Fecal Microbiota Transplant (FMT) After Treatment for a First Episode of Clostridium Difficile Infection (CDI)
Phase: PHASE1
Conditions: Clostridium Difficile Infection
Sponsor: Boston Medical Center

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Fecal Microbiota Transplantation (FMT) after treatment for primary 
Clostridium difficile infection 
 
ClinicalTrials.gov number: NCT03795233 
Protocol Version Number: 1.7 
Protocol Version Date: February 18, 2020 
Funding Mechanism:  This project was supported by the National Center for Advancing Translational 
Sciences, National Institutes of Health, through BU-CTSI Grant Number 1UL1TR001430. 
IND / IDE  
Sponsor:  Tamar F Barlam MD 
IND / IDE number: 18616 
Principal Investigator: Tamar Barlam 
 
Phone: 617-414-5190 
 
E-mail: Tamar.Barlam@bmc.org 
 
 
 
Statistical Plan pages 16-18 
 
 
 
 
 
 
CONFIDENTIAL 
 
This document is confidential and the property of Boston Medical Center. No part of it may be 
transmitted, reproduced, published, or used by other persons without prior written authorization 
from the study sponsor. 
 
 

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TABLE OF CONTENTS 
1 List of Abbreviations .............................................................................................................. 3 
2 Protocol Summary .................................................................................................................. 3 
3 Background/Rationale & Purpose ....................................................................................... 4 
3.1 
Background Information .............................................................................................. 4 
3.2 
Rationale and Purpose ................................................................................................ 5 
4 Objectives ................................................................................................................................ 5 
4.1 
Study Objectives ........................................................................................................... 5 
4.2 
Study Outcome Measures ........................................................................................... 5 
4.2.1 
Primary Outcome Measures ......................................................................... 5 
4.2.2 
Secondary Outcome Measures ................................................................... 6 
5 Study Design ........................................................................................................................... 6 
6 Potential Risks and Benefits ................................................................................................. 7 
6.1 
Risks ............................................................................................................................... 7 
6.2 
Potential Benefits .......................................................................................................... 8 
6.3 
Analysis of Risks in Relation to Benefits ................................................................... 8 
7 Study Subject Selection ........................................................................................................ 8 
7.1 
Subject Inclusion Criteria ............................................................................................. 8 
7.2 
Subject Exclusion Criteria ........................................................................................... 9 
8 Study Intervention .................................................................................................................. 9 
9 Study Procedures ................................................................................................................... 9 
10 Assessment of Safety and Data Safety Monitoring Plan (DSMP) ................................ 12 
10.1 Definitions .................................................................................................................... 12 
10.2 Safety Review ............................................................................................................. 14 
10.3 Reporting Plans .......................................................................................................... 14 
10.4 Stopping Rules ............................................................................................................ 15 
11 Data Handling and Record Keeping .................................................................................. 15 
11.1 Confidentiality .............................................................................................................. 15 
11.2 Source Documents ..................................................................................................... 16 
11.3 Case Report Forms .................................................................................................... 16 
11.4 Study Records Retention .......................................................................................... 16 
12 Statistical Plan ...................................................................................................................... 16 
12.1 Study Hypotheses ...................................................................................................... 16 
12.2 Sample Size Determination ...................................................................................... 17 
12.3 Statistical Methods ..................................................................................................... 18 
13 Ethics/Protection of Human Subjects ................................................................................ 18 
14 Literature References .......................................................................................................... 19 
15 Appendix ................................................................................................................................ 21 

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1 
List of Abbreviations 
 
Abbreviation 
Abbreviation definition 
FMT 
Fecal Microbiota Transplantation  
BMC 
Boston Medical Center 
CDI 
Clostridium difficile infection  
CTU 
Clinical Trials Unit 
OTU 
Operational taxonomic units 
 
2 
Protocol Summary 
Title: 
Fecal Microbiota Transplant (FMT) after treatment for primary 
Clostridium difficile Infection (CDI)  
Population: 
Patients >= 18 years hospitalized at Boston Medical Center (BMC) 
with a first documented episode of CDI.  
Intervention: 
30 FMT capsules administered orally under direct observation 
within 7 days of completion of 10-14 day treatment of oral 
vancomycin for CDI.  
Objectives: 
1. To characterize the microbial diversity in stool samples 
from subjects with a primary episode of CDI before and 
after oral vancomycin and determine the impact of FMT 
after completion of oral vancomycin course. 
2. To characterize the feasibility and tolerability of FMT after 
completion of a course of oral vancomycin therapy for 
primary CDI, and to describe 30-day hospital readmission 
and gastrointestinal symptomatology and/or CDI 
recurrence during 60-day and 6 month follow-up.  
Design/Methodology: 
15 subjects will be enrolled who are diagnosed at BMC for a primary 
episode of CDI.  A discard aliquot from baseline stool samples 
obtained clinically for diagnosis will be frozen.  Subjects will receive 
the standard of care treatment (oral vancomycin for 10-14 days) 
and within 7 days following completion will receive oral FMT during 
a 2 hour visit in the ID Clinical Trials Unit. An additional 5 subjects 
will be enrolled as controls. Stool samples will be collected at time 
of CDI diagnosis and again 3 weeks after FMT for intervention group 
and 4 weeks after completion of oral vancomycin treatment for 
control subjects.  The post-treatment samples will be obtained by 
the patient using RNAlater kits and mailed to the BMC CTU where it 
will aliquoted, centrifuged and frozen. 
Samples will be processed at a collaborating lab at Tufts for DNA 
extraction, 16SrDNA amplicon generation, and MiSeq sequencing 
will characterize the fecal microbiome pre- and post oral FMT.   
Study personnel will contact participants via telephone 60 days and 
6 months after oral vancomycin completion to administer a follow-

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up survey (including questions on residual symptoms. CDI 
recurrence, re-hospitalization, adverse events and FMT 
acceptability).  
Subjects will be compensated for study participation with $100 gift 
cards provided for attendance of the clinic visit for FMT and $25 gift 
card after receipt of fecal sample post-treatment. 
Total Study Duration: 
Anticipated time: 12 months 
Subject Participation Duration: We anticipate a period of 1) 1-2 hours while an inpatient for the 
screening and consent process or 2) 1-2 hours if you are screened 
and consented as an outpatient, plus transit time to the visit, 2 
hours for the CTU visit for FMT and 20-30 minutes responding to a 
follow up telephone survey. Total time in the study from enrollment 
to completion of follow-up will be approximately 6 months and will 
include 10-14 days of CDI treatment with oral vancomycin (per 
standard of care treatment), the FMT administration and a 60-day 
and 6-month follow up. 
 
