to an intensive care unit within prior 7 days for any reason 
 
Previously undergone FMT 
 
Severely immunocompromised patients 


 
11
o HIV infection (any CD4 count) 
o AIDS-defining diagnoses  
o Inherited/primary immune disorder 
o Immunosuppressant medications 
 
Current or recent (<3 months) treatment with anti-neoplastic 
agents  
 
Current or recent (<3 months) treatment with calcineurin 
inhibitors (tacrolimus, cyclosporine) 
 
Current or recent (<3 months) treatment with 
mycophenolate mofetil 
 
Current or recent (<3 months) treatment with monoclonal 
antibodies to B and T-Cells, anti-TNF, glucocorticoids, 
antimetabolities (azathioprine, 6-mercaptopurine)  
 
Neutropenia with absolute neutrophil count (ANC) <0.5 x 109/L 
 
Active gastroenteritis due to infectious cause other than CDI 
 
Short gut syndrome 
 
Colostomy 
 
Ascites 
 
End-stage liver disease  
 
Untreated, in-situ colorectal cancer 
 
Irritable bowel syndrome  
 
Inflammatory bowel disease including Crohn’s disease and ulcerative 
colitis 
 
Microscopic colitis including collagenous colitis and lymphocytic colitis  
 
Severe food allergy (anaphylaxis) that cannot be confirmed as having 
been excluded from a donor’s diet within the five days prior to donation 
 
Anorectal disorder/severe rectal sphincter tone abnormality or inability to 
retain enema material  
 
Unable or unwilling to tolerate colonoscopy/sigmoidoscopy, colonoscopy 
prep, or enema for any reason at discretion of MD 
 
Severe underlying disease that the patient is not expected to survive for 
the subsequent 12 months at the discretion of the MD. 
 
Any conditions for which, in opinion of MD, the treatment may pose a 
health risk 
Target enrollment and rationale 
The longitudinal cohort sample size (n=150) was chosen to allow recruitment in a 
suitable time frame. Strong consideration will be given to resubmitting documentation 
including IND amendment and IRB ammendment for a larger sample size and/or 
prolonged follow-up at the completion of the study. 
 
 




 
13
IV. Therapeutic Administration: Fecal Microbiota Transplantation 
Protocol 
Donor selection, laboratory screening, and monitoring  
A. Donor Clinical Assessment: OpenBiome utilizes adult, healthy, universal donors 
who have given informed consent with oversight from Massachusetts Institute of 
Technology (MIT) IRB/COUHES.  Refer to the OpenBiome Master File (BB-MF 
15543) for donor selection details. An accompanying authorization letter from 
OpenBiome can be found in Appendix 1. The OpenBiome Donor Risk Factor 
Questionnaire (Appendix 2) is detailed elsewhere. Briefly, the Donor Risk Factor 
Questionnaire is modified from a similar questionnaire developed by the 
American Association of Blood Banks for blood donors. The stool donor 
questionnaire is used to exclude potential donors with infectious disease risk 
factors (eg. high risk sexual behavior, risk factors for variant Creutzfeldt-Jakob 
disease, high risk travel), medications that may mediate the microbiome, and a 
wide array of medical conditions that may have a theoretical link the 
microbiome. 
 
B. Donor Laboratory Screening: Refer to the OpenBiome Master File (BB-MF 
15543) for donor laboratory screening details. An accompanying authorization 
letter from OpenBiome can be found in Appendix 1. Briefly, both serologic and 
stool test are completed on prospective donors. All tests are outsourced to third-
party Clinical Laboratory Improvement Amendments (CLIA)-certified testing 
facilities. As a condition for participation in this program, donors are required to 
submit written authorization for the disclosure of these test results to the 
OpenBiome, in compliance with the Health Insurance Portability and 
Accountability Act (HIPAA). 
 
C. Donor Monitoring: Refer to the OpenBiome Master File (BB-MF 15543) for donor 
monitoring details. An accompanying authorization letter from OpenBiome can 
be found in Appendix 1. Briefly, the OpenBiome Donor Prospective Health Status 
Questionnaire (Appendix 3) details the routine questions asked before processing 
of a specimen, with initiation of clinical assessment by the Chief Medical Officer 
if a positive answer disclosed by a donor.  
 
 
 
 
 
Subject preparation: determining eligibility, pre-treatment, and special considerations 
A. Subject Clinical Assessment  
1. Potential subjects will undergo a medical interview to determine 
eligibility for the study and a physical exam will be performed at the 
screening visit. 


 
14
2. Baseline status, at MD discretion, will be conducted:  HIV (type 1 and 2), 
hepatitis A (IgM, G), hepatitis B (HBsAg, anti-HBcIgM and anti-HBcIgG), 
hepatitis C (HCV antibody), and Treponema pallidum screening (EIA with 
reflex to RPR). 
3. Baseline complete blood count, liver function panel, creatinine and 
random blood glucose, and stool C. difficile PCR will be documented prior 
to FMT.  
 
B. Pre-Treatment Medications 
1. Subjects will complete at least a 10 day course of vancomycin or 
fidaxomicin for the most recently diagnosed acute CDI prior to 
undergoing FMT.  
2. To minimize symptoms, antibiotic therapy will be continued by 
subjects until 3 days prior to the FMT procedure then subsequently 
discontinued. 
3. Any probiotics will be continued by subject until 3 days prior to 
the FMT procedure then subsequently discontinued. 
4. The day before the procedure, the subject will be prepped with 
standard bowel prep if undergoing colonoscopic/sigmoidoscopy 
delivery at MD discretion. Patients receiving FMT via enema 
administration need not undergo a bowel prep at MD discretion. 
 
C. Specimen Collection 
 
Patients must be willing and able to provide stool samples for microbial 
characterization via high-throughput 16S rRNA sequencing. Patients will 
be asked to provide a baseline stool sample prior to the FMT procedure 
(Day 0), as well as follow-up stool samples collected at Day 3, Week 6, 
Month 6 and Month 12 after FMT. 
 
Samples will be collected with a standard stool collection hat. 
Immediately after passage, approximately 1 gram of stool will be 
suspended in 5 ml of RNALater (Life Technologies) using a para-pak 
collection vial with embedded scoop (Meridian Biosciences). Samples will 
then be shipped at room temperature to the OpenBiome study site where 
they will be frozen within 7 days of passage, consistent with the technical 
specifications for stabilization of nucleic acids in RNAlater. To avoid 
contamination of the OpenBiome production facility, sample processing 
and sequencing will take place at the Massachusetts Institute of 
Technology in a BL-2 laboratory with appropriate containment.  
 
 
 
D. Female Subjects:  
1. Females of childbearing potential will have a urine pregnancy test on 
the day of enrollment and be excluded if pregnant.  


 
15
2. Female subjects must not be and should not become pregnant nor 
breast-feed an infant while in this study. In order to reduce the risk of 
pregnancy, the subject or her partner should use one or more of the 
acceptable methods of birth control listed below, regularly and 
consistently, while enrolled in this study. Acceptable methods of birth 
control, which will continue throughout the study and for one month after 
the study, include: 
 
 
Approved oral contraceptive/birth control pill 
 
Intra-uterine device (IUD) 
 
Hormone implants 
 
Contraceptive injections (Depo-Provera) 
 
Barrier method (diaphragm with spermicidal gel or condoms) 
 
Transdermal contraceptives (birth control patch) 
 
Vaginal contraceptive ring (birth control rings) 
 
Sterilization (tubal ligation, hysterectomy or vasectomy) 
 
Abstinence 
 
3. If subjects become pregnant or suspect that they are pregnant during 
this study, they will be instructed to immediately inform the study 
personnel. If subjects become pregnant or suspect that they are pregnant 
while in this study, a pregnancy test will be performed. If pregnancy is 
confirmed, they will be withdrawn from the study if FMT therapy has not 
yet occurred. If FMT has occurred and conception occurs within the study 
period, the study physician will assist the patient in getting obstetrical care 
and will follow the progress of the pregnancy. The study physician will 
request access to subject and/or infant’s medical records for up to 8 
weeks after delivery. 
 
