Improvements Through the Use of a Rapid Multiplex PCR Enteric Pathogen Detection Kit in Children With Hematochezia
Phase: NA
Conditions: Diarrhea Bloody
Sponsor: University of Calgary

 
 
 
 
Ethics ID: REB17-1916 
Title: Improvements Through the Use of a Rapid Multiplex PCR Enteric Pathogen Detection Kit in Children with 
Hematochezia 
PI: Dr. Stephen Freedman 
Version 5.0 – May 16, 2018 
Previous REB Approved Version Date: January 12, 2018 
 
 
 
 
Page 1 of 19 
 
 
 
Improvements Through the Use of a Rapid Multiplex PCR 
Enteric Pathogen Detection Kit in Children with Hematochezia 
 
 
Lead: 
Dr. Stephen Freedman  
Associate Professor of Pediatrics 
ACHF Professor in Child Health and WellnessTheme Lead 
Alberta Children’s Hospital Research Institute 
University of Calgary 
Tel: 403-955-7740 
E-mail: stephen.freedman@ahs.ca 
 
Co-Leads: 
Otto Vanderkooi  
Associate Professor 
Pediatric Infectious Diseases, Medical Microbiology and Tropical Medicine 
Program Director, Pediatric Infectious Diseases Residence Training Program  
Alberta Children's Hospital 
University of Calgary  
Tel: 403-955-7407 
 E-mail: ovanderk@ucalgary.ca 
 
Byron Berenger 
Clinical Assistant Professor 
University of Calgary 
Tel: 403-770-3808 
E-mail: byron.berenger@ahs.ca  
 

Ethics ID: REB17-1916 
Title: Improvements Through the Use of a Rapid Multiplex PCR Enteric Pathogen Detection Kit in Children with 
Hematochezia 
PI: Dr. Stephen Freedman 
Version 5.0 – May 16, 2018 
Previous REB Approved Version Date: January 12, 2018 
 
 
 
 
Page 2 of 19 
 
Collaborators: 
 
 
Angelo Mikrogianakis 
Associate Professor 
Department of Paediatrics 
Alberta Children’s Hospital 
University of Calgary 
Tel: 403-955-7916 
E-mail: angelo.mikrogianakis@ahs.ca 
 
Silviu Grisaru 
Associate Professor 
Department of Paediatrics 
Alberta Children’s Hospital 
University of Calgary 
Tel: 403-955-7759 
E-mail: silviu.grisaru@ahs.ca 
 
Susan Samuel 
Associate Professor 
Department of Paediatrics 
Alberta Children’s Hospital 
University of Calgary 
Tel: 403-955-7950 
E-mail: s.samuel@ahs.ca 
 
Dan Gregson 
Associate Professor 
Departments of Pathology & Laboratory Medicine and Medicine 
University of Calgary 
Tel: 403-770-3762 
E-mail: dgregson@ucalgary.ca 
 
 
 
 

Ethics ID: REB17-1916 
Title: Improvements Through the Use of a Rapid Multiplex PCR Enteric Pathogen Detection Kit in Children with 
Hematochezia 
PI: Dr. Stephen Freedman 
Version 5.0 – May 16, 2018 
Previous REB Approved Version Date: January 12, 2018 
 
 
 
 
Page 3 of 19 
 
 
Linda Chui 
Professor 
Division of Diagnostic and Applied Microbiology, Molecular Microbiologist 
Program Leader Molecular Diagnostics 
University of Alberta 
Tel: 780-407-8951 
E-mail: lchui@ualberta.ca 
 
Gillian Currie 
Associate Professor 
Departments of Paediatrics & Community Health Sciences 
University of Calgary 
Tel: 403-955-7404 
E-mail: currie@ucalgary.ca 
 
Brent Hagel 
Associate Professor 
Departments of Paediatrics & Community Health Sciences 
University of Calgary 
Tel: 403-955-7403 
E-mail: brent.hagel@ahs.ca 
 