3 
Background/Rationale & Purpose 
 
3.1 
Background Information 
Clostridium difficile infection (CDI) is one of the most urgent health threats in the U.S. associated with 
antibiotic use, according to the Centers for Disease Control and Prevention [1]. An antibiotic course can 
affect the gut microbiome for years [2], and patients with CDI have additional dysbiosis of their gut flora 
for similarly prolonged periods [3,4]. After an episode of CDI, the risk of serious complications, including 
recurrent CDI, and mortality are high for 3-6 months, particularly in patients who are older or have 
comorbid illness [5-8]. Preventing CDI recurrence and restoring the microbiome is a high public health 
priority.  Oral vancomycin, an effective therapy for CDI, perturbs the gut microbiome further [9]. CDI 
recurrences and relapses can occur in 20-30% of people treated with oral vancomycin [10]. In contrast, 
fidaxomicin preserves the intestinal microbiome during and after treatment of CDI, minimizing 
additional dysbiosis.  Likely as a result, fidaxomicin has been shown to reduce recurrence of CDI when 
compared with standard treatment with oral vancomycin, 15.4% vs 25.3%, respectively [10].   
Use of fidaxomicin as primary treatment for CDI has been advocated.  However the agent is costly and 
there is still a significant risk of recurrence after disease.   
 
Fecal Microbiota Transplant (FMT) has been studied as an effective treatment for recurrent CDI [11].  
Konijeti et al performed a decision analysis comparing metronidazole, oral vancomycin, fidaxomicin and 
FMT for first-line treatment of recurrent CDI and found FMT colonoscopy was the most cost effective 
[12]. FMT has had very limited study for a primary episode of CDI and an endoscopic procedure for all 
those cases would be invasive, costly and associated with risk. However, FMT is now available via frozen 
oral capsules and has been shown to be non-inferior to FMT via colonoscopy in randomized controlled 
trials [13].   
  
The FDA allows FMT without an IND only for treatment of recurrent episodes of CDI. At this time, FMT is 
not indicated for primary CDI therapy. We propose oral FMT following standard of care treatment for a 
first episode of CDI as a way to prevent recurrences and improve symptoms post infection. Potential 
benefits include reduced re-admissions from recurrent CDI, improved quality of life and resolution of 

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symptoms sooner.  
 
This study will be conducted in compliance with the protocol, applicable regulatory requirements, and 
BMC/BU Medical Campus Human Research Protection policies and procedures. 
 
3.2 
Rationale and Purpose  
Our study is based on the current understanding of the long-term impacts of antibiotic use and 
CDI.  Medications such as antibiotics or proton-pump inhibitors, CDI itself, and the antibiotic treatment 
of CDI all adversely impact the diversity of the gut microbiome.  Recurrent courses of CDI treated with 
antibiotics alone leads to progressive decreased microbial diversity in the gut [14]. The competition with 
and presence of other organisms in the gut are key to mucosal integrity and overall gut health [11]. Gut 
dysbiosis has not only been associated with recurrent CDI [15] but also increased risk of chronic 
gastrointestinal (GI) disorders, obesity, and non-C. difficile enteric infections [16] which FMT has been 
shown can improve [17].  Recent data suggests that antibiotic-resistant flora after a course of antibiotics 
can be reversed with FMT [18].  A regimen of CDI therapy followed by FMT may reduce recurrence rates 
beyond current available treatments as well as provide other health benefits.  Fidaxomicin and 
bezlotoxumab, a human monoclonal antibody directed against C. difficile toxin B both have been shown 
to reduce CDI recurrence compared with oral vancomycin [10,19], but neither reverse dysbiosis or 
provide other potential health benefits. 
 
There has been sparse study of FMT for primary CDI.  Because of the potential health benefits, 
this cost-effective approach deserves further study.  Our pilot study examines the effect of FMT on gut 
microbial diversity after CDI and oral vancomycin compared with gut microbial diversity in those 
subjects with CDI who receive oral vancomycin alone.  Evidence of reduction of dysbiosis as well as 
information on GI symptomatology (e.g., diarrhea, abdominal pain, bloating), CDI recurrence and 
healthcare utilization, would provide preliminary data to support a randomized controlled, multicenter 
clinical trial to test this approach. 
 
4 
Objectives 
 
4.1 
Study Objectives 
 
Objective 1: To characterize the microbial diversity in stool samples from subjects with a primary 
episode of CDI before and after oral vancomycin and determine the impact of FMT after completion of 
oral vancomycin course. 
Objective 2: To characterize the feasibility and tolerability of FMT after completion of a course of oral 
vancomycin therapy for primary CDI, and to describe 30-day hospital readmission and gastrointestinal 
symptomatology and/or CDI recurrence during 60-day follow-up.   
 
Study Outcome Measures 
 
4.1.1 Primary Outcome Measures 
 
Microbial community abundance and patterns of the stool dysbiosis will be compared before and after 
treatment for primary CDI with or without end of therapy FMT. Primary analyses will consist of direct 

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comparisons of the abundance of individual microbiomes, often represented by operational taxonomic 
units (OTUs), between conditions. We will also identify differences between samples pre- and post-
treatment in FMT-treated subjects and controls using measures of biodiversity (α- and β-diversity, i.e., 
Shannon index, Jenson-Shannon Distance), summary meta-statistics such as principal components and 
coordinate analysis, and the PICRUSt software will be used to generate predicted pathway-level profiles 
for comparison between conditions. 
 
4.1.2 Secondary Outcome Measures 
• At ~60 days after completion of oral vancomycin treatment, secondary outcomes 
measured by telephone survey are: 
o Feasibility and tolerability of administering FMT after completion of a course of 
oral vancomycin therapy 
o Post CDI, post-treatment gastrointestinal symptomatology and response to 
therapy 
• CDI recurrence within 60 days, defined as retreatment for CDI with vancomycin or 
fidaxomicin, and hospital admission within 30-day after treatment as determined by 
chart review. 
• At ~6 months after completion of oral vancomycin treatment, secondary outcomes 
measured by telephone survey are: 
o CDI recurrence or hospital admission 
o Post CDI, post treatment gastrointestinal symptomatology and response to 
therapy 
o Any new onset chronic medical conditions that may represent theoretical 
association with prior FMT 
 
5 
Study Design 
 
We propose an open label pilot study examining the effectiveness of oral FMT at restoring the 
microbial diversity following a primary episode of CDI.  
Subjects who are diagnosed with CDI at Boston Medical Center via normal clinical processes 
(i.e., providers have a clinical concern for CDI, send a stool sample for diagnosis which is positive) will be 
screened for inclusion in the study until 10 subjects are enrolled in the FMT-treatment arm and 5 
subjects are enrolled as controls.  The assignment is not randomized for this pilot study to expedite 
recruitment and because the primary outcome, analysis of the gut microbiome diversity for each subject 
pre- and post CDI and FMT treatment, uses each subject as their own control.  If the patient has a 
preference, they will be allowed to choose the treatment or control group if there are still slots open in 
the group preferred.  If the patient does not have a preference, they will be assigned in a 2:1 ratio (2 
patient receiving FMT for each control subject).  All subjects will receive the standard of care for CDI 
treatment – oral vancomycin 125 mg orally for 10-14 days.  The ten subjects in the FMT-treatment arm 
will receive oral FMT capsules within one week of oral vancomycin completion. Oral FMT frozen 
capsules DE (OpenBiome, Somerville, MA), a formulation designed to deliver the product to the large 
intestine, is the product currently used by BMC for recurrent CDI and will be used for this study.  FMT 
will be stored and dispensed by the Investigational Pharmacy Service and administered in an outpatient 
setting supervised by the staff of the Clinical Trials Unit (CTU) within BMC’s Center for Infectious 
Diseases. Study participants who receive FMT will attend one clinic visit (within 7 days of completion of 