 
Preparation and handling of stool for FMT infusion 
1. Frozen material will thaw for 4 hours at room temperature. After thawing, 
material may remain at room temperature for up to 4 hours, or be kept 
refrigerated/on ice for up to 8 hours.  
2. Standard protocol for handling biohazardous material will be employed in 
order to avoid contamination and risk to healthcare handlers. Sterile 
microbiological technique will be employed during material transfer peri-
procedure. 
FMT procedure guidelines 
 
1. Colonoscopy/sigmoidoscopy 
 
The procedure will be completed by a trained gastroenterologist or 
general surgeon in a standard endoscopy unit. 
 
Colonoscopy/sigmoidoscopy will be performed under direct visualization, 
aiming to reach the most proximal part of the colon using safe 
endoscopic maneuvers and minimal insufflation. 
 
Once at the most proximal location, 250 mL of the fecal material will be 
passed through the endoscope channel using a sterile syringe. 


 
16
 
Standard normal saline flush(s) will be utilized after all the fecal material 
has been infused. 
2. Enema  
 
The procedure will be completed by a trained Registered Nurse, Physician 
Assistant, or physician in an outpatient clinic, endoscopy recovery area, or 
standard endoscopy unit. 
 
Fecal material will be transferred into conventional enema bottle/bag. 
 
Patient will be placed in left lateral decubitus position with foot of bed 
elevated to facilitate retention. 
 
Lubricated enema nozzle will be inserted into rectum and contents 
expelled into the distal colon with the subject requested to retain material 
for at minimum 30 minutes but targeting 1 hour. 
 
The patient will be asked to transfer to right lateral decubitus position after 
15 minutes. If unable to retain material for 1 hour, total retention time will 
be recorded.  
 
Baseline assessment and follow-up  
1. Subjects will be instructed to contact the clinical team if any symptoms of 
diarrhea occur or any adverse events occur. Follow-up assessments will focus on 
capturing any serious adverse events, new medical conditions, or changes in 
patients’ pre-FMT medical conditions since last study contact. 
2. After passing eligibility assessment, subjects will be assessed at baseline (Day 0 = 
day of procedure) to capture and document clinical and FMT characteristics 
(Appendix 4). 
3. A study representative will contact subjects at Day 3 (+/- 2 days) post-FMT 
(Appendix 4). 
4. An in-person clinical assessment between Week 6 (+/- 2 weeks) post-FMT will be 
performed by FMT provider (Appendix 4). If this visit cannot be arranged, the 
patient’s primary care provider may complete the assessment under the guidance 
and instruction of the FMT provider. 
5. A study representative will contact subjects at Month 6 (+/- 2 weeks) and Month 
12 (+/- 2 weeks) post-FMT (Appendix 4). 
6. A study representative will contact subjects to remind them to collect and mail 
stool samples. 
7. A full Subject Contact Schedule (Appendix 5) and Informed Consent (Appendix 
6). 
In the event of clinical non-response 
1. Colonoscopy/sigmoidoscopy: If patients experience clinical non-response and 
require vancomycin/fidaxomicin within 6 weeks post-FMT, after a 10 day course 
subject may opt to undergo a second FMT via colonoscopy with an alternate donor 
at the physician’s discretion. Subjects will be followed per protocol for duration of 
study. 
2. Enema: If patients experience clinical non-response and require 
vancomycin/fidaxomicin within 6 weeks post-FMT, after a 10 day course subject 
may opt to undergo a second FMT via colonoscopy with an alternate donor at the 
physician’s. Subjects will be followed per protocol for duration of study. 
 


 
17
Withdrawal 
1. All reasons for study withdrawal will be reported.  
2. Classification of withdrawal: 
 
Post-enrollment but pre-FMT 
 
Post-FMT but lost to follow-up at: 
o Day 3 
o Week 6 
o Month 6 
o Month 12 
 
All reasonable efforts will be exhausted to contact subjects, 
and data analysis will be based on the last available 
assessment.  
 
Adverse event or serious adverse event mediating withdrawal 
 
Rescind informed consent 
 
Procedure-related ineligible patients 
o Patients undergoing colonoscopy/sigmoidoscopy and discovered 
to be ineligible due to newly discovered exclusionary criteria (eg. 
in-situ colorectal cancer). FMT will not be performed as part of this 
study and no data will be collected.  
 
 
V. Study Observations and Measurements  
 
Study Endpoints 
 
Safety Endpoints 
Safety will be assessed by short and long-term safety summaries classified into: 
1. Serious Adverse Events (SAE)  
2. Adverse Events (AE) 
3. New medical conditions 
4. Changes in pre-existing medical conditions 
 
Efficacy Endpoints 
Efficacy will be captured through: 
 
1. Clinical cure rate of first FMT at 6-week follow-up 
 
Colonoscopy 
 
Sigmoidoscopy 
 
Enema 
2. Clinical cure rate of second FMT at 6-week follow-up, in patients with clinical 
non-response 
 
Colonoscopy/sigmoidoscopy non-response: Alternate donor FMT by 
colonoscopy 
 
Enema non-response: Alternate donor FMT by colonoscopy 
 
Microbiome Endpoints 


 
18
Sequencing data targeting the composition of the gut microbiome will be used to 
assess whether there are consistent microbial signatures associated with clinical cure, 
clinical non-response, or safety endpoints. 
Data Collection  
Case Report Forms will be utilized to capture and collect data (Appendix 4):  
 
1. Baseline Assessment (Day 0 – Clinical Assessment) 
Section 1: Clinical Evaluation  
 
Study patient identifier  
 
Date 
 
Demographics: Age, sex, race/ethnicity 
 
Pre-existing medical conditions 
 
Concomitant medications  
 
Vital signs including height, weight, calculated body mass index (BMI) 
 
CDI History, including: 
o Date of initial CDI diagnosis 
o Number of CDI recurrences since initial diagnosis  
o Baseline clinical symptoms and severity related to CDI 
o CDI treatments received to date  
 
Section 2: FMT Procedure 
 
Study patient identifier  
 
Date 
 
Description of FMT procedures, including: 
o Route of administration (colonoscopy/sigmoidoscopy or enema) 
o Location in colon where FMT delivered 
o Sedation (if applicable) 
o Prep quality (if applicable) 
o Findings of colonoscopy (if applicable) 
 
Dose 
 
Unit ID/Lot# of each treatment 
 
Expiration Date 
 
Storage Condition  
 
FMT retention time (if less than 1 hour) 
 
Immediate adverse events 
 
2. Short-term assessment I (Day 3 – Telephone Assessment) 
 
Study patient identifier 
 
Date 
 
Any change in baseline clinical symptoms related to CDI 
 
Assessment of SAEs, including: 
o Any complications related to FMT administration 
o Any potential infectious complications such as transmissible enteric 
infections, sepsis, peritonitis, or bacteremia 
o Any non-serious newly diagnosed infections (specific organism and body 
site) 


 
19
o Any new medical conditions / new medications with associated clinical 
course 
o Any changes in pre-existing medical conditions with associated clinical 
course 
 
3. Short-term assessment II (Week 6 – Clinical Assessment) 
 
Study patient identifier 
 
Date 
 
Vital signs including height, weight, calculated body mass index (BMI) 
 
Any change in baseline clinical symptoms related to CDI including 
documentation of clinical cure or clinical non-response  
 
Assessment of SAEs, including: 
o Any complications related to FMT administration  
o Any potential infectious complications such as transmissible enteric 
infections, sepsis, peritonitis, or bacteremia. 
 
