Serial Fecal Microbiota Transplant (FMT) Plus Fidaxomicin in the Treatment of Severe or Fulminant Clostridium Difficile Infection, With Detailed Characterization in Microbiota, Metabolomics and Host Immune Response
Phase: PHASE2
Conditions: Clostridium Difficile
Sponsor: University of Alberta

 
 
Serial fecal microbiota transplant plus fidaxomicin in the treatment of severe or fulminant 
Clostridium difficile infection (CDI) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Principal Investigator:            Dina Kao, MD 
                                                 University of Alberta (U of A) 
                                                 Edmonton, Alberta 
 
 
Co- Investigators: 
 
 
 
 
Lindsey Russell, MD, U of A  
 
 
 
 
Karen Wong, MD, U of A 
 
 
 
 
Christine Lee, MD, U of British Columbia 
 
 
 
 
Thomas Louie, MD, U of Calgary 
 
 
 
 
Haili Wang, MD, U of A 
                                                Wendy Sligl, MD U of A 
                                                Ryan Snelgrove MD U of A 
 
 
 
 
Lynora Saxinger, MD, U of A 
                                                Tanya Monaghan, MD, U of Nottingham 
 
 
 
 
Gane Wong, MD, U of A 
 
 
 
 
Jens Walter, PhD, U of A 
 
 
 
 
Julian Marchesi, PhD, Imperial College London 
 
 
 
 
 
 
 
 
 
 
Study product:                        Human biotherapy 
Protocol#:  
 
 
Pro00081229 
Version:  
 
 
April 21 , 2020 
 

 
2
Table of Contents 
 
 List of acronyms and abbreviations 
3 
Study Synopsis 
4 
1.0 Introduction 
6 
2.0 Objectives 
7 
3.0 Methods 
7 
3.1 Study Design  
7 
3.2 Study Setting, Patient Recruitment and Sample Size 
8 
3.3 Study Population 
8 
3.31 Inclusion Criteria 
8 
3.32 Exclusion Criteria 
8 
3.4 Stool donor selection and testing 
8 
3.41 Donor stool collection and processing 
9 
3.5 Intervention and follow up 
9 
3.6 Subject Withdraw or Termination 
10 
3.7 Outcome Measures  
10 
3.71 Primary Outcomes 
10 
3.72 Secondary Outcomes 
10 
3.73 Exploratory Outcomes 
         a) Metagenomics of Microbiota in Stool samples 
         b) Stool microbiota and metagenomics analysis 
         c) Urine and Stool (bile acids) Metabolomics analysis 
         d) Blood immunological profiling 
10 
10 
10 
11 
12 
4.0 Proposed Analysis 
12 
5.0 Data Safety and Monitoring 
6.0 Significance 
12 
12 
Figure 1.0 
Figure 2.0 
Figure 3.0 
13 
14 
15 
Figure 4.0 
Appendix A Donor Screening 
Appendix B Donor Sample Collection                                                                                                      
Appendix C Donor Questionnaire                                                                                                 
Appendix D Donor Consent                                                                                                                       
References                                                                                                                                            
16 
17 
19 
21 
24 
25 
 
 
 
 
 
 
 
 
 
 

 
3
 
List of Abbreviations and Acronyms 
 
BA                    bile acid 
BID 
 
 twice a day  
BSC                  biological safety cabinet 
C&S                 culture and sensitivity 
CDI                  Clostridium difficile infection 
CRP                  C reactive protein 
FMT                 fecal microbiota transplantation 
HAV                 hepatitis A virus 
HBV                 hepatitis B virus 
HCV                 hepatitis C virus 
Hgb                  hemoglobin 
HIV                  human immunodeficiency virus 
HTLV               human T lymphotrophic virus 
IBD                   inflammatory bowel disease 
IBS                   irritable bowel syndrome 
LFT                   liver function test 
O&P                 ovum and parasite 
PLT                   platelet 
RIW 
 
Resource Intensity Weights 
SCDI  
Severe Clostridium difficile infection 
SOP                 Standard Operating Procedure 
UAH                University of Alberta Hospital 
WBC                white blood cell 
 
 
 
 

 
 
4
Study Synopsis 
 
Title 
Serial fecal microbiota transplant (FMT) plus 
fidaxomicin in the treatment of severe or 
fulminant Clostridium difficile infection (CDI) 
Investigational Product and Indication for 
Use 
FMT and fidaxomicin for severe or fulminant 
CDI 
Study Purpose 
To determine the safety and efficacy of serial 
FMT and fidaxomicin in the treatment of 
severe or fulminant CDI  
Study Design 
Prospective, open label, multi-center study  
Study Population 
Patients with severe or fulminant CDI  
Sample Size 
30 
Dosing 
Each cycle consists of: 
1. Serial FMT:  720 cc (1st treatment), 
360 cc (2nd Rx), and 180 cc (3rd Rx) of 
fecal slurry  
2. Fidaxomicin: 200 mg oral BID x7-10 
days 
Masking 
None 
Study Outcomes 
Primary outcomes 
      CDI resolution, defined as < 3 unformed 
bowel movements/ 24 hrs or a return to 
baseline bowel habit, 2 weeks after the last 
FMT treatment 
 