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initial CDI therapy) where they will be administered 30 FMT capsules over ~2 hours, under direct 
observation.   
Subjects will complete a baseline survey after completion of oral vancomycin.  The survey will 
take 10-15 minutes and include questions on residual or recurrent symptoms, any new antibiotics, 
probiotics or other medications and any new or additional hospital admissions.  Subjects in the 
treatment arm will complete the survey at the time of their FMT visit.  Controls will be contacted by 
phone to complete a similar survey within a week of completing oral vancomycin.  Study personnel will 
contact all participants via telephone 60 days and 6 months after the initial survey to administer a 
follow-up survey which will take 15-20 minutes. The 18-question survey was adapted from the validated 
Rome III questionnaire [17] and can be found in the Appendix. Subjects will be compensated for study 
participation with $100 gift cards for the CTU visit for FMT and with a $25 gift card after the post 
treatment fecal sample is received.  
Sample processing and analysis: two stool samples will be analyzed. The first sample is collected 
in all subjects as part of routine clinical care and the microbiology laboratory will provide a discard 
aliquot for the study.  The second sample will be collected by the subject at home 30 days after 
completion of oral vancomycin treatment, and mailed to investigators using a collection kit subjects 
have been provided.  All stool samples will be aliquoted into 2 tubes, centrifuged and the pellets stored 
at -80⁰C until all samples are received.  Those samples will then be sent to Dr. Anne Kane at Tufts. Her 
laboratory will extract DNA from the stool samples and the V4 region of the 16S rRNA will be amplified 
and sequenced using an Ilumina MiSeq [16]. Sequences will be de-multiplexed, pre-processed for quality 
control, and aligned to the Greengenes database to generate a table of OTU abundances and taxonomic 
lineages. That data will then be analyzed by study investigator, Dr. W. Evan Johnson in BU Division of 
Computational Biomedicine. Microbial community abundance and patterns will be assessed between 
the treatment and control groups using: (i) direct comparisons of individual OTUs, (ii) measures of 
biodiversity (α- and β-diversity, i.e., Shannon index, Jenson-Shannon Distance), (iii) summary meta-
statistics such as principal components and coordinate analysis, and (iv) the PICRUSt software to 
generate predicted pathway-level profiles.  
 
Please see the Appendix for a schematic of the study design.  
 
6 
Potential Risks and Benefits 
 
6.1 
Risks 
There will be 10 subjects who receive FMT and 5 controls. 
For controls, care will be standard clinical care with no additional risks. However, they will be asked to 
submit a stool sample (a mailer will be provided so this can be done from home) and may feel some 
discomfort. Potential risk of confidentiality violation will be avoided because investigators will follow 
accepted procedures to prevent this. 
 
For subjects who receive FMT, in addition to the above risks, they will be given oral capsules that may 
have side effects. Only capsules for subjects who are alert and can swallow will be given. 
Common, mild adverse events: transient diarrhea (70%), transient abdominal cramps/discomfort 
(20%) and nausea (<5%) in the 24 hours post FMT. Transient fever, abdominal distension/bloating, 
belching, vomiting, dehydration, borborygmus have also been reported. Constipation (20%) and excess 
flatulence (25%) have been  
reported in follow- up. There is also a theoretical risk of small intestinal bacterial overgrowth. 
Rare, serious adverse events: The following risks should be considered: 

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Infection: Although this material has been screened for bacteria, viruses, fungi and parasites there is a 
risk of transmission of known and unknown infectious organisms contained in the donor stool. Post-FMT 
bacteremia (e.g. E. coli), sepsis and fatal events may rarely occur. 
Inflammatory bowel disease (IBD) flare in those with underlying IBD (excluded from our study 
population but previously undiagnosed subjects may inadvertently be enrolled). 
Allergy/Anaphylaxis to antigens in donor stool; 
Non-infectious disease transmission: There is a theoretical risk of developing disease that may be 
related to donor gut microbiota. These include obesity, metabolic syndrome, cardiovascular disease, 
autoimmune conditions, allergic/atopic disorders, neurologic disorders, psychiatric conditions and 
malignancy. Persons with these known conditions are excluded from donating stool. 
Aspiration: Capsule administration carries a risk of aspiration 
 
Additional potential risks or discomfort include the extra visit to the hospital which will take ~2 hours 
that would not be necessary if they were not study participants. They will also have to submit stool 
samples and participate in a phone interview as controls and have same potential risk or discomfort. 
Patients receiving FMT will be required to fast, which may risk hypoglycemia in diabetic patients. Fasting 
may also result in lightheadedness, dizziness or fainting.   
6.2 
Potential Benefits 
 
For controls, there are no benefits to the patient other than satisfaction for participating in a study to 
improve patient care. 
 
For subjects receiving FMT, potential benefit includes possible restoration of gut microbiome which has 
been linked to reduced recurrence of CDI; microbiome restoration may reduce the risk of post-infectious 
irritable bowel syndrome described after CDI. The recurrence rate for primary CDI is high (20-30%) after 
treatment with oral vancomycin and FMT may reduce that risk.  Improved quality of life, fewer missed 
days of work and psychological upset due to recurrent CDI are potential benefits of oral FMT following 
primary CDI. 
 
6.3 
Analysis of Risks in Relation to Benefits 
 
The recurrence rate of CDI following treatment with oral vancomycin (the current standard of care) is 
20-30%, and the risks of oral FMT via frozen capsules are primarily temporary diarrhea, 
cramping/bloating and nausea. If we can decrease that with one time oral FMT then the benefits of 
avoiding recurrent CDI (and the morbidity, mortality and economic impacts therein) would outweigh the 
risks. 
 
7 
Study Subject Selection 
 
7.1 
Subject Inclusion Criteria 
 
In order to be eligible to participate in this study, an individual must meet all of the following criteria: 
 
Inclusion 

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• 
Adult (18 years and older) hospitalized patients with primary C. difficile infection for which oral 
vancomycin 125 mg every 6 hours for 10-14 days is standard of care therapy (CDI that is non-
severe or severe e.g., leukocytosis ≥15,000 cells/mL and serum creatinine >1.5 mg/dl). 
 