Any new non-serious diagnosed infections (specific organism and body site) 
 
Any new medical conditions / new medications with associated clinical course 
 
Any changes in pre-existing medical conditions with associated clinical course 
 
4. Long-term assessment I (Month 6 – Telephone Assessment) 
 
Study patient identifier 
 
Date 
 
Self-reported height and weight, resulting calculated BMI 
 
Any recurrence of CDI with description of clinical course, including additional 
treatments received 
 
Assessment of any new SAEs 
 
Any new medical conditions / new medications with associated clinical course 
 
Any changes in pre-existing medical conditions with associated clinical course 
 
5. Long-term assessment II (Month 12 – Telephone Assessment) 
 
Study patient identifier 
 
Date 
 
Self-reported height and weight, resulting calculated BMI 
 
Any recurrence of CDI with description of clinical course, including additional 
treatments received 
 
Assessment of any new SAEs 
 
Any new medical conditions / new medications with associated clinical course 
 
Any changes in pre-existing medical conditions with associated clinical course 
 
Data Analysis 
 
Primary Study: Safety and Efficacy (Aims 1-3) 
 
Safety endpoints will be collected with raw events and crude rates reported for overall 
cohort. Clinical cure rates will be calculated at each site. Both unweighted pooled 
clinical cure rates and weighted pooled clinical cure rates will be calculated with 
corresponding 95% confidence interval (CI) for overall study, as well as pre-defined 


 
20
subgroups including: age, gender, modified Horn index, delivery modality, donor, 
retention time, site. Weighted pooled rates will be analyzed using the random effects 
model (DerSimonian-Laird method). Heterogeneity across sites will be assessed using 
the I2 statistics and the Cochran Q test, where I2 > 50% or Cochrane Q < 0.10 will be 
considered to indicate significant heterogeneity. Statistics will be performed using Stata 
SE 12.0 (StataCorp, College Station, TX). 
 
Secondary Study: Microbiome (Aim 4) 
 
DNA will be extracted from each frozen specimen collected from both donors 
and recipients. The V4 region of the 16S rRNA gene will be amplified using 
multiplexed bar-coded primers as previously described.27 Up to 192 samples will 
be pooled onto a single lane of an Ilumina MiSeq for high-throughput 
sequencing. Quality trimmed and filtered sequence data will be clustered into 
Operational Taxonomic Units (OTUs) for computational analysis. Each recipient 
will receive an engraftment score based on their similarity (as quantified by the 
Jensen-Shannon Divergence) to the donor used in their FMT. Recipients will also 
be assessed for the long-term stability of their community and it’s similarity to 
samples collected and previously published from both healthy and dysbiotic 
patients. 
 
 
VI. Adverse Event Monitoring 
 
We will capture both short and long-term safety profiles, including: 
1. FMT procedure-related adverse events from colonoscopy/sigmoidoscopy or enema 
(e.g. perforation, sedation related) 
2. FMT material-related adverse events (e.g. transmissible infection, allergic reaction) 
3. Short-term safety: Both solicited and unsolicited adverse events will be recorded by, 
telephone follow-up at Day 3 (+/- 2 days) and clinical assessment at Week 6 (+/- 2 
weeks). 
4. Long-term safety evaluations will focus on any new medical conditions/medications, 
which may be related or unrelated to FMT. This information will be elicited at the 6 
month and 12 month follow-up telephone call and documented.  
VI-A. Adverse Events Reporting 
 
A. Definitions  
 
1. Adverse Event (AE) 
An AE is any untoward medical occurrence in a patient or study subject administered 
FMT that does not necessarily have a causal relationship with this treatment. An AE can 
therefore be any unfavorable and unintended sign (including an abnormal laboratory 
finding), symptom, or disease temporally associated with the use of a FMT, whether or 
not related to the FMT.  


 
21
• AEs may be new events or may be pre-existing conditions that have become 
aggravated or have worsened in severity or frequency.  
• AEs may be clinically significant changes from baseline in physical examination, 
laboratory tests, or other diagnostic investigation (e.g. laboratory results, radiographic 
findings).  
Pregnancy is not an AE; however, if a female subject becomes pregnant during the 
conduct of the study, the Investigator will notify Sponsor who is responsible for notifying 
the FDA.  
 
2. Serious Adverse Event (SAE)  
A serious adverse event is any adverse experience occurring during or after FMT that 
results in any of the following outcomes:  
 
• Death 
 
• Life-threatening experience 
Note: An adverse event or suspected adverse reaction is considered “life-threatening” 
if, in the view of either the Investigator or Sponsor, its occurrence places the patient or 
subject at immediate risk of death. It does not include an adverse event or suspected 
adverse reaction that, had it occurred in a more severe form, might have caused 
death.  
 
• Requires inpatient hospitalization or prolongation of existing hospitalization 
Note: Adverse events requiring hospital admissions that are less than 24 hours in 
duration do not meet this criterion. A scheduled hospitalization for a pre-existing 
condition that has not worsened during participation in the study does not meet this 
criterion. Pre-planned hospitalizations for an elective medical/surgical procedure or 
routine check-ups do not meet this criterion. Admission for CDI relapse will not be 
included in this definition. 
 
• Results in persistent or significant disability or incapacity 
 
• Results in a congenital anomaly or birth defect 
 
• Results in an important medical event 
Note: Important medical events are those that may not be immediately life threatening 
or result in death or hospitalization but may jeopardize the patient or may require 
intervention to prevent one of the outcomes listed in the definition above.  
 
3. Planned Hospitalization  
A hospitalization planned prior to FMT is to be considered a therapeutic intervention 
and not the result of a new SAE. If the hospitalization or procedure is executed as 
planned, it will be recorded in the subject’s medical history or procedures. However, if 
the event/condition worsens during the study, it must be reported as an AE.  
 
4. Adverse reaction 


 
22
An adverse reaction means any adverse event caused by FMT.  Adverse reactions are 
a subset of all suspected adverse events for which there is reason to conclude that FMT 
caused the event. 
 
5. Suspected Adverse Reaction  
Suspected adverse reaction means any adverse event for which there is a reasonable 
possibility that FMT caused the adverse event. For the purposes of IND safety reporting, 
‘reasonable possibility’ means there is evidence to suggest a causal relationship 
between FMT and the adverse event. A suspected adverse reaction implies a lesser 
degree of certainty about causality than adverse reaction, which means any adverse 
event caused by a therapy. 
 
6. Unexpected 
An adverse event or suspected adverse reaction is considered “unexpected” if it is not 
listed in the Investigator brochure or is not listed at the specificity or severity that has 
been observed; or is not consistent with the risk information described in the general 
investigational plan or elsewhere in the current application.  
 
VI-B. Solicited Adverse Events 
 
Short-term 
The following short-term AEs and their associated intensities will be solicited:  
 
 
Fever 
o Mild 37.7-38.6°C 
o Moderate 38.7-39.3°C 
o Severe 
 39.4-40.5°C 
o Potentially Life Threatening >40.5°C 
 
 
Chills 
 
Mild: No or minimal interference with usual social and functional 
activities 
 
Moderate: Greater than minimal interference with usual social and 
functional activities 
 
Severe: Inability to perform usual social and functional activities 
 
 
Fatigue/Malaise 
 
Mild: No or minimal interference with usual social and functional 
activities 
 
Moderate: Greater than minimal interference with usual social and 
functional activities 
 
Severe: Inability to perform usual social and functional activities 
 
Potentially Life Threatening: Incapacitating fatigue/malaise 
symptoms causing inability to perform basic self-care functions 
 
 
Anorexia (loss of appetite) 
 
Mild: Loss of appetite without decreased oral intake 


 
23
 
Moderate: Loss of appetite with decreased oral intake but without 
significant weight loss 
 
Severe: Loss of appetite with decreased oral intake associated with 
significant weight loss 
 
Potentially Life Threatening: Life-threatening consequences or 
aggressive intervention indicated (TPN or tube feeding) 
 
 
Abdominal pain 
 
 
Mild: Pain causing no or minimal interference with usual social and 
functional activities  
 
Moderate: Pain causing greater than minimal interference with 
usual social and functional activities  
 
Severe: Pain causing inability to perform usual social and functional 
activities  
 
Potentially Life Threatening: Disabling pain causing inability to 
perform basic self-care functions OR hospitalization (other than an 
emergency room visit) indicated 
 
 
Bloating 
o Mild: No or minimal interference with usual social and functional 
activities  
o Moderate: Greater than minimal interference with usual social and 
functional activities 
o Severe: Inability to perform usual social and functional activities 
 
 
Gas/Flatulance 
o Mild: No or minimal interference with usual social and functional 
activities 
o Moderate: Greater than minimal interference with usual social and 
functional activities 
o Severe: Inability to perform usual social and functional activities 
 
 
Constipation 
o Mild: Irregularity of BMs not requiring dietary modification, laxative, 
or enema 
o Moderate: Persistent constipation requiring regular use of dietary 
modifications, laxatives, or enemas  
o Severe: Obstipation with manual evacuation indicated 
o Potentially Life Threatening: Life-threatening consequences (e.g. 
obstruction) 
 