Secondary outcomes 
1. Sustained CDI resolution, defined as a 
lack of CDI recurrence 8 weeks after 
the last FMT treatment 
2. All serious adverse events up to and 
including week 8. A serious adverse event 
is any event which results in any of the 
following after the final FMT: 
a. Death 
b. Colonic perforation 
c. Proven infection related to 
FMT 
d. Subsequent hospitalization due 
to CDI within study period 
3. Need for colectomy 
 
 

 
 
5
Exploratory outcomes 
1. All direct medical costs for CDI 
treatment 
2. Changes in microbial composition, 
metabolomics and host immune 
response after treatment 
Inclusion Criteria  
 
1. Age > 18 years with severe1 or 
fulminant2 CDI, without an adequate 
response to metronidazole IV 500 mg 
q8H and vancomycin 500 mg PO q6h 
for at least 2 days or after a single 
Fecal Microbiota Transplant (FMT). 
An adequate response is defined as a 
decrease in stool frequency or 
inflammatory markers (WBC or C 
reactive protein) by at least 10% over 
48 hours 
2. Those with ability to provide informed 
consent or an alternative decision 
maker providing consent 
1- Severe CDI defined as WBC > 15,000 
cells/mm3 or serum creatinine level >1.5mg/dL or 
1.5x premorbid level 
2- Fulminant CDI defined as defined as having any 
of the following attributable to CDI: Hypotension 
or shock, ileus, megacolon. An abdominal CT 
scan should be strongly considered to rule out 
perforation 
Exclusion Criteria 
1.  Those with bowel perforation 
2. Those taking chemotherapy or 
radiation treatment with absolute 
neutrophil count of < 1000 cells/mm3 
3. Those with colostomy or ileostomy, or 
colonic strictures 
4. Those with subtotal colectomy or 
planning to have a colectomy  
5. Those with significant ileus 
Study Duration 
 Approximately 20 weeks 
Study Sponsor 
University of Alberta 
Edmonton, Alberta 
 
 
 

 
 
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1.0 Introduction 
 
Clostridium difficile infection (CDI) is the leading cause of hospital-acquired infectious diarrhea, 
representing a significant healthcare burden worldwide. Severe CDI is associated with a high 
morbidity and mortality rate despite medical and/or surgical intervention(1). Around 8% develop  
fulminant CDI resulting in toxic megacolon, multi-organ failure and death (2). In a meta-analysis 
of primary studies published in 2002 and 2007, the annual cost of CDI in the United States 
adjusted to 2012 dollars was estimated at $1.5 billion (3). Recent surveillance data from Alberta 
Health Services (AHS) found that Edmonton has the highest rate of healthcare associated CDI 
and the highest CDI mortality in the province of Alberta (Figure 1).  
 
Mild and moderate cases of CDI respond very well to vancomycin, which has recently become 
the first line therapy(4). Unfortunately, vancomycin is not a narrow spectrum antibiotic, and 
could also promote the development of vancomycin resistant Enterococcus (VRE) (5). Another 
antibiotic fidaxomicin, a narrow spectrum antibiotic, is non inferior to vancomycin in clinical 
efficacy in the treatment of mild to moderate infection and is shown to significantly reduce the 
rate of CDI recurrence (6); however, the high cost prohibits its use as a first line therapy. 
Furthermore, it has not been used in treating patients with severe or fulminant CDI. Other 
therapies including monoclonal antibodies against C Diff Toxin B, bezlotoxumab, have been 
shown to reduce recurrence in CDI but the mechanism of action and long term effects are still 
not understood (7). Tigecycline is another broad spectrum antibiotic of the glycylcycline class 
that has been shown to significantly reduce the toxin production in hypervirulent strains of C 
difficile, however it can lead to further disruption to the gut microbiome (7). Overall these newer 
therapies are expensive with little knowledge of the mechanism of action in the treatment of CDI 
(7).  
 
Severe CDI is defined as white blood cell count > 15,000 cells/mm3 and fulminant CDI is 
defined as having any of the following attributable to CDI:  hypotension or shock, ileus, and 
toxic megacolon (4). Severe and fulminant CDI require combined metronidazole with 
vancomycin; surgical intervention is indicated when medical therapy fails (4). 
 
Fecal microbiota transplantation (FMT), which restores intestinal dysbiosis, is highly efficacious 
in treating mild to moderate recurrent CDI. There is also emerging evidence that FMT may be 
effective in severe or fulminant CDI, a situation where the only alternative is colectomy (8). 
FMT can be administered by difference routes, including enema, colonoscopy and oral capsules 
(9).  However, Health Canada does not allow oral capsule to be administered outside the context 
of a clinical trial. Although few studies have described the efficacy of FMT in severe and 
fulminant CDI, it appeared efficacious and safe. For example, our group has successfully treated 
6 patients with fulminant CDI with serial FMT plus vancomycin over the past year (Figure 2.0). 
These patients had avoided colectomy as a result. In addition, Fischer and colleagues 
demonstrated 17/19 (91%) clinical cure rate in these critically ill patients using combined serial 
FMT and vancomycin (10). In this protocol, Fischer described using colonoscopy-delivered 
FMT to treat fulminant CDI after failing at least 5 days of combined metronidazole and 
vancomycin. At the time of colonoscopy to deliver FMT, if pseudomembranes were noted, 
vancomycin was restarted for at least 5 days before the second colonoscopic FMT was 
administered. If pseudomembranes persisted, vancomycin was again given for 5 more days 

 
 
7
before the third FMT was administered, and this cycle of FMT and vancomycin continued until 
pseudomembranes resolved. However, there are questions and uncertainties with this approach. 
First, vancomycin is not a narrow spectrum antibiotic, and it seems counterintuitive to give FMT 
and vancomycin at the same time. Second, the presence of pseudomembranes cannot be used for 
daily clinical monitoring, as it requires an invasive test. Furthermore, it remains unknown how 
many FMT each patient requires, what the most convenient or safest way to administer the FMT 
is, what the best strategies for monitoring is, or what the optimal frequency of FMT is. The 
success of Fischer’s protocol thus also highlights the gaps in knowledge in mechanisms of action 
for FMT in severe or fulminant CDI.   
 