7.2 
Subject Exclusion Criteria 
 
Exclusion 
An individual who meets any of the following criteria will be excluded from participation in this study: 
• 
Previous history of CDI 
• 
Patients <18 years of age  
• 
History of ongoing antibiotic use (e.g., nitrofurantoin for UTI prophylaxis) 
• 
Diagnosis of cancer not in remission and/or actively being treated 
• 
Personal or family history of Inflammatory Bowel Disease or unexplained GI illness 
• 
History of irritable bowel syndrome, excessive gas, bloating, lymphocytic colitis, idiopathic 
chronic constipation, chronic use of laxatives or chronic diarrhea 
• 
Use of probiotics or any over the counter aids to regulate digestion 
• 
Major immunosuppressive medications, e.g., calcineurin inhibitors, exogenous glucocorticoids, 
biologic agents, etc. 
• 
Systemic anti-neoplastic agents 
• 
Pregnant and breastfeeding women 
• 
History of dysphagia: oropharyngeal, esophageal, functional, neuromuscular (e.g. stroke, 
multiple sclerosis, ALS), or patient shows evidence of dysphagia when the ‘safety test’ capsule is 
administered 
• 
History of aspiration, intestinal obstruction or gastroparesis 
• 
History of severe food allergies  
• 
History of allergy to sodium chloride, glycerol, theobroma oil, hide bovine gelatin, sodium lauryl 
sulfate, colorants FD&C, or titanium dioxide   
• 
Hospitalized at the time oral vancomycin therapy is completed 
• 
Fulminant CDI (hypotension, ileus, and/or megacolon) 
• 
Non-English speaking 
 
8 
Study Intervention 
 
The main study intervention will be a one-time course of 30 FMT capsules after treatment with oral 
vancomycin for primary CDI.  
 
9 
Study Procedures 
 
See the Appendix for the schedule of events and procedures.  
 
1. Patient admitted to BMC has a positive stool PCR test for C. difficile.  (The stool is sent as part of 
clinical care). 
a. Per BMC protocol, the microbiology laboratory reports the positive result in the 
electronic medical record for Infection control review in real time.   
b. A member of Infection Control, reviews this list daily, Monday through Friday and 
notifies study investigator of possible case.   

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c. The study investigator (under IRB-approved HIPAA waiver) reviews the patient chart to 
determine if this is a primary episode of CDI. She will also determine if there are study 
exclusions already recorded in the chart.  
d. If the patient has primary CDI and no documented exclusions, a member of the study 
team will contact the patient’s provider to ask permission to discuss the study with the 
patient if the patient is hospitalized at the time; outpatients will be contacted directly 
via the contact number listed in the electronic medical record. 
e. A study team member either visits the patient if inpatient or calls the patient if 
outpatient, explains briefly the purpose of the visit, and reviews the Brief Screening 
Agreement. If the patient agrees, they are formally screened for inclusion/exclusion 
criteria.  No identifying information will be kept after screening, consent will be 
obtained as soon as possible after screening.  If consent is delayed, the screening data 
will not be kept in an identifiable manner.   
f. If eligible and interested, the patient undergoes the informed consent process.  
i. Patients who are recruited in the outpatient setting will agree by phone to receive the 
informed consent documents via email (preferred) or mail.  If interested and eligible, a 
study visit will be arranged in the CTU to complete the consent process. 
ii. Consent will be obtained during the hospital stay for inpatients. They will be given up 
to 48 hours to decide whether to participate and consent.  
iii. Patients who consent will be designated either a control or treatment subject.  If the 
patient has a preference, they will be allowed to choose the treatment or control group 
as long as there are still slots open in the group preferred. If not, they will be assigned in 
a 2:1 ratio (2 patient receiving FMT for each control subject). 
g. Subjects in the treatment group who are females of childbearing potential will have a 
pregnancy test that will be reviewed and negative before enrollment.  Those subjects 
must agree to use an effective form of contraception from the time of enrollment 
through at least 30 days after FMT. 
h. The patient will receive the standard of care treatment for CDI, vancomycin 125 mg po 
q6h for 10-14 days, per their physician. 
i. 
The laboratory will provide a discard aliquot of the stored stool (on which the initial PCR 
test was done), which will be labeled by study ID number and stored at -80⁰C by the 
CTU.  There will be adequate stool sample remaining for any additional clinical testing 
the laboratory might need to do.  
j. 
At outpatient enrollment or prior to hospital discharge, subjects will be provided an 
RNALater kit for stool collection with instructions on how to obtain the sample.  They 
will be provided a prepaid, addressed mailer. For treatment subjects, an appointment 
for CTU will be confirmed for 3-7 days after completion of oral vancomycin is planned.  
k. Subjects will be given written instructions related to FMT treatment (clear liquid diet 
after midnight the day of the appointment, fasting 2 hours prior to arrival to 
appointment)  
l. 
All subjects will be given instructions on how to clean their bathroom at home. 
2. Subjects with worsening CDI infection during oral vancomycin therapy who require escalation of 
care will be withdrawn from the study. All medical management will be by the subject’s primary 
provider. 
3. Subjects in the FMT-group will receive a phone call reminder two days prior to CRU appointment 
and instructions (i.e., clear liquid diet and fasting prior to visit) will be reviewed.   

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4. Phone call to controls within 7 days of completion of CDI therapy for a survey of symptoms that 
should take 10-15 minutes to complete. 
5. The Investigational Pharmacy will handle storage and labeling for research of all FMT capsules.  
10 courses of 30 capsules will be obtained from OpenBiome.  OpenBiome will also provide 
either a small aliquot of stool for each treatment course, or genetic analysis of the microbiome, 
of the donor stool.  As the microbiome can vary for each treatment, even for a single donor, this 
information is necessary for analysis.  The oral FMT capsules are stored in a tamper evident 
plastic bottle inside packaging on dry ice, they are stored at -20 degrees C and removed from 
freezer and must be consumed within 90 minutes. OpenBiome FMT DE capsules contain: Frozen 
human fecal microbiota (filtered to 330 microns), theobroma oil, glycerol, hide bovine gelatin, 
sodium lauryl sulfate, colorants FD&C and titanium dioxide. Per OpenBiome’s product 
information: “FMT Capsule  DE is physically stable at room temperature for more than 30 days. 
However to preserve microbial viability, providers should strictly maintain the cold chain 
without allowing freeze-thaw cycles. Material should be delivered within 90 minutes of removal 
from frozen storage.” Prior to swallowing the 30 capsules that constitutes oral FMT the patient 
will swallow an empty “placebo” capsule to confirm they can swallow the capsule without 
difficulty [20]. 
6. At CTU appointment (within 7 days after completion of oral vancomycin): 
a. Confirm fasting and liquid diet compliance 
b. In person survey on symptoms completed with patient by CTU personnel.  
c. CTU personnel goes through check list (reviewing contraindications and exclusion 
criteria) 
d. For subjects who are females of childbearing potential, a pregnancy test will be 
administered, reviewed and negative prior to administration of FMT.  If positive, the 
subject will be excluded from the study. 
e. One test placebo capsule administered.  If patient is able to swallow without a problem, 
administration proceeds.  
f. 30 pills FMT removed from freezer and given to patient (protocol) over 90 minutes 
(Study specific).  If patient vomits, the procedure will be aborted. 
g. Patient instructed to fast for 1 hour post FMT. Patient given post-FMT instruction sheet 
and number to call to report any concerns or adverse events.  
h. Patient will be given a diary to record daily symptoms for a total of 7 days after FMT.  
Patients will be given an addressed, stamped envelope to return the diary to the 
investigators.   Patients who cannot read the diary will have someone complete the 
form with them or the investigators will arrange to phone daily for one week to 
complete the form. 
i. 
The patient will receive a phone call to determine if there had been any adverse events 
after the FMT within 48-72 hours. Investigators will confirm that the patient is 
completing the symptom diary and will do so for a total of 7 days. 
j. 
Patient reminded to submit stool sample in 20 days using RNALater kit and instructions 
reviewed again.  If patient has misplaced RNALater kit, a new one will be provided.  
7. Any serious, life-threatening adverse event that is unexpected and possibly related to the 
research will result in suspension of the trial.  All adverse events will be reported to OpenBiome 
using their adverse event reporting form: www.OpenBiome.org/adverse-events, as well as to 
the IRB.  
8. Control subjects will provide stool sample ~30 days post completion of oral vancomycin; FMT-
treated subjects will provided stool sample ~20 days after oral FMT administration. 