 
Diarrhea 
o Mild: Transient or intermittent episodes of unformed stools OR 
increase of ≤3 stools over baseline per 24-hour period 
o Moderate: persistent episodes of unformed to watery stools OR 
increase of 4-6 stools over baseline per 24-hour period 
o Severe: Bloody diarrhea OR increase of ≥7 stools per 24-hour period 
OR IV fluid replacement indicated 


 
24
o Potentially Life Threatening: Life-threatening consequences (e.g. 
hypotensive shock) 
 
 
 
Nausea 
o Mild: Transient (<24 hours) or intermittent nausea with no or minimal 
interference with oral intake  
o Moderate: Persistent nausea resulting in decreased oral intake for 
24-48 hours 
o Severe: Persistent nausea resulting in minimal oral intake for >48 
hours OR aggressive rehydration indicated (intravenous fluids)  
o Potentially Life Threatening: Life-threatening consequences (e.g. 
hypotensive shock) 
 
 
 
Vomiting 
o Mild: Transient or intermittent vomiting with no or minimal 
interference with oral intake  
o Moderate: Frequent episodes of vomiting with no or mild 
dehydration 
o Severe: Persistent vomiting resulting in orthostatic hypotension OR 
aggressive rehydration indicated (intravenous fluids) 
o Potentially Life Threatening: Life-threatening consequences (e.g. 
hypotensive shock) 
 
Long-term 
The following long-term AEs and relevant details will be solicited:  
 
Gastrointestinal disorders 
 
Inflammatory bowel disease  
 
Irritable bowel syndrome  
 
Celiac disease  
 
Colon cancer 
 
Chronic infectious diseases (no therapy, outpatient, inpatient, ICU care) 
 
Autoimmune diseases 
 
Inflammatory arthritis  
 
Thyroid disease  
 
Connective tissue diseases 
 
Idiopathic thromobocytopenic purpura 
 
Metabolic disease 
 
Diabetes Type 2 (diet controlled, oral hypoglycemic, insulin, 
uncontrolled/complication associated) 
 
Metabolic syndrome (including obesity independently) 
 
Non-alcoholic fatty liver disease  
 
Neuro-psychiatric disease  
 
Multiple sclerosis 
 
Depression/Anxiety 
 
Cardiovascular disease 
 
Myocardial infarction (NSTEMI/STEMI) 


 
25
 
Stroke  
 
Cardiovascular related death 
VI-C. Monitoring of Adverse Events 
 
Each subject will be monitored for the occurrence of AEs, including SAEs. Each subject 
will be followed for safety monitoring at each follow-up interval.  
 
Subjects will be questioned at each follow-up interval regarding stool 
patterns, abdominal pain, fevers, and subjective well-being. First, subjects 
will be questioned about the possible occurrence of adverse events in a 
generalized way, such as, "How have you been feeling since your last 
visit?” Subsequent solicited questions and relevant details will be asked if 
there is a positive response. Site Investigators will be contacted by study 
representative if there is a suspected or reported AE 
 
Subjects having AEs will be monitored with relevant clinical assessments 
and laboratory tests, as determined by the site Investigator 
 
AEs, actions taken as a result of AEs, and follow-up results will be recorded 
as well as in the subject's source documentation. Follow-up laboratory 
results should be filed with the subject's source documentation  
 
Subjects will receive a follow-up phone call 6 and 12 months post-FMT to 
record any SAEs, new medical conditions/new medications, or changes in 
pre-existing medical conditions  
  
VI-D.  Assessment of Adverse Events 
1.  Assessment of Severity 
The severity of AEs will be assessed according to the following definitions: 
•Mild: the AE is noticeable to the patient and/or the Investigator, but does not 
interfere with routine activity 
•Moderate: the AE interferes with routine activity, but responds to symptomatic 
therapy or rest 
•Severe: the AE significantly limits the patient’s ability to perform routine activities 
despite symptomatic therapy 
 
2.  Assessment of Causality 
The Investigator must assess the relationship of any AE (including SAEs) to FMT, as 
related or not related, based on clinical judgment and using all available 
information, and may include consideration of the following factors: 
• Possible alternative causes of the AE, including the disease under treatment, 
pre-existing conditions, concomitant use of other drugs, and presence of 
environmental or genetic factors 
• The temporal association between FMT exposure and onset of the AE 
• Whether the manifestations of the AE are consistent with known actions or 
theoretical toxicity of FMT 
 
The causal relationship between FMT and the AE will be assessed using one of 
the following categories: 
 


 
26
 
Not Related: An AE is not associated with FMT if: 
• Temporal relationship is lacking (e.g., the event did not occur within a 
reasonable time frame following administration of FMT); or 
• Other causative factors more likely explain the event (e.g. pre-existing 
condition, other concomitant treatments);  
Related: An AE is attributed to FMT if: 
• There is a positive temporal relationship (e.g. the event occurred within a 
reasonable time frame following FMT); and 
• The AE is more likely explained by FMT than by another cause  
 
VI-E.   Reporting Safety Observations by the Investigator to the Sponsor 
1.  Reporting of Non-serious AEs 
All AEs, regardless of seriousness, severity, or causal relationship to FMT, will be 
recorded in the AE section of the subject case report form. 
 
2.  Reporting of FMT Exposure during Pregnancy 
If a female subject or the female partner of a male subject becomes pregnant 
during the course of study, the Investigator must report this occurrence to the 
Sponsor within 72 hours of becoming aware of the pregnancy.  The Investigator is 
required to follow up on the pregnancy until it has completed. The outcome of 
the pregnancy and the status of the newborn (if applicable) will be reported 
within 24 hours of becoming aware. If the female partner of a male subject 
becomes pregnant, the Investigator must obtain consent to collect pregnancy 
information (including the status of the newborn, if applicable). 
 
3. Reporting of Safety Observations by the Investigator 
Any occurrence of the following events or outcomes must be reported 
expeditiously by the Investigator or qualified designee to the local IRB and to the 
Sponsor, who will be responsible for reporting the event to the FDA:  
1. SAE related to FMT material 
2. Death of a subject related to FMT material  
 
The site Investigator is to report any safety observations from the list above to the 
Sponsor within 72 hours of becoming aware of the event, who in turn, will notify 
the FDA. Any observation that is also an AE will be recorded on the subjects case 
report form along with any actions taken. If not all information is available at the 
time of initial report, follow up SAE reports will be completed and submitted.  
 
Any occurrence of the following events or outcomes must be reported by the 
Investigator or qualified designee to the local IRB and to the Sponsor, who will be 
responsible for reporting the event to the FDA:   
 
1. SAE unrelated to FMT material 
2. Death of subject unrelated to FMT material 
3. Hospitalization related to CDI 
4. New or worsening medical condition, or new medications  


 
27
 
The Investigator is to report any safety observations from the list above to the 
Sponsor within 7 days of becoming aware of the event. The Sponsor will notify 
the FDA in its Annual Report. Any observation that is also an AE will be recorded 
on the subjects case report form along with any actions taken. If not all 
information is available at the time of initial report, follow up SAE reports will be 
completed and submitted.  
 
The site Investigator is required to follow SAEs until resolution, regardless of 
whether the subjects are still participating in the study. Resolution is defined as:  
 
Resolved with or without residual effects 
 
Return to baseline for a pre-existing condition 
 
Fatal outcome; if autopsy is performed, the autopsy report must be 
provided to the Sponsor. 
 
VI-F. Monitoring the study database and submitting safety reports 
The Sponsor will notify FDA of any unexpected fatal or life-threatening suspected 
adverse reaction related to FMT material as soon as possible, but no later than 7 
calendar days after the Sponsor's initial receipt of the information.  The Sponsor 
will notify the FDA and all participating Investigators in an IND safety report of 
potentially serious risks from this study as soon as possible, but no later than 15 
calendar days after the Sponsor receives the safety information and determines 
that the information qualifies for reporting.  Participating Investigators include all 
Investigators enrolling subjects under the Sponsor’s IND.  In addition, the Sponsor 
will identify in each IND safety report, all IND safety reports previously submitted 
to FDA concerning a similar suspected adverse reaction and will analyze the 
significance of the suspected adverse reaction in light of previous, similar reports 
or any other relevant information. The Sponsor will evaluate a suspected adverse 
reaction in the context of other related reports or adverse events.  The Sponsor 
will periodically review and analyze the entire safety database, for IND safety 
reporting purposes, and also to update Investigator brochures with new safety 
information. An IND safety report will be submitted when any of the following 
criteria are met:  
 
A. Serious and unexpected suspected adverse reaction related to FMT material 
A serious and unexpected adverse reaction is a SAE as determined by the 
Investigator or Sponsor, as defined previously, in combination with unexpected, 
defined as not being listed in the Investigator Brochure. At the request of the 
FDA, it will also include any documented infectious disease, defined as a new 
onset infection in the recipient after FMT and the infection present in the safety 
aliquot, which is a sample of the exact specimen that was transferred to the 
patient.  
 