In this prospective, open-label, multi-center study, we aim to determine the efficacy and safety of 
using a combined serial FMT by enema plus fidaxomicin to treat patients who have severe or 
fulminant CDI not responding to maximal medical therapy.  Our hypothesis is the combination 
of FMT plus fidaxomicin can reduce the number of FMT required and hospital length of stay 
compared to FMT plus vancomycin from a historical cohort. 
 
2.0 Objectives 
 
Primary Objectives: To determine the clinical efficacy of combined serial FMT and Fidaxomicin 
in severe or fulminant CDI 2 weeks after the last FMT 
 
Secondary Objectives:  
a) To determine sustained CDI resolution, defined as a lack of CDI recurrence 8 weeks after 
the last FMT treatment 
b) To describe serious adverse events up to and including week 8 after the final FMT. A 
serious adverse event is any event which results in any of the following: 
i) Death 
ii) Colonic perforation 
iii) Proven infection related to FMT 
iv) Subsequent hospitalization due to CDI within study period 
c) To determine colectomy rate in this patient population 
 
 
Exploratory Objectives:  
a) All direct Medical costs for CDI treatment 
b) Changes in microbial composition, metabolomics and host immune response after 
combined serial FMT plus fidaxomicin 
 
3.0 Methods 
 
3.1 Study Design 
 
Prospective, open-label, multi-center study. Sites include: 
 
University of Alberta Hospital, Edmonton AB 

 
 
8
 
Foothills Medical Centre, Calgary AB 
 
Royal Jubilee Hospital, Victoria BC 
3.2 Study Setting, Patient Recruitment and Sample Size 
In-patients with severe or fulminant CDI who fulfill study criteria will be screened for potential 
enrollment into this study. We aim to recruit 30 patients over 2 years. As this is a pilot study with 
a unique patient population, the sample size is based on estimate of patient volume seen 
previously at all participating sites combined.  
 
3.3 Study Population 
 
Patients admitted to hospital who fulfill the inclusion/exclusion criteria will be considered.  
 
 
3.31 Inclusion Criteria 
 
 Age > 18 years with severe1 or fulminant2 CDI, without an adequate response 
to metronidazole IV 500 mg q8H and vancomycin 500 mg PO q6h for at least 
2 days or after Fecal Microbiota Transplant (FMT). An adequate response is 
defined as a decrease in stool frequency or inflammatory markers (WBC or C 
reactive protein) by 10% over 48 hours 
 Those with ability to provide informed consent or an alternative decision 
maker providing consent 
1- Severe CDI defined as WBC > 15,000 cells/mm3 or serum creatinine level >1.5mg/dL or 1.5x 
premorbid level 
2- Fulminant CDI defined as defined as having any of the following attributable to CDI: 
Hypotension or shock, ileus, megacolon (4). An abdominal CT scan should be strongly considered 
to rule out perforation 
 
 
3.32 Exclusion Criteria 
 Those with bowel perforation 
 Those taking chemotherapy or radiation treatment with absolute neutrophil 
count of < 1000 cells/mm3 
 Those with colostomy or ileostomy or colonic strictures 
 Those with subtotal colectomy or planning to have a colectomy 
 Those with significant ileus 
 
3.4 Stool donor selection and testing 
 
Five universal stool donors registered with the Edmonton FMT program will provide the starting 
material, which is raw stool for patients recruited in Edmonton. These donors do not work in 
healthcare fields, and are known personally to the investigators. They are instructed not to provide a 
stool collection if they have any symptoms suggestive of an infection as per donor collection SOP 
and checklist in Appendix B. 

 
 
9
They are rescreened every 2 months. The other sites included in the study have established stool 
donors in their facility and will provide their own FMT using identical protocol for donor 
screening and FMT manufacturing.   
 
3.41 Donor stool collection and processing 
 
Each donor will provide a fresh stool specimen, weighing approximately 100g, as per donor stool 
collection SOP (Appendix B). The stool specimen is stored at 4-80C after collection and brought 
into the lab within 12 hours of collection. Donor stools should have the appearance of type 2-5 
on the Bristol Stool Scale, and be free of blood, mucus or urine contamination. No pooling of 
stools will occur. Once received by the lab, it will be processed as per protocol for enema. Each 
donation of 100 g of stool will produce approximately 360 cc of fecal slurry, which will be 
stored frozen at -800C as per our manufacturing protocol. 
 
See Appendix A for donor screening SOP, Appendix C for donor questionnaire and Appendix D 
for donor consent. 
 