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a. A study team member will phone 2 days prior to planned date for stool submission 
b. A study team member will phone 2 days after stool submission was planned to confirm 
it had been sent 
c. All stool samples will be collected at home per manufacturer’s protocol, mailed to PI. A 
study team member will aliquot the sample, centrifuge, decant the RNALater media, 
and the pellets will be stored by CTU at -80oC till further processing.  All baseline and 
follow-up stool samples will be shipped to Tufts together for batch processing. 
9. Sixty days after initial survey (± 1 week) - Phone survey of cases and controls that will take 15-
20 minutes and include questions on residual symptoms. CDI recurrence, 30 day readmission, 
adverse events as well as their impression of burden and acceptability of FMT post-vancomycin 
treatment.   
10. Six months after the initial survey (± 1 week) - Phone survey of cases and controls that will take 
15-20 minutes and include questions on residual symptoms. CDI recurrence, 30 day 
readmission, adverse events. They will also be questioned about any serious adverse events 
and/or new onset of chronic medical conditions that may represent theoretical risks of FMT 
(e.g., immune-mediated diseases, obesity, metabolic syndrome, gastrointestinal disorders, 
cardiovascular disease, neuropsychiatric disorders, and malignancies). 
11. Frozen stool samples, all labeled only by study ID number, and sent to Tufts for batch processing 
and analyses. The V4 region of the 16S rRNA will be amplified from DNA extracted from the 
stool samples and sequenced using an Ilumina MiSeq [16] under the direction of Dr. Anne Kane. 
Sequences will be de-multiplexed, pre-processed for quality control, and aligned to the 
Greengenes database to generate a table of OTU abundances and taxonomic lineages.  
12. Statistical analyses: BU Computational Biomedicine (Dr. Johnson’s laboratory) will perform these 
analyses. Microbial community abundance and patterns will be assessed between the treatment 
and control groups using: (i) direct comparisons of individual OTUs, (ii) measures of biodiversity 
(α- and β-diversity, i.e., Shannon index, Jenson-Shannon Distance), (iii) summary meta-statistics 
such as principal components and coordinate analysis, and (iv) the PICRUSt software to generate 
predicted pathway-level profiles. 
 
For each patient total time involved should be less than 180 days:  
1-3 days for screening and informed consent process 
10-14 days for CDI treatment 
Oral FMT within 7 days of completion of oral vancomycin 
60 days follow-up after completion of oral vancomycin course  
6 month follow-up after completion of oral vancomycin course 
 
10 Assessment of Safety and Data Safety Monitoring Plan (DSMP) 
 
10.1 Definitions 
 
The following definitions will be used in the assessment of safety: 
 
Adverse Event (AE) is any untoward or unfavorable medical occurrence in a human subject, including any 
abnormal sign (for example, abnormal physical exam or laboratory finding), symptom, or disease, 
temporally associated with the subject’s participation in the research, whether or not considered 
related to the subject’s participation in the research. 
 

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AEs will be evaluated for relationship to study intervention per the physician investigator who will 
determine the relationship as definite, probable, possible, and not related to the intervention. 
This is defined as: 
Definitely related: The adverse event is clearly related to the FMT material – i.e., an event that follows a 
reasonable temporal sequence from administration of the FMT material, follows a known or expected 
response pattern to the FMT material, that is confirmed by improvement on stopping and reappearance 
of the event on repeated exposure and that could not be reasonably explained by the known 
characteristics of the patient’s clinical state. 
 
Probably related: The adverse event is likely related to the FMT material – i.e., an event that follows a 
reasonable temporal sequence from administration of the FMT material, follows a known or expected 
response pattern to the FMT material, that is confirmed by improvement on stopping and reappearance 
of the event on repeated exposure and that is unlikely to be explained by the known characteristics of 
the patient’s clinical state. 
 
Possibly related: An adverse event that follows a reasonable temporal sequence from administration of 
the FMT material, follows a known or expected response pattern to the FMT material, but that could 
readily have been produced by a number of other factors.  
 
Not related: The adverse event is clearly not related to the FMT material, i.e. another cause of the event 
is most plausible; and/or a clinically plausible temporal sequence is inconsistent with the onset of the 
event and the study intervention and/or causal relationship is considered biologically impossible. 
 
AEs will be graded by severity as: 
1- Mild AE (not requiring treatment) 
2- Moderate AE (resolved with treatment) 
3- Severe AE (inability to carry on normal activities/required professional medical attention) 
4- Life threatening or disabling AE 
5- Death 
 
Serious Adverse Event (SAE) is any adverse event that  
(1) results in death; 
(2) is life-threatening; 
(3) results in inpatient hospitalization or prolongation of existing hospitalization; 
(4) results in a persistent or significant disability/incapacity; 
(5) results in a congenital anomaly/birth defect; or 
(6) based upon appropriate medical judgment, may jeopardize the subject's health and may require 
medical or surgical intervention to prevent one of the other outcomes listed in this definition 
(examples of such events include allergic bronchospasm requiring intensive treatment in the 
emergency room or at home, blood dyscrasias or convulsions that do not result in inpatient 
hospitalization, or the development of drug dependency or drug abuse).  
 
Life-threatening means that the event places the subject at immediate risk of death from the event as it 
occurred.  
 
 

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Unanticipated Problem is defined as an event, experience or outcome that meets all three of the 
following criteria:  
• 
is unexpected; AND 
• 
is related or possibly related to participation in the research; AND 
• 
suggests that the research places subjects or others at a greater risk of harm (including physical, 
psychological, economic, or social harm) than was previously known or recognized.  
 