B. Findings from other sources  
The Sponsor will also expeditiously report any findings from clinical, 
epidemiological, or pooled analysis of multiple studies or any findings that 


 
28
suggest a significant risk in humans exposed to FMT and thought related to the 
material. 
 
 
C. Increased occurrence of serious suspected adverse reactions related to FMT 
material 
The Sponsor will report any clinically important increase in the rate of a serious 
suspected adverse reaction over that listed in the protocol. 
 
 
VI-G. Halting Rules 
 
Specific safety findings will result in temporarily suspending enrollment until a safety 
review is convened, the objective of which is to determine whether the study should 
continue per protocol, proceed with caution, be further investigated, be discontinued, 
or be modified and then proceed. Suspension of enrollment, for a particular group, 
such as colonoscopy group, or for the entire study, is another potential outcome of a 
safety review. 
 
Subsequent review of serious, unexpected, and related AEs by the Data and Safety 
Monitoring Board (DSMB), IRB, the Sponsor, or the FDA or relevant local regulatory 
authorities may also result in suspension of further trial interventions/administration of 
FMT at a site. The FDA and study Sponsor(s) retain the authority to suspend additional 
enrollment and study interventions/administration of FMT for the entire study, as 
applicable. 
 
Findings that will trigger a safety review are  
 
Death related to FMT material  
 
Confirmed transmission of a clinically concerning infection from donor to subject 
via FMT 
 
If one or more subjects experiences a serious, unexpected adverse event related 
to FMT material  
 
Significant increase in frequency of events theoretically related to FMT material 
(specifically, new diagnoses of inflammatory bowel disease). Discussion on 
threshold will be discussed with DSMB and will be on a disease specific basis.  
 
For individual subjects enrolled in the study: any subject who has an SAE related to FMT 
will be ineligible for a second FMT during the course of the study.   
 
FDA/CBER will be notified by phone or fax within 48 hours if the study is halted for review.  
VII. Minimizing Risks to Human Subjects 
VII-A. Human Subjects Involvement, Characteristics and Design 
1. IRB approval for stool donor program has been obtained; Local site IRB approval from 
each study center will be obtained subsequent to required FDA approval of IND.  


 
29
2. Subjects will be given informed consent using the standard consent process for 
colonoscopy/enema. Additional theoretical risk for transmission of infectious agents, 
and other diseases and conditions will also be discussed.  
3. Donors will be given informed consent and will tested for most common infectious 
agents. Individuals with communicable disorders or a history of high-risk behaviors will 
not be permitted to donate.  
4. Protections against risk are provided in detail below.  
 
A sample size of 150 patients will be recruited. These patients will have been treated at 
or referred to one of the study centers for management of recurrent CDI.  
 
Additional sites may be asked to join this study as needed to expeditiously achieve the 
targeted enrollment. The initial study sites will include: 
 
1. Brown University and Women’s Medicine Collaborative 
Providence, Rhode Island 
 
2. Indiana University Health University Hospital  
Indianapolis, Indiana 
 
3. University of Virginia Health Systems 
Charlottesville, Virginia   
 
4. Edward Hospital 
Naperville, Illinois 
 
This will be an open-label, longitudinal cohort study with follow-up at Day 3, Week 6, 
Month 6 and Month 12. The estimated duration of this study is 24 months.  No patient will 
be excluded from recruitment on the basis of ethnicity, race, or disability. Subjects are 
expected to range in age from 18-75. Subjects will all be outpatients and will likely have 
comorbidities. All subjects will be fully informed about the purposes of the study, and 
risks and benefits of the study, and informed consent for all phases of the study will be 
obtained. Patients who agree to participate will be free to withdraw from the project at 
any time.  
 
The intervention, FMT, will be administered directly through the endoscope at time of 
colonoscopy/sigmoidoscopy or by retention enema at the discretion of the MD. The 
material, donor stool 
, will be given as a single dose of 250mL. 
Lower gastrointestinal delivery was chosen based on a systematic review and meta-
analysis that in subgroup analysis suggested lower gastrointestinal delivery was superior 
to upper delivery.21 
 
We are collaborating with Investigators at the Massachusetts Institute of Technology 
(MIT) who will perform microbiome analysis on the stools collected from donors and 
subjects as part of this study. The samples can be mailed via conventional mail to 
OpenBiome who will locally transport them to MIT on dry ice after sample inspection 
and logging. 
 


 
30
VII-B. Potential Risks to Subjects 
There are 3 areas of risk to subjects associated with participation in the 
proposed study.  
 
These include:  
1) Physical risks related to the colonoscopy/sigmoidoscopy  
2) Theoretical risks (infectious and otherwise) related to FMT and  
3) Psychological or other risks related to confidentiality and loss of privacy.  
 
Risks of ingestion of the colon preparation include the risk of dehydration and minor 
electrolyte imbalances. Standard colonoscopy/sigmoidoscopy risks include the risk of 
bowel perforation, bleeding, and adverse cardiopulmonary events related to sedation. 
The infusion of the liquid fecal matter will prolong the colonoscopy by less than 5 
minutes and adds no additional risk to the colonoscopy. Many adverse effects of 
colonoscopy/ sigmoidoscopy resolve shortly after the procedure has been completed, 
but in some cases abdominal discomfort and gaseous side pain can persist for several 
hours. The use of retention enema is widely accepted to be safe but may include the 
risk of bowel perforation or bleeding. 
 
There have been no confirmed infectious complications directly attributable to FMT 
reported in the literature to date aside from a speculation about a norovirus case 
although the donor was untested for this.29 There have been two FMT cases of 
bacteremia, although the strength of the association is unclear, and both patients did 
well on antibiotic therapy. However, since the process involves infusion of one person’s 
“body fluids” into another person, transmission of an infectious agent or other disease or 
condition remains a theoretical possibility. From infusion of donor microbiome, the 
patient/subject could potentially acquire risk factors for chronic diseases such as 
diabetes or  inflammatory bowel disease. Risks will be minimized by a rigorous donor 
selection process and evaluative studies on stool and blood of the donor to exclude 
transmission of infectious agents prior to FMT. Subjects will be informed that the 
treatment or procedure may involve these theoretical risks and additional risks that are 
currently unforeseeable. There have been reported cases of inflammatory bowel 
disease flares post-FMT, but it is unclear if this was attributable, and these patients have 
been excluded from this study. Infusion of fecal material containing a potential agent 
to which the subject is allergic is also a concern and so patients with a history of severe 
(anaphylactic) food allergy will be excluded from this study. 
VII-C. Potential Risks to Donors 
This study also involves individuals (donors) recruited to donate stool for FMT. 
There are 3 areas of risk to the potential stool donor.  
 
These include:  
1) Physical risks related to laboratory testing and the stool collection protocol  
2) Psychological risks related to both revealing and potentially learning sensitive 
information during donor screening process  
3) Risks related to confidentiality and loss of privacy.  


 
31
 
OpenBiome volunteer donors will be utilized. In order to exclude donors at high 
risk of passing infection, a Donor Risk Factor Questionnaire (Appendix 2) will be 
administered. This questionnaire contains sensitive and potentially embarrassing 
questions 
. 
Additionally, prospective donors will be asked questions about their baseline 
health status and co-morbidities. Laboratory tests drawn as part of the screening 
process will include testing for HIV, viral hepatitis, and syphilis. These serologic 
results, if found to be abnormal, could cause psychological distress to the donor. 
However, the benefit of being made aware of a previously undiagnosed 
infectious condition outweighs this risk. Potential donors may experience 
psychological distress if they are excluded from donating stool based on these 
screenings.  
 