3.5 Intervention and follow up 
 
Patients who fulfill inclusion and exclusion criteria will be screened. Once consent has been 
obtained, metronidazole and vancomycin will be discontinued and 2L of Golytely will be 
administered the night prior to FMT. Each participant will undergo a large volume enema of 
720cc delivered FMT on day 1, consisting of 200g of donor stool followed by two more days of 
small volume FMT enema on day 2 (360 cc) and day 3 (180 cc). This constitutes as cycle 1. In 
the event that a colonoscopy is indicated to rule out other pathology, then the scheduled enema 
can be delivered at the time of colonoscopy. On day 1, the patient will also start a 7-10 day 
course of fidaxomicin, as long as no clinical deterioration or complication such as bowel 
perforation has occurred. Following the first cycle of FMT treatment and fidaxomicin, careful 
clinical monitoring including vitals, requirement for vasopressors (if applicable), abdominal pain 
and distension, stool frequency and inflammatory markers including white blood cell (WBC) and 
C reactive protein (CRP) will be performed daily (Figure 3.0). If the patient has improvement in 
clinical parameters and inflammatory markers, and has reached a plateau without further changes 
over 24-48 hours after the first cycle, a second cycle of enema delivery of FMT will be 
administered. The FMT and fidaxomicin combined cycles will continue until clinical resolution 
of diarrhea and/or return of inflammatory markers to baseline prior to CDI. At that point, a final 
FMT 180cc enema will be administered. The maximal number of cycles is 4.  At any time when 
a patient’s conditions worsens, the study team will refer patient for surgery. 
 
Each patient will receive FMT from a single donor with multiple collections obtained over time. 
  
Baseline characteristics including age, sex, duration and response to CDI therapy, medical 
history (including inflammatory bowel disease, irritable bowel syndrome, baseline bowel habit, 
bowel resection, chemotherapy or radiation, number of CDI in the previous 12 months) and 
medication use will be collected. Blood work including CBC, electrolytes, creatinine, ALT, 
ALP, albumin, CRP, INR, HIV, and viral hepatitis serology will be drawn at screening visit 
(Figure 4.0). In addition, baseline collection of blood, and stool samples will be collected and 

 
 
10 
biobanked. Repeat stool and blood samples will be collected after each cycle of treatment, and 
again at week 1, 2, 4 and 8 after the final FMT.  
 
Clinical status as well as monitoring for adverse events will be assessed daily up to 2 weeks after 
the final FMT while a patient is hospitalized, then weekly to week 8 after the final FMT. Patients 
are instructed to call as soon as there is recurrence of persistent diarrhea, and an unscheduled 
visit will take place if needed.  Given COVID-19 pandemic, study follow-up may be 
conducted by phone if participants are unable to or do not want to attend study visits or if the 
visit is deemed non-essential by the principal investigator (PI).  Due to this some of these 
sample collections may not be possible. 
 
3.6 Subject Withdraw or Termination 
 
Subjects will be discontinued from the trial in the following situations: 
 Withdrawal of consent. The subject is free to withdraw from the trial without 
prejudice to further medical care 
 Need for urgent colectomy 
3.7 Outcome Measures  
 
 
3.71 Primary Outcome 
CDI resolution, defined as < 3 unformed bowel movements/ 24 hrs or a return to baseline bowel 
habit, 2 weeks after the last FMT treatment 
 
 
 
 
3.72 Secondary Outcomes 
 
a)  Sustained CDI resolution, defined as a lack of CDI recurrence 8 weeks after the 
last FMT treatment 
Clinical status and biological samples will be taken to ensure that CDI has not recurred at 8 
weeks 
 
 
b) Adverse events 
All serious adverse events will be recorded up to and including week 8 after the final FMT. A 
serious adverse event is any event which results in any of the following: 
 Death 
 Colonic perforation 
 Proven infections related to FMT 
 Subsequent hospitalization due to CDI within study period 
 
c) Proportion of patients requiring colectomy 
 
 
 
3.73 Exploratory Outcomes 
 
 
 
a) Costs for CDI Treatment  
Direct Medical costs for CDI treatment will be obtained from administrative database to include 
length of hospital stay, procedures and tests performed, ICU stay and medication use. 

 
 
11 
 
 
b) Stool microbial composition analysis by metagenomics  
 
Stool samples will be subjected to physical disruption using a bead-beating kit and microbial 
DNA extracted using the Qiagen QIAamp DNA stool kit and stored at -20oC. DNA will be used 
as input for the Illumina Nextera® XT DNA Sample Preparation Kit to construct indexed paired-
end DNA libraries.  The pooled and indexed library set will be denatured and sequenced on an 
Illumina MiSeq.  
 
 
 
Whole-genome shotgun sequencing will be performed and taxonomic classification of reads 
from each library will be conducted with Kraken. The database used consisted of all bacteria, 
archaea, viruses, fungi, and protozoa full-length genomes from NCBI RefSeq, the human 
genome assembly GRCh38, and reference bacterial assemblies from the Human Microbiome 
Project. Read assignments were filtered with Kraken-filter using a threshold of 10%.     
 
 
 c) Stool Metabolomics analysis  
 
 
 
Stool samples will be lyophilized for 24 hours using a VirTis Benchtop BTP 8ZL freeze dryer 
(BPS, UK).  Dried samples will be weighed, and bile acids extracted using a 2:1:1 (vol) mixture 
of water, acetonitrile and 2-propanol in a Biospec bead beater with 1.0 mm Zirconia beads. 
Following centrifugation (16,000 x g, 20 minutes) the supernatant will be filtered using 0.45 μm 
microcentrifuge filters (Costar, Corning). Quality control (QC) samples will be prepared using 
equal parts of the fecal filtrates. QC samples will be used as an assay performance monitor, and 
as a proxy to remove features with high variation. QC samples will also be spiked with mixtures 
of bile acid standards (55 bile acid standards including 36 non-conjugated, 12 conjugated with 
taurine, seven conjugated with glycine (Steraloids, Newport, RI, USA)) and will be analyzed 
along with the stool samples to determine the chromatographic retention times of bile acids and 
to aid in metabolite identification.  
  