Possibly related means there is a reasonable possibility that the incident, experience, or outcome may 
have been caused by the procedures involved in the research 
 
Unexpected means the nature, severity, or frequency of the event is not consistent with either: 
• 
the known or foreseeable risk of adverse events associated with the procedures involved in the 
research that are described in (a) the protocol–related documents, such as the IRB-approved 
research protocol, any applicable investigator brochure, and the current IRB-approved informed 
consent document, and (b) other relevant sources of information, such as product labeling and 
package inserts; or 
• 
the expected natural progression of any underlying disease, disorder, or condition of the 
subject(s) experiencing the adverse event and the subject’s predisposing risk factor profile for the 
adverse event. 
 
In addition, any adverse events will be reported to the FDA, as appropriate. 
 
10.2 Safety Review 
 
Both the risks listed in Section 6.1 and unknown risks will be monitored as follows:  
Events:  
• 
Study participants will be monitored during FMT test dose, the 90 minutes of FMT 
administration, and 30 minutes after FMT administration to ensure no reactions occur such as 
nausea or vomiting, abdominal pain or diarrhea.  
• 
Following administration of oral FMT study participants will receive a handout with contact 
information to report any adverse symptoms or concerns.  
• 
Subjects will be contacted within 48-72 hours after FMT to check on patient status. 
• 
Study participants will be contacted at 60 days following their initial survey at which point a 
detailed survey will be administered on symptoms, side effects and any re-admissions. 
• 
Physicians will go over any significant adverse events to determine if they are related, possibly 
related or unrelated to oral FMT administration 
• 
Patients will be contacted six months after FMT to determine if there were any further serious 
adverse events and/or new onset of chronic medical conditions that may represent theoretical 
risks of FMT (e.g., immune-mediated diseases, obesity, metabolic syndrome, gastrointestinal 
disorders, cardiovascular disease, neuropsychiatric disorders, and malignancies). 
 
10.3 Reporting Plans 
 
The Principal Investigator at BMC/BU Medical Campus will report Unanticipated Problems, safety 
monitors’ reports, and Adverse Events to the BMC/BU Medical Center IRB in accordance with IRB 
policies: 

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• 
Unanticipated Problems occurring at BMC/BU Medical Campus involving a fatal or life-threatening 
event will be reported to the IRB within 2 days of the investigator learning of the event. 
• 
Unanticipated Problems occurring at BMC/BU Medical Campus not involving a fatal or life-
threatening event will be reported to the IRB within 7 days of the investigator learning of the 
event. 
• 
Adverse Events (including Serious Adverse Events) will be reported in summary at the time of 
continuing review, along with a statement that the pattern of adverse events, in total, does not 
suggest that the research places subjects or others at a greater risk of harm than was previously 
known. 
• 
OpenBiome will receive serious adverse event reports within 24 hours and review with 
investigators to assess whether it needs to be reported to the FDA.  The IRB will be informed.  
 
10.4 Stopping Rules 
 
• 
The study will be suspended if one or more serious or life-threatening adverse event that is 
unexpected and possibly related to the research occur in the study population.   In addition, the 
following events would trigger a study pause and safety review: Any single new potentially 
transmitted infection assessed as at least possibly related to FMT, i.e., infection cannot be 
assessed as unrelated to FMT with certainty in the judgment of the investigator alone or in 
consultation with OpenBiome. 
• 
Two or more FMT recipients develop a new medical condition or immune-mediated disease 
assessed by the investigator and/or OpenBiome as definitely related to FMT. 
• 
Three or more FMT recipients experience a non-serious Grade 3 (severe) AE of the same type 
assessed as definitely related to FMT (e.g., vomiting, dehydration, abdominal pain, fever). 
 
11 Data Handling and Record Keeping 
 
11.1 Confidentiality 
• 
The information that will be obtained from and/or about subjects and potential subjects is the 
minimum necessary to conduct the study; and 
• 
If any interventions and interactions occur with subjects and potential subjects, they will take 
place in private settings.  
• 
All patient data will be linked to a study ID and the master list of the study ID will be kept in a 
separate, password-protected file and stored within a locked BMC office in a HIPAA-compliant 
BMC-controlled computer that meets BMC's standard for storage of PHI. 
Clinical specimens (two stool samples) will be labeled with the study ID only and sample number 
only.  
• 
Microbiome analyses will be labeled with the study ID only.  
• 
Study participants will be identified by a unique Study ID linked to subject identifiers via a 
master code list as described, only available to study investigators and research staff. No other 
persons will have access to the master code list.  
• 
The master code list will be kept for 7 years to allow the option for further follow-up of the 
subjects, for which additional IRB approval would be obtained. After 7 years and/or when no 
further changes need to be made to the underlining raw data, the master code list will be 
destroyed.  
 

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11.2 Source Documents 
 
Data generated by the methods described in the protocol will be recorded in the subjects' medical 
records if part of routine patient care only. Otherwise, all records will be in each subjects Case Report 
Forms and/or study progress notes. Data may be transcribed legibly on CRFs supplied for each subject or 
directly inputted into an electronic system or any combination thereof. 
 
11.3 Case Report Forms 
 
The study Case Report Form (CRF) will be the primary data collection instrument for the study. All data 
requested on the CRF will be recorded. All missing data will be explained. If a space on the CRF is left 
blank because the procedure was not done or the question was not asked, “N/D” will be written. If the 
item is not applicable to the individual case, “N/A” will be written. All entries will be printed legibly in 
black ink. If any entry error has been made, to correct such an error, a single straight line will be drawn 
through the incorrect entry and the correct data will be entered above it. All such changes will be 
initialed and dated.  There will be no erasures or white-out on CRFs.  For clarification of illegible or 
uncertain entries, the clarification will be printed above the item, then initialed and dated. The following 
source data will be recorded directly on the CRFs: brief screening agreement, eligibility check list, 
informed consent, phone survey interviews, telephone contact documentation with reminders, Adverse 
event tracking, subject deviation/violation/withdrawal from the study. Duration of oral vancomycin and 
date of completion; antibiotic administration (other than for CDI) and dates; date of oral FMT 
administration; dates of stool specimens; patient diary; and any unscheduled contacts with subject will 
be recorded/included in the CRF folder for each subject. 
 
See the Appendix for templates for the CRFs including documentation of eligibility and consent, 
documentation for the CTU visit, and adverse event tracking. 
 
11.4 Study Records Retention 
 
Study records will be retained by the PI in a locked BMC office, identified by Study ID only.  The master 
list of the study ID will be kept in a in a HIPAA-compliant BMC-controlled computer that meets BMC's 
standard for storage of PHI for at least 7 years. Documentation of informed consent of subjects be 
retained for at least seven years after the study is closed electronically in a password-protected file on a 
HIPAA compliant BMC computer accessible only to the investigators and available for inspection and 
copying by authorized individuals.  
 
 
Drug/Biologics: 
• 
FDA requirements, records and reports will be kept for 2 years after a marketing application is 
approved for the drug; or if an application is not approved for drug, until 2 years after shipment 
and delivery of the drug for investigational use is discontinued and the FDA so notified. 
 