Physical Risks to Donor:  
Very Likely  
• Blood drawing: pain, bruising, feeling faint, slight risk of infection  
 
Nonphysical Risks to Donor:  
Less Likely  
• Embarrassment or psychological distress from answering questions 
 
asked during the donor screening process.  
• Psychological distress (guilt and embarrassment) if screening questionnaire or 
laboratory test results exclude the person from donation.  
 
Unlikely  
• There is also risk of compromising donor privacy through breach of data 
confidentiality of sensitive protected health information such as results of HIV or viral 
hepatitis testing. 
VII-D. Protection against Risks to Subjects and Donors  
 
 
Recruitment and informed consent 
 
Participants will be recruited from study sites previously outlined. After a patient has 
been referred for recurrent CDI and determined to meet inclusion criteria, the clinician 
will introduce him/her to the research assistant, if applicable. The research staff or 
clinician will carefully explain all aspects of the study to a potential recruit, including the 
risks and benefits, and obtain participant’s written informed consent.  
 
 
The research assistant or clinician will orally describe the material written in the 
informed consent document and answer any questions the participant may 
have. 
 
Participants will be reminded that they are not required to participate in the 
study and that they will receive the standard care provided by their physician 


 
32
regardless of whether or not they choose to participate. Participants who give 
their consent will sign a copy of the document and will be given a signed copy 
of the informed consent document. 
 
Recruits will be informed of the treatment commitment, amount and general types of 
assessments, the follow-up telephone interview procedures, and clinic visits. They will be 
given detailed descriptions of the colonoscopy or enema with FMT procedure. They will 
be informed of the requirements to submit stool specimens up to 3 days before, as well 
as 6 weeks, 6 months, and 12 months after the FMT procedure. 
 
Potential donors who are healthy will be recruited to serve in a pool through an IRB-
approved process. Each potential donor will then be contacted by the research staff 
and asked to participate in the donor consent process. Research staff will carefully 
explain all aspects of the study to a potential donor, including the risks and benefits, 
and obtain participants’ written informed consent. Recruits will be informed of the 
commitment including a detailed description of the donor screening process and a 
stool donation protocol. The research staff will explain that a thorough clinical 
assessment and screening questionnaire will be completed. Volunteers will be informed 
that the questionnaire asks potentially sensitive questions 
 
. They will be informed of the requirements to have laboratory 
blood work, including HIV testing, and stool studies and to submit stool specimens. The 
research assistant will orally describe the material written in the informed consent 
document and answer any questions the potential donor may have. Potential donors 
will be reminded that they are not required to participate in the study. Participants who 
give their consent will sign a copy of the document and will be given a signed copy of 
the informed consent document. 
 
Protection against risk 
 
Data and safety monitoring will take place to assure the safety of subjects. All 
participants will be reminded that their participation is voluntary and that they can 
withdraw at any time without penalty.  
 
Additionally, the risks described above will be minimized by the following procedures:  
 
1. We will minimize the risk of potential coercion by following standard procedures for 
obtaining informed consent from both subjects and stool donors. We will begin this 
process during the intake, where we will clarify the nature of the study and possible 
alternatives upfront. Prior to enrolling subjects in the research, we will fully explain the 
study procedures, risks, benefits, and alternatives, emphasizing that the subject’s 
participation has no impact on the other services they receive. Also, subjects who do 
not consent or who withdraw during the study period will continue to receive 
appropriate treatment if needed. All subjects and donors will be reminded that there is 
no penalty for those who choose to not participate or to withdraw from the study and 
that their decision to participate does not impact the standard services they receive 
through the hospital. Subjects will not receive financial compensation for their 
participation. Volunteer donors will be paid a small stipend of $40 per stool donation (to 
cover their time & burden of screening/stool collection).  


 
33
 
2. We will minimize potential risks due to loss of confidentiality by having all information 
collected and handled by research staff trained to deal appropriately with sensitive 
clinical issues. Potential donors will also be informed about the risk of being ineligible to 
donate stool due to positive results on screening questionnaires or laboratory testing. 
Results of donor medical interview, screening and laboratory testing will be kept 
separate from subject data and only deidentified data will be available to the subject 
or his/her most responsible physician. All study information will be treated as confidential 
and will be available only to research staff. Hardcopies will be kept in locked file 
cabinets, and computer data files will be encrypted and available only to authorized 
personnel – with no storage of names or obvious identifying information. No participant 
will be identified in any report of the project. Further, when contacting participants for 
follow-up, no identifying information other than the first name of the research assistant 
will be used when leaving messages or speaking to anyone other than the participant 
him/herself. Written consent will be obtained to contact other persons for the purpose 
of locating the participant for follow-up and participants can refuse or revoke such 
consents. No information about participants will be released without their permission or 
where required by law.  
 
3. We will minimize the theoretical risks of infectious disease or other conditions possibly 
transmitted through FMT by using donor screening protocols modeled after blood banks 
and organ transplant programs. Potential volunteer donors will undergo thorough 
screening to determine eligibility for donation. The primary purpose of the donor 
examination and interview is to ensure that the donor is in good health, and to identify 
risk factors for diseases transmissible by stool. The donor interview will be used to identify 
risks for diseases and conditions for which there are no laboratory tests, for which tests 
are not sensitive enough to detect infectious disease agents, and for tests unable to 
identify early stage or window period infections. Potential donors will be interviewed 
using a Donor Risk Factor Questionnaire (Appendix 2). Health status of donors will be 
monitored prospectively, and donors will be rescreened every 60 days. 
 
4. We will minimize the risk of severe CDI recurrence by maintaining close clinical 
contact with all subjects. Subjects will be encouraged to contact the clinical team if 
they experience recurrence of diarrhea, fever, or abdominal pain – so that stool can be 
tested for C. difficile toxin if indicated, and antibiotics can be resumed if necessary. 
Subjects will be contacted via telephone by a Clinical Research Coordinator on Day 3, 
Week 6, Month 6 and Month 12.  A clinical assessment will take place between Week 6 
+/- 2 weeks. If any patient experiences recurrent CDI via FMP250 or FMP250E they would 
be eligible for a second FMT by colonoscopy using a different donor.  
 
VII-E. Safety Monitoring Protocol 
 
Safety monitoring plan 
 
An external Data and Safety Monitoring Board (DSMB) will be assembled to evaluate 
the data and the safety of subjects enrolled in the study. The DSMB will consist of 3 
physicians with specialization in gastroenterology and/or infectious disease. Initially, the 


 
34
Board will convene with the Principal Investigator to review the study protocol and 
guidelines for data and safety monitoring. This initial review will include establishing 
standard procedures for monitoring. At each quarterly meeting the DSMB will evaluate 
recruitment, the progress of the study, subject retention, data quality and 
confidentiality. The DSMB will review subjects' clinical status, adverse events and 
whether or not there have been any changes in risk to participating subjects. This 
quarterly review will ensure that subject risk does not outweigh study benefits. A report 
generated from each of these meetings will be retained at the study sites and will be 
forwarded to the IRBs and to the FDA (in accordance with Investigational New Drug 
(IND) regulations). The DSMB will be available to convene outside of the appointed 
meeting schedule if necessary, due to concerns regarding a particular subject, or due 
to any adverse events during the study. The DSMB will make appropriate 
recommendations for changes in the study protocol, if needed.  
 
The safety of participants will be monitored for the duration of the study at each 
contact. Both anticipated and unanticipated adverse events and problems will be 
formally monitored and recorded. Unanticipated serious adverse events related to FMT 
material will be reported to the hospital and IRBs (as per local reporting requirements) 
and the FDA (within 15 days; or 7 days for unexpected fatal or life threatening events 
related to FMT material). Anticipated and less serious adverse events, as previously 
outlined, will be submitted annually in reports to the IRBs and FDA. The Principal 
Investigators will be responsible for monitoring the safety and efficacy of this study, 
executing the DSMB plan, and complying with the reporting requirements. The DSMB 
report will include the participants’ sociodemographic characteristics, any quality 
assurance or regulatory issues that occurred during the past year, a summary of 
adverse events and serious adverse events, and any actions or changes with respect to 
the protocol.  
VIII. Data Monitoring Plan  
 
Data will be collected using standardized paper forms and will only be identified with 
the study’s ID of the subject. The codes that link the name of the subject and the study 
ID will be kept confidential by the Site Investigator in a secured cabinet. Collected 
forms will be transported to the Site Investigator data entry center. Data will be entered 
in the REDCap computer database independently by trained data entry staff, and 
discrepancies will be corrected by the Site Investigator, based on source documents. 
Data quality will be monitored once per month by random inspection of the completed 
forms by a Clinical Research Coordinator; any problems detected will be discussed with 
the Principle Investigator. Telephone follow-up will be conducted by a Clinical Research 
Coordinator with data directly entered immediately into the REDCap computer 
database. Descriptive statistics will be computed periodically and reported in 
aggregate as part of the study monitoring process. 
 