Bile acid analysis of fecal extracts will be performed using ACQUITY UPLC (Waters Ltd, 
Elstree, UK) coupled to a Xevo G2 Q-ToF mass spectrometer equipped with an electrospray 
ionization source operating in negative ion mode (ESI-), using the method described by Sarafian 
and colleagues. Waters raw data files will be converted to NetCDF format and data will be 
extracted using XCMS (v1.50) package with R (v3.1.1) software. Probabilistic quotient 
normalisation6 will be used to correct for dilution effects and chromatographic features with 
coefficient of variation higher than 30% in the QC samples will be excluded from further 
analysis. The relative intensities of the features will be corrected to the dry weight of the fecal 
samples.  
 
 
 d) Blood Immunological profiling 
 
Immunophenotypic analysis of peripheral immune cell populations through peripheral blood 
mononuclear cells (PBMCS) that will be isolated from buffy coats and stained with optimized 
combinations of fluorochrome-conjugated mAbs. Frequencies of T- and B-regulatory cells, as 
well as Th1, Th2 and Th17 populations will be ascertained using established flow cytometric 
antibody panels and isotype controls. Relevant signature cytokines and transcription factors will 

 
 
12 
also be included in an already established 9-colour FACS panel. We will also examine effector 
and memory B and T cell populations, in addition to plasma and dendritic cell populations plus 
their relevant signature cytokines and transcription factors (all pending sufficient availability of 
cells). 
 
 
 
4.0 Proposed Analysis 
 
The primary outcome, defined as CDI resolution at week 2, will be analyzed using the per 
protocol approach for all patients. Adverse events will be tabulated in each predetermined 
outcome.  
To measure the direct medical costs, the utilization of different health care services, including 
pharmaceuticals, physician services, Emergency Department visits, inpatient hospitalizations, 
and Intensive Care Unit time will be summed for each individual patient. In order to attempt to 
attach a dollar value to the resource utilization, CIHI’s Resource Intensity Weights (RIW) will be 
used. The RIW methodology derives estimated resource utilization compared to an ‘average 
typical hospital stay’, the cost of which has been calculated for the Edmonton Zone in each fiscal 
year from 2010/11 through 2014/15. RIW values are calculated based on the patient’s 
Comprehensive Ambulatory Classification System (CACS) group, age and based on the patient’s 
Case Mix Group (CMG) code, age, and comorbidity level.   
 
5.0 Data Safety and Monitoring 
 
An independent data safety monitoring board (DSMB) will be appointed and following the 
project till completion. The members of the DSMB are not involved in this trial, and will not 
have potential patients who might be enrolled in this study.  
 
6.0 Significance  
 
This will be the first study examining the clinical efficacy of combined FMT and fidaxomicin in 
the treatment of severe or fulminant CDI refractory to medical therapy. Longitudinal clinical 
samples collected during this study will undergo detailed analysis to gain better understanding of 
the complex interaction between microbiome and human host in this context. Results from this 
study have the potential to stimulate further studies aimed at elucidating mechanisms of action of 
FMT in order to develop rational microbiome based therapy. The knowledge gained during this 
trial will help us optimize treatment algorithm for patients suffering from treatment refractory 
CDI, and ultimately improve patient experiences and outcomes while simultaneously decrease 
health care costs.  
 
 
 
 
 
 

 
 
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14 
Figure 2.0- Pilot data: 
Over the past year, we have successfully treated 6 patients with fulminant CDI with serial FMT 
plus vancomycin at the University of Alberta. These patients had avoided colectomy as a result. 
Below are endoscopic images of before (image on the left, showing severe pseudomembranous 
colitis) and after serial FMT treatment (image on the right, showing normal colon). 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
15 
Figure 3.0 
Sequential fecal microbiota transplant protocol proposed.  
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
16 
Figure 4.0 
 
Proposed sampling for each patient given the protocol seen in Figure 3.0 
 
 
 