 
12 Statistical Plan 
 
12.1 Study Hypotheses 
 

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Objective 1: To characterize the microbial diversity in stool samples from subjects with a primary 
episode of CDI before and after oral vancomycin and determine the impact of FMT after completion of 
oral vancomycin course. 
Hypothesis 1a: At the time of the CDI diagnosis (baseline), there will be dysbiosis of the gut 
microbiome compared to published microbiome data of healthy patients. 
Hypothesis 1b: One month after completion of oral vancomycin therapy, the microbiome will be 
similar to baseline in subjects who do not receive FMT. 
Hypothesis 1c: There will be a significant reduction in dysbiosis following FMT compared to 
subjects who do not receive FMT. 
Objective 2 : To characterize the feasibility and tolerability of FMT after completion of a course of oral 
vancomycin therapy for primary CDI, and to describe 30-day hospital readmission and gastrointestinal 
symptomatology and/or CDI recurrence during 60-day follow-up. 
Hypothesis 1a: FMT after completion of oral vancomycin treatment will not be unduly 
burdensome for subjects and will be well tolerated. 
Hypothesis 1b: GI symptomatology will be improved in subjects who receive FMT compared to 
those subjects who do not, and other outcomes may show improvement. 
 
Study Outcome Measures 
 
12.1.1 Primary Outcome Measures 
 
Microbial community abundance and patterns of the stool dysbiosis will be compared before and after 
treatment for primary CDI with or without end of therapy FMT. Primary analyses will consist of direct 
comparisons of the abundance of individual microbiomes, often represented by operational taxonomic 
units (OTUs), between conditions. We will also identify differences between samples pre- and post-
treatment in FMT-treated subjects and controls using measures of biodiversity (α- and β-diversity, i.e., 
Shannon index, Jenson-Shannon Distance), summary meta-statistics such as principal components and 
coordinate analysis, and the PICRUSt software will be used to generate predicted pathway-level profiles 
for comparison between conditions. 
 
12.1.2 Secondary Outcome Measures 
• 
Descriptive data using survey responses and comparing control and treatment groups re:  
o Feasibility and tolerability of administering FMT after completion of a course of oral 
vancomycin therapy 
o Post CDI, post-treatment gastrointestinal symptomatology and response to therapy  
o  CDI recurrence within 60 days  
• 
30-day hospital readmission after treatment – percent of subjects readmitted within 30 days of 
treatment per patient history and by chart review.  
Secondary outcomes will be compared between treatment and control groups using Fisher’s 
exact testing for small sample sizes. 
 
12.2 Sample Size Determination 
 
This is a pilot study for proof of concept i.e. that this approach is feasible and acceptable to subjects and 
that FMT after oral vancomycin therapy improves dysbiosis.  We will likely lack statistical power for our 
secondary outcome analyses, given the small sample size. Further there will be no matching of controls 

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to intervention.  Boston Medical Center had over 150 cases of primary CDI in 2017 which should be 
ample number to recruit 15 subjects. 
 
12.3 Statistical Methods 
 
Quality control of sequencing reads will be performed with the FastQC software [21]. Dr. Johnson’s 
PathoScope and PathoStat software frameworks [22-25] will be used to preprocess the data, align the 
reads to the Greengenes database [26], and analyze the microbial composition of RNA-seq data analysis. 
This will generate a table of OTU-level relative abundances with associated taxonomic lineage, microbial 
function, gene coverage and expression, etc. The microbial communities will be characterized at 
multiple taxonomic levels (e.g., phylum, genus, and species). PathoStat [27] will be used for diversity 
estimates (alpha and beta), dimension reduction using PCA and PCoA, and differential abundance and 
expression using DeSeq2 [28] and other tools implemented into our PathoStat tool. Expression 
differences for microbial genes will be assessed also with DeSeq2, including relevant sets of pathways 
available in the KEGG database using PICRUSt, enrichment analysis and ASSIGN profiling. 
 
Sample size: Since our sample size proposed here is small, we recognize that sample size will limit our 
ability to develop confirmed biomarkers for FMT treatment efficacy, so our study will be focused on 
exploration and to justify larger studies to make more concrete conclusions. However, our previously 
published work and microbiome work from others [29,30], have demonstrated that small sample sizes 
can yield significant results for discriminating diseased samples from controls. Our group size should be 
more than sufficient for our explorations and justify a larger study with larger sample size. We can 
estimate detectable effect sizes for the microbiome and host expression data based on a combination of 
the Human Microbiome Project (HMP) [31] and our previous work [29]. In our previous study, we 
observed an average of 8 species per sample with relative species abundance >1%, with corresponding 
standard deviation quartiles of [0.081, 0.10, 0.15] in the HMP, and another 47 rarer species our dataset 
(>0.01% relative abundance), with corresponding HMP standard deviation quartiles of [0.016, 0.027, 
0.058]. Using the human microbiome project data as a baseline, we anticipate to have 80% power to 
detect a ~0.07-unit difference in rare taxon abundance and a ~0.25-unit difference in common taxa 
abundance, using a Bonferroni correction (0.05/300=0.00017). The corresponding detection limit for 
~250 metabolic pathways allows for power of 80% for a ~0.25-unit difference in relative abundance, 
again employing a Bonferroni false positive cutoff. Analyses that evaluate diversity of organisms and 
pathways will be powered similarly as described above for common taxa abundances. This although our 
sample sizes are relatively small in each group, it is clear that we will have sufficient power to detect any 
large species-, higher level taxa-, and pathway-level differences present between the treatment and 
control groups.  
 
13 Ethics/Protection of Human Subjects 
 
This study is to be conducted according to applicable US federal regulations and institutional policies 
(which are based in federal regulations, guidance, and ICH Good Clinical Practice guidelines). 
 
This protocol and any amendments will be submitted to the Boston Medical Center and Boston 
University Medical Campus IRB, for formal approval of the study conduct. The decision of the IRB 
concerning the conduct of the study will be made in writing to the investigator. A copy of the initial IRB 
approval letter will be provided to the sponsor before commencement of this study.  
 

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All subjects for this study will be provided a consent form describing this study and providing sufficient 
information for subjects to make an informed decision about their participation in this study. The 
consent form will be submitted with the protocol for review and approval by the IRB. The consent of a 
subject, using the IRB-approved consent form, must be obtained before that subject is submitted to any 
study procedure. Consent will be documented as required by the IRB.   
 