 
35
IX. Educational Training 
 
Researchers and clinician Investigators involved in the study will need to have 
successfully completed an institutional human subjects research program/module.  The 
principle Investigator will maintain documentation for all study personnel. For sites 
utilizing enema delivery, a Clinical Research Coordinator will be responsible for 
overseeing a brief enema session with site study personnel. 
X. Potential Limitations  
 
1. Correlational Safety Data 
 
This open-label, longitudinal cohort will not have an untreated arm. Thus, it will be 
difficult to compare the probability of a newly acquired adverse event 
compared to a control. Given that the patient population with recurrent CDI 
tends to be older with core comorbidities, they are at high risk for new medical 
conditions, new medications, and poor outcomes, which may or may not be 
related to FMT. Thus, it is difficult to discern if there is an increased probability over 
baseline risk. This study has purposefully limited the age to 50 and designed 
exclusionary criteria (e.g. severe underlying disease that the patient is not 
expected to survive for the next 1 year) to reduce noise and strengthen any 
signal. Where possible age matched, prognostic factor matched population 
data will be considered to determine if FMT material may present a statistically 
significant increase in risk of developing a serious adverse event. However, this 
will not be the primary aim, and the focus will be on capturing count data/rates 
for serious adverse events.  
 
2. Stool Collection 
 
Patients will be collect his or her own stool samples and there may be potential 
error carrying out this procedure. Our study minimizes this risk with clear 
instructions, simplified process, clear labels, significant contact with study 
representatives, and reminder phone calls. 
XI. Investigator Credentials and Associated Body Contact 
Information  
 
Principal Investigator:  
Zain Kassam MD, MPH 
Address:  
 
 
196 Boston Ave, Suite 2100, Medford, MA, 02155 
 
Co-Investigator:  
 
Mark Smith PhD 
Address: 
 
 
196 Boston Ave, Suite 2100, Medford, MA, 02155 
 
Co-Investigator:  
 
Eric Alm PhD 
Address: 
77 Massachusetts Ave, MIT-NE47-379, Cambridge, MA, 02139 
Co-Investigator: 
 
Colleen Kelly MD 
Address: 
 
 
146 West River St, Providence, RI, 02904 


 
36
 
Co-Investigator:  
 
Monika Fischer MD 
Address: 
550 North University Blvd, Suite 1710, Indianapolis, IN, 46202 
 
Co-Investigator: 
 
Rachel Ann Hays MD 
Address: 
 
 
1215 Lee St, Charlottesville, VA, 22908 
 
Co-Investigator: 
 
Darren Kastin MD 
Address: 
 
 
1243 Rickert Dr, Naperville, IL, 60540 
 
IRB Name: 
 
 
MIT IRB/COUHES 
IRB Address:  
 
77 Massachusetts Ave, E25-143B, Cambridge, MA, 02139 
 
Principal Investigator CV:  Appendix 7 
 
 
XII. ClinicalTrials.gov Requirements 
 
As required by law, this trial will be registered in ClinicalTrials.gov no later than 21 days 
after the first subject is enrolled. The reporting of summary results information (including 
adverse events) will be done no later than 1 year after the study completion date. The 
principle investigator will be responsible for registering the trial and results reporting. 
 
 
7. CHEMISTRY, MANUFACTURING, AND CONTROLS INFORMATION 
 
Refer to the OpenBiome Master File (BB-MF 15543) for Chemistry, Manufacturing, and 
Controls Information. An accompanying authorization letter from OpenBiome can be 
found in Appendix 1. 
 
Addendum: Stability of fecal microbiota during long-term frozen storage 
 
We are able to integrate evidence from human trials, animal models and laboratory 
studies to inform our understanding of the long-term stability of gut microbial 
communities during long-term cryopreservation. This evidence supports the long-term 
stability of fecal microbiota preparations, potentially for periods of up to several years. 
However, direct human experience at these extended time scales is still lacking and a 
test of potency will be helpful for continuing to evaluate the limits of viability in these 
communities. As a result, in addition to current evidence supporting the investigational 
use of microbiota preparations subject to long-term storage, here we present our plans 
for future work that we hope will further address this question. 
 
Evidence from studies in humans 


 
37
First, we have direct clinical experience using frozen fecal preparations for FMT with 
human subjects suffering from recurrent C. difficile infections. From this body of clinical 
experience, a few published reports in particular are worth highlighting. Hamilton et al 
provided the first published illustration of the clinical utility of frozen preparations.28 In an 
open-label, non-randomized case series, they report that frozen material stored in 
glycerol for up to 56 days at -80°C was similarly efficacious for the treatment of 
recurrent CDI as freshly prepared material. Following up on this study, our group 
contributed to an open-label randomized trial comparing nasogastric with 
colonoscopic delivery using frozen material. This material was stored for up to 156 days 
at -80°C prior to successful use.22  
 
Evidence from in-vitro and animal studies 
 
In addition to this direct clinical experience using fecal microbiota for the treatment of 
recurrent CDI, we have a body of microbiological experience successfully culturing 
bacteria that have been cryopreserved for many years and in some cases decades.29–
31 For example, one group found less than 1 log-fold decrease in microbial viability after 
more than 4 years of storage at -70°C.32. Another group found that not only are cells 
viable after 2 years of storage at -80°C, they remain capable of causing infections in an 
animal model at similar rates to samples stored for shorter periods.33 Standard 
microbiological practice and extensive laboratory experience supports the long-term 
viability of the gut microbiota during cryopreservation. 
 
Investigational plan  
 
Nonetheless, to supplement this data, we plan to conduct a small-scale clinical trial 
comparing the efficacy of material stored for either 6, 12 or 24 months at -80°C. We will 
supplement clinical experience, the most relevant indicator of viability in this case, with 
a differential fluorescent staining assay (Live/Dead, Life Technologies) to detect the 
relative ratio of intact versus ruptured cells in each sample. We have quarantined 60 
samples for long-term storage and have saved additional aliquots from each sample 
that we can interrogate with our live-dead assay at 3-month intervals. By comparing 
measured viability with clinical outcomes at 6, 12 and 24 months, it will be possible to 
both establish the long-term stability of microbiota preparations stored under these 
conditions and to establish a surrogate marker of viability that does not require clinical 
evaluation. We hope that such a validated tool could facilitate the standardization of 
best practices for storing and processing of FMT. 
 
To control for the extreme intra-subject and inter-subject variability that characterizes 
the gut microbiome, we will use treatments collected not only from the same donor, 
but from the same bowel movement for each treatment arm. For example, a single 
bowel movement that produces sufficient material for 3 treatments will have one unit 
included in the 6-month cohort, one in the 12-month cohort and one in the 24-month 
cohort. The average efficacy of material derived from each cohort will be compared 
to determine whether there are meaningful differences between the groups. This study 
will be powered to detect a difference of more than 25% between groups (α = 0.05 β= 
0.2). Due to the need to store samples for an extended period of time prior to clinical 
work, we do not endeavor to include a formal proposal for this investigation at this time. 


 
38
Nonetheless, given the time delay required, we felt it would be useful to include our 
plans in this application to solicit comments now. These comments can be used to 
inform the design of this study while it is still feasible to make modifications that will not 
substantially delay the investigation. 
 
 
8. PHARMACOLOGY AND TOXICOLOGY INFORMATION 
 
FMT grew from empirical clinical practice instead of a conventional drug development 
program. Accordingly, salient non-clinical data is not particularly informative, 
particularly in context that the human microbiome has co-evolved with its host since 
birth.   
 