17 
 
Appendix A: Universal stool donor testing/screening SOP 
 
Universal stool donors will provide the raw material for FMT. They are screened at baseline and 
then every 2 months at a minimum for the duration of the trial. They underwent initial detailed 
history and physical exam, and had been screened with a donor questionnaire, which did not 
identify any high risk behaviors. They tested negative for all the following potential infections as 
listed below. They have provided stools for over 300 patients since 2012, and none of the 
recipients have developed any known infectious complications. All donors are personally known 
to the investigators. It is simply not practical or cost effective to keep testing them each and 
every time when there is a scheduled FMT. Since there is no consensus on the mandatory 
required tests for stool donor, we have chosen the recommendations published by Khoruts et al 
in the American Journal of Gastroenterology in 2012 as well as the FMT Safety Notice from 
Health Canada dated  July 16,2019 pertains to MDRO testing, while Mar 25, 2020 pertains to 
COVID-19 
These universal donors have been in the same positions for years are unlikely to move away or 
stop donating. We have not encountered a situation when a universal donor could not donate on a 
day when HBT was scheduled to treat patients with recurrent Clostridium difficile infections 
since Oct 2012. In the unlikely event that he or she can no longer donate, a second donor will be 
assigned to a particular patient.  
We will hold donor FMT materials in quarantine until a donor has passed 2 consecutive 
screening tests. 
On initial history, the donors must fulfill the following inclusion and exclusion criteria: 
Donor inclusion criteria: 
1. Able to provide and sign informed consent (Appendix D). 
2. Able to complete and sign the stool donor questionnaire (Appendix C). 
3. Able to adhere to fecal transplantation stool collection standard operating procedure 
(appendix B).   
 
Donor exclusion criteria: 
1. History of any type of active cancer or autoimmune disease (eg multiple sclerosis, 
connective tissue disease), metabolic syndrome, chronic pain syndrome, and atopic 
diseases. 
2. History of risk factors for acquisition of HIV, syphilis, Hepatitis B, Hepatitis C, prion or 
any neurological disease as determined by the donor questionnaire (Appendix 2).  
3. Gastrointestinal comorbidities, e.g., inflammatory bowel disease, irritable bowel 
syndrome, chronic constipation or diarrhea, gastrointestinal malignancy or known 
polyposis. 
4. Tattoo or body piercing within 6 months of stool donation. 
5. Incarceration or history of incarceration. 
6. Receipt of blood transfusion from a country other than Canada in preceding 6 
months. 
7. Antibiotic use, systemic immunosuppressive or biological agents, systemic 
antineoplastic agents and exogenous glucocorticoids in the preceding 3 months prior 
to stool donation. 
8. Receipt of any type of live vaccine within 3 months prior to stool donation. 

18 
 
9. Ingestion of nut or shell fish 3 days preceding donation if the recipient has known 
allergies to these food. 
10. Any current or previous medical or psychosocial condition or behaviors which in the 
opinion of the investigator may pose risk to the recipients or the donor. 
11. Travel to areas of the world where diarrheal illnesses or BSE/TSE are endemic (within 
6 months of stool donation). 
12. High risk of multi-drug resistant organisms, including healthcare workers, recent 
hospitalization and medical tourism. 
13. Travel history outside of Canada within 14 days of screening  
14. Exposure to individuals diagnosed with or suspected to have COVID-19 within 14 
days of screening 
15.  History of a fever, cough, sore throat or any other signs of respiratory or other 
infectious illness in the past 14 days 
16. Diagnosis of COVID-19 within the past 3 months 
 
Initial blood work and stool testing, which will be repeated at a minimum of every 2 months..  
However, to minimize COVID-19 transmission risk, the quarantine period is at least 5 weeks as 
per Health Canada directions. 
 
Blood:   
 
CBC, electrolytes, creatinine, AST,  ALT and ALP 
 
Human Immunodeficiency virus (HIV) 1/2  
 
hepatitis A IgM Ab 
 
hepatitis B: HBVsAg,  HBVsAb, HBVc Ab (IgM and IgG) 
 
hepatitis C antibody  
 
RPR  (syphilis) 
 
human T- lymphotrophic virus (HTLV) I/II 
Nasal Swab: 
 
MRSA 
Stool:  
 
 
enteric pathogens: Salmonella, Shigella, E.coli O157 H7, Yersinia, Campylobacter (see 
Appendices 3-8 for lab SOP) 
 
C. difficile toxin by EIA 
 
ova and parasites  
 
VRE 
 
CRE 
 
ESBL  
 
 
 
 
 
 
 

19 
 
 
 
 
Appendix B:  Stool collection instruction for donors (to be filled out with each stool collection) 
 
 
Stool collection for human biotherapy/ fecal microbiota 
transplantation  
 
1. Do not take any medications (laxatives, anti-diarrheal drugs, mineral oil, bismuth, 
magnesium, or kaolin) for at least 3 days prior to and during the specimen collection. If 
you have been taking antibiotics in the last 90 days, you should not be collecting stool. 
 
2. Make sure you have not had fever, vomiting, diarrhea or other symptoms of infection 
within the last 30 days. Inform the investigator if you have any of these symptoms, as 
you should not donate stool. Please hand in this form with your stool collection with 
each donation. 
 
Day of FMT Procedure/donor Checklist 
Donor Name: 
Date: 
 
Yes 
No 
Do you feel well and healthy today? 
Have you had any fever, vomiting, diarrhea, cough, sore throat or other 
symptoms of infection within the last 30 days? 
If yes, specify:   
 
Antibiotics within past 90 days?  
Any medications (laxatives, anti-diarrheal drugs, mineral oil, bismuth, or 
magnesium, kaolin) in the past 3 days 
Since your last donation have you started working as a healthcare worker 
or worked in a long term care facility? 
Since your last donation have you been hospitalized or been treated in a 
long term care facility? 
Since your last donation, have you had medical treatment requiring 
frequent, regular attendance at outpatient medical or surgical clinics?  If 
yes, please list dates and reason: 
 

20 
 
Since your last donation, have you engaged in medical tourism i.e. 
travelled outside Canada for medical treatment? 
Since your last donation, have you had contact with anyone who has 
been diagnosed or suspected to have COVID-19? 
Since your last donation, have you travelled outside of Canada? 
Since your last donation, have you been diagnosed with COVID-19? 
 