14 Literature References 
References 
1. Centers for Disease Control and Prevention (CDC), “Antibiotic Resistance Threats in the United States. 
2013,” available at http://www.cdc.gov/drugresistance/threat-report-2013/   (cited March 29, 2018). 
2. Dethlefsen L, Relman DA. Incomplete recovery and individualized responses of the human distal gut 
microbiota to repeated antibiotic perturbation. Proc Natl Acad Sci USA 2011; 108 Suppl 1:4554-61. 
3. Perez-Cobas AE, Artacho A, Ott SJ, Moya A, Gosalbes MJ, Latorre A. StruCRUral and functional changes 
in the gut microbiota associated to Clostridium difficile infection. Front Microbiol 2014;5:335 
4. Skraban J, Dzeroski S, Zenko B, Mongus D, Gangl S, Rupnik M. Gut microbiota patterns associated with 
colonization of different Clostridium difficile ribotypes. PLoS One 2013;8(2):e58005 
5. Murphy CR, Avery TR, Dubberke ER, Huang SS. Frequent hospital readmissions for Clostridium difficile 
infection and the impact on estimates of hospital-associated C. difficile burden. Infect Control Hosp 
Epidemiol 2012;33 (1):20-8. 
6. Rodriguez-Pardo D, Almirante B, Bartolome RM, et al. Epidemiology of Clostridium difficile infection 
and risk factors for unfavorable clinical outcomes: results of a hospital-based study in Barcelona, Spain. J 
Clin Microbiol 2013; 51 (5):1465-73. 
7. Louie TJ, Miller MA, Crook DW, et al. Effect of age on treatment outcomes in Clostridium difficile 
infection. J Am Geriatr Soc 2013; 61 (2):222-30. 
8. Abou Chakra CN, Pepin J, Sirard S, Valiquette L. Risk factors for recurrence, complications and 
mortality in Clostridium difficile infection: a systematic review. PLoS ONE 2014;9 (6):e98400. 
9. Louie T, Cannon K, Byrne B, Emery J, Ward L, Eyben M, Krulicki W. Fidaxomicin preserves the intestinal 
microbiome during and after treatment of Clostridium difficile infection (CDI) and reduces both toxin re-
expression and recurrence of CDI. Clin Infect Dis 2012;55 (suppl 2) S132-142. 
10. Louie T, Miller, M, Mullane K et al. Fidaxomycin versus Vancomycin for Clostridium difficile. N Engl J 
Med 2011; 364(5);422-31.  
11.  Khoruts A, Sadowsky MJ. Understanding the mechanisms of faecal microbiota transplantation. Nat 
Rev Gastroenterol and Hepatol 2016; 13 (9): 508-516. 
12. Konijeti G, Sauk J, Shrime M, Gupta M, Ananthakrishnan A. Cost effectiveness of Competing 
Strategies for Management of Recurrent Clostridium difficile infection: A Decision Analysis. Clin Infect 
Dis 2014;58 (11): 1507-14. 
13. Kao D, Roach B, Silva M, et al. Effect of oral capsule- vs colonoscopy-delivered fecal microbiota 
transplantation on recurrent Clostridium difficile infection: a randomized clinical trial. JAMA 
2017;318(2):1985-93 
14. Cheng J et al Decreased diversity of fecal microbiome in recurrent Clostridium difficile associated 
diarrhea. J infect Dis. 2008 Feb 1;197(3) 435-8 
15. Seekatz AM, Rao K, Santhosh K, Young VB. Dynamics of the fecal microbiome in patients with 
recurrent and nonrecurrent Clostridium difficile infection. Genome Med 2016;8:47. 
16.DuPont AW, DuPont HL. The intestinal microbiota and chronic disorders of the gut. Nat Rev 
Gastroenterol Hepatol 2011;8:523-31. 
17. Konturek PC, Haziri D, Brzozowski T et al. Emerging role of fecal microbiota therapy in the treatment 
of gastrointestinal and extra-gastrointestinal diseases. J Physiol Pharmacol 2015; 66(4):483-91. 

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18. Davido B, Batista R, Michelon H et al. Is faecal microbiota transplantation an option to eradicate 
highly drug-resistant enteric bacteria carriage? J Hosp Infect 2017;95(4):433-37. 
19. Wilcox MH, Gerding DN, Poxton IR et al. Bezlotoxumab for prevention of recurrent Clostridium 
difficile infection. N Engl J Med 2017;376(4):305-317. 
20. OpenBiome Oral FMT G3 handling instructions 
21. https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ 
22. Byrd AL, Perez-Rogers JF, Manimaran S., et al. Clinical PathoScope: rapid alignment and filtration for 
accurate pathogen identification ni clinical samples using unassembled dsequencing data.  BMC 
Bioinformatics 2014; 15:262. Doi: 10.1186/1471-2105-15-262. 
23. Francis, O., Bendall, M. & Manimaran, S. Pathoscope: Species identification and strain attribution 
with unassembled sequencing data. Genome 2013; 1–10. doi:10.1101/gr.150151.112.Freely 
24. Hong, C. Manimaran S, Shen Y, et al.. PathoScope 2.0: a complete computational framework for 
strain identification in environmental or clinical sequencing samples. Microbiome 2014:2,:33. 
25. Hong, C., Manimaran, S. & Johnson, WE. PathoQC: Computationally Efficient Read Preprocessing and 
Quality Control for High-Throughput Sequencing Data Sets. Cancer Informatics 2014; 13: 167–76. 
26. http://greengenes.secondgenome.com/ 
27. Solaiappan Manimaran et al. PathoStat: PathoStat Statistical Microbiome Analysis Package. 2017. 
https://rdrr.io/bioc/PathoStat 
28. Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq 
data with DESeq2. Genome Biol. 2014;15, 550. 
29. Castro-Nallar, E., Shen Y, Freishtat RJ, et al. Integrating microbial and host transcriptomics to 
characterize asthma-associated microbial communities. BMC Med. Genomics 2015; 8:50 (2015). 
30. Botero, L. E. et al. Respiratory tract clinical sample selection for microbiota analysis in patients with 
pulmonary tuberculosis. Microbiome 2014;2:29. 
31. Consortium, T. H. M. P. Structure, function and diversity of the healthy human microbiome. Nature 
2012;486: 207. 
 
 

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15 Appendix 
 
Schedule of Events for Overall Study 
 
Months 
1-3 
Months 
4-8 
Months 
9-11 
Months 
11-12 
Months 
13-15 
Protocol development and IRB application, Clinical 
trials.gov registration, IND submission 
X 
 
 
 
 
Patient enrollment 
 
XX 
 
 
 
Stool sample collection 
 
X 
 
 
 
Fecal Microbiota Transplant Capsules Administration 
 
X 
 
 
 
Symptom Survey  
 
 
XX 
 
 
Complete 60 day follow-up 
 
 
X 
 
 
Complete six-month follow-up  
 
 
 
X 
X 
Stool sample processing and analysis 
 
 
X 
 
 
Manuscript Preparation 
 
 
 
XX 
 
1. Study design schematic 
2. Telephone surveys – baseline and 60-day follow-up 
3. Inclusion/exclusion criteria screening form 
4. Case Report Forms 
 
Schedule of Events for Individual Subject 
 
Day 
Stool sample positive for C. difficile 
0 
Patient enrollment 
0-3 
Completion of oral vancomycin therapy 
10-14 
Baseline survey within 1 week of completing 
vancomycin therapy 
10-21 
 Fecal Microbiota Transplant Capsules 
Administration for treatment group 
14-21 
Stool sample 30 days after completing vancomycin 
40-45 
Complete 60 day follow-up 
70-90 
Complete six-month follow-up  
~180