 
 
 
CONFIDENTIAL 
This document is confidential and the property of individual.  No part of it may be transmitted, reproduced, 
published, or used by other persons without prior written authorization from the author. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Ethics ID: REB17-1916 
Title: Improvements Through the Use of a Rapid Multiplex PCR Enteric Pathogen Detection Kit in Children with 
Hematochezia 
PI: Dr. Stephen Freedman 
Version 5.0 – May 16, 2018 
Previous REB Approved Version Date: January 12, 2018 
 
 
 
 
Page 4 of 19 
 
Table of Contents 
 
 
1 
INTRODUCTION/SIGNIFICANCE .................................................................................. 5 
2 
KNOWLEDGE GAPS .......................................................................................................... 6 
3 
STUDY OBJECTIVES ......................................................................................................... 7 
4 
PATIENTS AND METHODS ............................................................................................. 8 
4.1 
STUDY DESIGN .................................................................................................................. 8 
4.1.1 
Outcome Variables.................................................................................................. 10 
4.2 
SUBJECT SELECTION AND WITHDRAWAL ........................................................................ 11 
4.2.1 
Inclusion Criteria .................................................................................................... 11 
4.2.2 
Exclusion Criteria ................................................................................................... 11 
4.2.3 
Withdrawal .............................................................................................................. 11 
4.3 
STUDY PROCEDURES ....................................................................................................... 12 
4.4 
STATISTICAL PLAN .......................................................................................................... 13 
4.4.1 
Sample Size Determination ..................................................................................... 13 
4.4.2 
Statistical Methods .................................................................................................. 13 
5 
ADVERSE EVENT REPORTING ................................................................................... 14 
6 
DATA HANDLING AND RECORD KEEPING ............................................................. 14 
6.1 
CONFIDENTIALITY ........................................................................................................... 14 
6.2 
RECORDS RETENTION ...................................................................................................... 15 
6.3 
REGULATORY BINDER ..................................................................................................... 15 
7 
BUDGET .............................................................................................................................. 15 
8 
PUBLICATION PLAN ...................................................................................................... 16 
9 
REFERENCES .................................................................................................................... 16 
10 ATTACHMENTS ............................................................................................................... 19 
 

Ethics ID: REB17-1916 
Title: Improvements Through the Use of a Rapid Multiplex PCR Enteric Pathogen Detection Kit in Children with 
Hematochezia 
PI: Dr. Stephen Freedman 
Version 5.0 – May 16, 2018 
Previous REB Approved Version Date: January 12, 2018 
 
 
 
 
Page 5 of 19 
 
1  Introduction/Significance 
This document is a clinical research protocol and the described study will be conducted in 
compliance with the protocol, Good Clinical Practices standards and associated Federal 
regulations (i.e. Health Canada), and all applicable institutional research requirements. 
 
Approximately 22,000 STEC infections occur in Canada annually at a cost of $404 million. 1 
The effects of an E. coli O157 infection (O157 is the most common and virulent STEC 
pathogen) range from asymptomatic to lethal with ~15% of infected children developing HUS 5 
to 13 days after diarrhea onset. 1 HUS is defined as the presence of azotemia, thrombocytopenia 
and anemia. In a recent study of 770 children with HUS, 57% of children required dialysis, 93% 
received transfusions, and 3% died. 2 Among HUS survivors, 12% develop end-stage renal 
disease 3 and in children with prolonged anuria, 100% suffer from chronic kidney disease. 4 
Currently, most STEC infections are diagnosed microbiologically several days after they present 
for care when they suffer from bloody diarrhea. Because of the time lag to diagnosis as well as 
development of HUS, monitoring of renal and hematologic parameters is recommended to 
enable the early identification of children who are developing HUS. Our team has recently 
quantified this time lag in Alberta’s children (Appendix 1). 5 We have also published guidelines 
that have been distributed by Alberta Health Services indicating the importance of repeating the 
blood tests until the HUS window has passed and of the importance of preventing dehydration 
(http://www.albertahealthservices.ca/assets/info/hp/diseases/if-hp-dis-ecoli-stec.pdf).  
 