 
3. Collect the specimen into the commode specimen collection system provided (Fisher 
07544208) at home. This system fits under the toilette seat in center of rear of bowl. Do 
not mix with urine, paper or water. Once stool has been collected, snap on the lid 
provided to seal the container. Make sure you collect at least 100 g of stool, which is 
about the size of 4 Timbits donuts.  
 
4. Place the container in the plastic bag supplied by the designate. Seal the bag tightly. 
 
5. Mark date and time of collection and your initials on the lid. 
 
6. Wash hands with soap and water when finished. 
 
7. Maintain stool sample at 20C to 8oC until specimen can be delivered to University of 
Alberta Hospital. Keep an ice pack in the bag with stool sample to keep it cold during 
delivery. 
 
8. Please return specimen within 12 hours of collection and give to the hospital staff 
designate.  
 
 
 
 
 
 
 
 
 
 

21 
 
 
 
 
 
Appendix C: Donor Questionnaire for human biotherapy/fecal microbiota transplantation 
 
Yes 
No 
Are You 
1. Feeling healthy and well today? 
 
 
2. Have you experienced a fever, cough, sore throat or any other sign 
of respiratory or other infectious illness in past 14 day? 
 
 
3. Currently taking an antibiotic? 
 
 
4. Currently taking any other medication for an infection? 
 
 
5. Currently taking any immunosuppressant medication by mouth or 
injection? 
 
 
6. Have you taken any antibiotics in the past 6 months? 
 
 
7. Have you had any fevers, vomiting, diarrhea or other symptoms of 
infection within the past 4 weeks? 
 
 
Do you have 
8. History of chronic diarrhea persisting > 10 days? 
 
 
9. History of blood in stool not related to hemorrhoid? 
 
 
10. History of change in bowel habit, alternating from constipation to 
diarrhea? 
 
 
11. Any type of active cancer that is not cured? 
 
 
12. Any active autoimmune diseases? 
 
 
In the past 12 weeks have you 
13. Have you been in contact with anyone who has been diagnosed or 
suspected to have COVID-19? 
 
 
14.  Have you travelled outside Canada? 
 
 
15. Had any vaccinations?  If yes, please indicate which one(s) 
 
 
16. Had contact with someone who had a smallpox vaccination? 
 
 
17. taken antibiotics, systemic immunosuppressive or biological agents, 
systemic antineoplastic agents and exogenous glucocorticoids? 
If you have, you should not be a stool donor. 
 
 
In the past 16 weeks have you 
18. Lived with a person who has hepatitis A? 
 
 
19. If yes, have you received vaccine against hepatitis A? 
 
 
In the past 12 months have you 
20. Had a blood transfusion? 
 
 

22 
 
21. Had a transplant such as organ, tissue or bone marrow? 
 
 
22. Had a graft such as bone or skin? 
 
 
23. Come into contact with someone else’s blood? 
 
 
24. Had an accidental needle-stick? 
 
 
25. Had sexual contact with anyone who has HIV/AIDS or has had a 
positive test for the HIV/AIDS virus? 
 
 
26. Had sexual contact with a prostitute or anyone else who takes 
money or drugs or other payment for sex? 
 
 
27. Had sexual contact with anyone who has ever used needles to take 
drugs or steroids, or anything not prescribed by their doctor? 
 
 
28. Had sexual contact with anyone who has hemophilia or has used 
clotting factor concentrates? 
 
 
29. Female donors: Had sexual contact with a male who has ever had 
sexual contact with another male? (Males: check “I am male”) 
 
 
  
I am male 
30. Had sexual contact with a person who has hepatitis? 
 
 
31. Had a tattoo? 
 
 
32. Had ear or body piercing? 
 
 
33. Had or been treated for syphilis, gonorrhea or Chlamydia? 
 
 
34. Been in juvenile detention, lockup, jail, or prison for more than 72 
hours? 
 
 
In the past three years have you 
35. Been outside the United States or Canada?  If yes, list the places(s) 
 
 
From 1980 to the present, have you 
36. Receive a blood transfusion in the United Kingdom or France? 
(Review list of countries in UK.) 
 
 
From 1977 to the present, have you 
37. Received money, drugs, or other payment for sex? 
 
 
38. Male donors: had sexual contact with another male, even once? 
(Females: check “I am female”) 
 
 
  
I am female 
Have you EVER 
39. Had a positive test for the HIV/AIDS virus? 
 