 
9. PREVIOUS HUMAN EXPERIENCE WITH THE INVESTIGATIONAL DRUG 
I. Indication 
Recurrent Clostridium difficile infection  
II. Background of Disease and Rationale  
 
Background 
Clostridium difficile infection (CDI) has recently been reported as the most common 
healthcare-associated infection in the United States, and the emergence of 
community-acquired CDI is particularly concerning.1 2 CDI is associated with a spectrum 
of symptoms ranging from diarrhea to abdominal pain, and severe infections may lead 
to toxic megacolon and death. The clinical burden of CDI is significant. In the United 
States, the incidence of CDI per 10,000 inpatient hospitalizations doubled between 2000 
and 2005 (5.5 versus 11.2 cases).3 A common management challenge is recurrent CDI, 
which occurs in 20% of patients after standard therapy. Patients with one recurrence 
have a 40% risk of further relapse and for those with two or more episodes of 
recurrence, the risk of subsequent relapse has been reported to exceed 60% with 
antibiotic treatment.12 15 16 Current standard therapies for multiple recurrent CDI are 
limited and many patients become dependent on vancomycin, as they develop CDI 
relapse when this antibiotic is discontinued.17 Counterproductively, ongoing antibiotic 
therapy is thought to continue perturbation of the microbial community and to 
establish an optimal environment for toxigenic Clostridium difficile.18 19 Additionally, 
continued treatment may contribute to antibiotic-resistant Clostridium difficile, 
especially as the vanB gene, which confers vancomycin resistance, has been isolated 
in Clostridia.7–10 34 
 
Rationale 
The treatment options for recurrent CDI are limited. Clinical guidelines by the American 
College of Gastroenterology conditionally recommend, based on low-quality 
evidence, that first CDI recurrence is managed with metronidazole or vancomycin, and 


 
39
second recurrence treated with a pulsed vancomycin regimen.17 FMT is conditionally 
recommended for management of a third CDI recurrence; the recommendation is 
provisional in part because further clinical trials are ongoing and there is a lack of 
standardized longitudinal follow-up in patients post-FMT.17 The rationale for this multi-
center, open-label, longitudinal cohort study is to improve certainty about the safety 
profile of FMT, and complement the established literature and on-going studies of the 
efficacy of FMT.  The current body of literature suggests that the risks of FMT are low, and 
are linked to complications associated with delivery such as 
colonoscopy/sigmoidoscopy or to theoretical infectious and non-infectious risks. Given 
the established benefits of FMT and the paucity of high-quality safety data, the 
potential benefits of this study outweigh the risks of using this investigational product.  
III. Summary of Previous Human Safety Experience  
III-A Observational Data 
 
A robust systematic review and meta-analysis of 11 observational studies with a total of 
273 CDI patients did not report any adverse events attributable to the FMT material in 
variable follow-up from 3 weeks to 8 years.21 However, 3 studies that used upper 
gastrointestinal FMT indicated that the nasogastric/nasojejunal tube itself could not be 
ruled out in contributing to a suspected adverse event: upper gastrointestinal bleed, 
peritonitis, and possibly enteritis.21 
 
A multi-center retrospective review (n=77) reported no definite FMT adverse events in 
follow-up between 3-68 months (mean 17 months). The authors did report 4 patients 
developing diseases of potential interest after FMT (peripheral neuropathy, Sjogren’s 
disease, idiopathic thromobocytopenic purpura, rheumatoid arthritis) and 7 non-FMT 
related deaths. Interestingly, the study noted post-FMT improvement in a patient with 
pre-existing allergic sinusitis and one with “arthritis”, with the type unreported.24 
 
Safety outcomes for FMT treatment of CDI have also been reported in 
immunocompromised patients in a multi-center retrospective review (n=80).25 In this 
special group, 12 patients (15%) had a serious adverse event within 12 weeks of FMT, of 
which 10 were hospitalized. Specifically, 2 deaths occurred within 12 weeks, one a result 
of aspiration at the time of colonoscopy for FMT, and the other unrelated to FMT 
(progressive pneumonia). There were 3 deaths reported 6 months after FMT due to 
chronic, progressive medical conditions unrelated to FMT. There was another 
procedure-related complication as a subject sustained a superficial mucosal tear 
during colonoscopy. Importantly, no patients experienced an infection definitively 
ascribed to FMT, although 2 subjects sustained unrelated infections (influenza, catheter 
line infection). Self-limited diarrheal illness occurred in 5 patients but no causal organism 
was identified.25 
 


 
40
 
III-B Randomized Clinical Trial Data 
 
In a small randomized controlled trial (n = 42) for recurrent CDI assessing FMT via 
duodenal infusion compared to vancomycin and bowel lavage, or vancomycin alone, 
adverse event were minor and short-lived.23 In the FMT arm (n=16), minor gastrointestinal 
symptoms occurred on the day of infusion including belching (n=3), nausea (n=1), 
abdominal cramps (n=5), and abdominal pain (n=2); however, these symptoms 
resolved in all patients within 3 hours. In follow-up, constipation occurred in three 
patients. There were no adverse events attributed to FMT at 10-week follow-up.23  
 
In another small, open-label, randomized controlled trial (n=20) for recurrent CDI 
comparing FMT by nasogastric tube versus colonoscopy, no adverse or unexpected 
events were documented in the 6-month follow-up period.22 
 
 
III-C Investigator Preliminary Data 
 
OpenBiome is a 501(c)(3) non-profit organization that is the first public FMT stool bank in 
the United States for C. difficile infection non-responsive to standard therapy. In 
compliance with the current FDA regulatory framework, we have partnered with more 
than 100 health care institutions in over 32 states to expand safe, affordable access to 
FMT.34  OpenBiome has sent out over 1000 treatments and has had limited adverse 
events.  
 
To date, there have been two anecdotal cases of transient bacteremia potentially 
attributable to FMT, which was successfully treated with antibiotics. One case involved 
a 52 year old man with Hepatitis C and alcoholic liver cirrhosis admitted for 
pneumococcal bacteremia, that subsequently developed severe, complicated CDI 
including ileus that was non responsive to standard therapy. The patient underwent a 
FMT via nasoduodenal tube, and although diarrhea improved, 4 days after FMT was 
admitted to the intensive care for septic shock. Blood cultures grew 2 strains of pan-
susceptible E. coli, and peritoneal fluid also contained E. coli. The patient was 
successfully treated with conventional sepsis therapy and improved.  
 
 
 




 
42
also 
IC 
varied 
94% (1-2x, sub-group 
with concurrent IBD) 
 
abdominal pain, 4 IBD 
flares. See discussion. 
AEs: 4 related, 5 possibly 
related. See discussion. 
Polak et 
al. 201144 
15 
rCDI 
NJT 
 
87% (1-2x) 
 
SAEs: 1 procedure-
related.  
No comment on other 
AEs. 
MacCon
nachie et 
al. 200945 
15 
rCDI 
NGT 
 
73% (1-2x) 
 
No material-related AEs. 
One upper GI 
hemorrhage, may be 
procedure-related. 
Aas et al. 
200346 
18 
rCDI 
NGT 
 
83%(1x) 
SAEs related to 
procedure or co-
morbidities only: 
peritonitis, pneumonia. 
 
Notes: 
* FMT product is fresh as opposed to frozen unless stated otherwise. 
**Note that follow-up time for examining efficacy was variable. Most studies continued for at 
least 1.5-2 months, and some for years, but at least one continued for only 2-3 weeks.  
*** This column focuses on responder AEs. Note that patients who were unresponsive to FMT 
treatment died of rCDI or other non-FMT-related causes in several studies. 
Abbreviations: 
CDI form: rCDI = recurrent Clostridium difficile infection; mix = mix of recurrent (repeated), 
refractory (not responding to treatment), and severe (e.g., hospitalization) CDI.  
CDI form and also Efficacy: IBD = inflammatory bowel disease; IC = immunocompromised.  
FMT route: CsC = colonoscopy; G-tube = gastrotomy tube; NGT = nasogastric tube; NDT = 
nasoduodenal tube; NJT = nasojejunal tube. 
Safety: SAE = serious adverse event; BM = bowel movement; IBS = irritable bowel syndrome. 
 
 
 


 
43
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