 
40. Used needles to take drugs, steroids, or anything not prescribed by 
your doctor? 
 
 
41. Used clotting factor concentrates? 
 
 
42. Had hepatitis? 
 
 
43. Had malaria? 
 
 
44. Had Chagas’ disease? 
 
 

23 
 
45. Had babesiosis? 
 
 
46. Received a dura mater (or brain covering) graft? 
 
 
47. Had sexual contact with anyone who was born in or lived in Africa? 
 
 
48. Have any of your relatives had Creutzfeldt-Jakob disease? 
 
 
49. Have you ever had any of the following gastrointestinal diseases or 
problems: 
Irritable bowel syndrome 
       Crohn’s disease 
       Ulcerative colitis 
              Gastrointestinal cancers 
Celiac disease 
 
 
50. Do you have any autoimmune diseases, for example rheumatoid 
arthritis, multiple sclerosis or lupus?  If yes, please list: 
 
 
51. Do you have any neurological diseases, for example Parkinsons, 
autism, ALS?  If yes, please list: 
 
 
52. Are you a healthcare worker or do you work in long term care 
facilities? 
 
 
53. Have you ever engaged in medical tourism?  That is, travelled 
outside of Canada for medical treatment? 
 
 
54. Have you ever had medical treatment requiring frequent, regular 
attendance at outpatient medical or surgical clinics?  If yes, please 
describe reason and timing: 
 
 
55. Have you ever been hospitalized or been treated in a long term care 
facility?  If yes, please list reason and date: 
 
 
 
This information will remain strictly confidential  
To the best of my knowledge, the above information is accurate and true. 
 
 
Donor’s Signature:                                                                       Date: 

24 
 
Appendix D: CONSENT TO DONATE STOOL for FMT (fecal microbiota transplantation) 
I, _____________________________________consent to undergoing collection of blood and stool for potential  
donation of my stool for FMT now and as requested. I agree to undergoing blood and stool tests, including Complete  
Blood Count, creatinine, sodium, potassium, chloride, alanine transaminase, alkaline phosphatase,  
Hepatitis A, B and C, HIV, HTLV I/II, RPR (Syphillis), MRSA and stool culture and susceptibility for enteric  
ova and parasites, VRE, CRE, ESBL and Clostridium difficile toxin. I have completed the FMT donor questionnaire  
truthfully and to the best of my knowledge.   
 
I understand that I will have to bring in fresh stool sample within 5 hours of collection to UAH laboratory for  
processing when requested.  I will notify Dr. Dina Kao whenever there is a change to my health status.  I will  
refrain from the donation when I experience fever, sore throat and/or diarrhea and any changes to my social  
status which may pose any potential risk to the recipient.  
 
I also agree for my stool samples to undergo culture and molecular testing to determine the type of bacteria  
and products they contain.  This information along with any related research can be submitted for  
presentation and publication.  I understand that no information which discloses my identity will be released or  
published.   
 
I have had the opportunity to ask questions about the process and have had my questions answered to my  
satisfaction.   I declare that I have read this form and understand it.   
 
 
 
Signature of Donor 
                 Print Name of Donor       Date (DD/MMM/YYYY) 
 
 
 
 
Signature of Health Practitioner      Print Name                  Date (DD/MMM/YYYY) 

25 
 
 
 
 
References 
 
 
1. 
Surawicz CM, Brandt LJ, Binion DG, Ananthakrishnan AN, Curry SR, Gilligan PH, et al. 
Guidelines for diagnosis, treatment, and prevention of Clostridium difficile infections. The 
American journal of gastroenterology. 2013;108(4):478-98; quiz 99. 
2. 
Adams SD, Mercer DW. Fulminant Clostridium difficile colitis. Current opinion in 
critical care. 2007;13(4):450-5. 
3. 
Zimlichman E, Henderson D, Tamir O, Franz C, Song P, Yamin CK, et al. Health care-
associated infections: a meta-analysis of costs and financial impact on the US health care system. 
JAMA internal medicine. 2013;173(22):2039-46. 
4. 
McDonald LC, Gerding DN, Johnson S, Bakken JS, Carroll KC, Coffin SE, et al. Clinical 
Practice Guidelines for Clostridium difficile Infection in Adults and Children: 2017 Update by 
the Infectious Diseases Society of America (IDSA) and Society for Healthcare Epidemiology of 
America (SHEA). Clinical infectious diseases : an official publication of the Infectious Diseases 
Society of America. 2018. 
5. 
Pepin J. Vancomycin for the treatment of Clostridium difficile Infection: for whom is this 
expensive bullet really magic? Clinical infectious diseases : an official publication of the 
Infectious Diseases Society of America. 2008;46(10):1493-8. 
6. 
Louie TJ, Miller MA, Mullane KM, Weiss K, Lentnek A, Golan Y, et al. Fidaxomicin 
versus vancomycin for Clostridium difficile infection. The New England journal of medicine. 
2011;364(5):422-31. 
7. 
Goyal H, Perisetti A, Rubayat Rehman R, Singla U. New and emerging therapies in 
treatment of Clostridium difficile infection. European Journal of Gastroenterology & 
Hepatology. 2018;30:589-97. 
8. 
Weingarden AR, Hamilton MJ, Sadowsky MJ, Khoruts A. Resolution of severe 
Clostridium difficile infection following sequential fecal microbiota transplantation. Journal of 
clinical gastroenterology. 2013;47(8):735-7. 
9. 
Kao D, Roach B, Silva M, Beck P, Rioux K, Kaplan GG, et al. Effect of Oral Capsule- vs 
Colonoscopy-Delivered Fecal Microbiota Transplantation on Recurrent Clostridium difficile 
Infection: A Randomized Clinical Trial. Jama. 2017;318(20):1985-93. 
10. 
Fischer M, Sipe BW, Rogers NA, Cook GK, Robb BW, Vuppalanchi R, et al. Faecal 
microbiota transplantation plus selected use of vancomycin for severe-complicated Clostridium 
difficile infection: description of a protocol with high success rate. Alimentary pharmacology & 
therapeutics. 2015;42(4):470-6.