The prevention of dehydration in children with STEC infection is gaining prominence due to 
recent publications that associate early intravascular volume expansion with improved outcomes. 
6-9 It is important to note that no therapy has been proven to prevent HUS 10 or reduce kidney 
injury severity after HUS is established. 11 To address this gap experts recommend the 
administration of intravascular volume expansion to all children with bloody diarrhea while 
awaiting culture results. 12 The rationale for this approach builds on evidence that in children 
with HUS, volume expansion may improve outcomes   6,7,9 and that dehydration and 
hemoconcentration are associated with adverse outcomes 2,8,13-15 including death (OR=5.1; 
95% CI 1.5, 17.6). 16 Therefore, the ability to rapidly and reliably identify children with STEC 
infection, and to identify other pathogens with alternative treatment, public health, and follow-up 
implications, has the potential to dramatically improve the care of children with hematochezia.  
 
The BioFire Gastrointestinal Panel FilmArray® 17 is a multiplexed nucleic acid (NA) based 
device that simultaneously identifies 22 enteric pathogens. It specifically tests for the presence of 
Shiga toxin and for O157. It requires 2 minutes of hands-on-time, returns results in ~1 hour, and 
is Health Canada and Food and Drug Administration approved. The device, which has been 
validated, 18-21 hopefully will enable the early identification of infected children and initiation (or 
withholding) of interventions directed by previously unavailable clinical data. It will enable us to 
bring a precision medicine approach into ED care. Our proposal is the first attempt to evaluate 
the ability of this technology to minimize testing and treating to those most likely to benefit. 
Such an evaluation that is needed by knowledge users to justify integration into care. 

Ethics ID: REB17-1916 
Title: Improvements Through the Use of a Rapid Multiplex PCR Enteric Pathogen Detection Kit in Children with 
Hematochezia 
PI: Dr. Stephen Freedman 
Version 5.0 – May 16, 2018 
Previous REB Approved Version Date: January 12, 2018 
 
 
 
 
Page 6 of 19 
 
2 Knowledge Gaps 
Each year, in the United States, 31 pathogens cause 9.4 million episodes of foodborne illness, 
55,961 hospitalizations, and 1,351 deaths. 22 While a small proportion of this overall burden is 
due to STEC infection, 3% of all children with diarrhea associated HUS die. 2 In a study that 
included over 30,000 participants, STEC accounted for 39% of all bacterial pathogens identified 
23 and children 5 – 9 years of age were the group with the highest prevalence of STEC infection. 
23 We are targeting children with bloody diarrhea as E. coli O157:H7 infection is 59 times more 
common (95% CI, 37 to 96) in visibly bloody stool specimens than in non-bloody specimens. 23 
Lastly, this issue is of paramount importance to Southern Alberta as the incidence of diarrhea-
associated HUS is two to threefold higher than other parts of Canada, North America and 
Europe. 24  
 
We intend to determine new knowledge:  
1. If use of the BioFire FilmArray, a rapid, multi-analyte enteric pathogen detection 
device, reduces resource utilization in a pediatric ED when employed on children with 
bloody diarrhea. We hypothesize that use of the test combined with behavioural 
changes by physicians will reduce the use of baseline blood tests, the administration 
of intravenous fluids and future health-care resource use (children are often brought 
back for repeat blood tests and fluids while awaiting culture results).  
 
2. If satisfaction is higher in individuals randomized to testing with the BioFire FilmArray 
arm compared with children in the standard care arm.  
 
Potential child health improvements: 
1. Children with STEC infection will be rapidly identified: We have shown that in the 
2014 STEC outbreak in Alberta, the median time from initial health care presentation to 
stool sample collection was 7 hours (IQR 2, 28) and to sample receipt at the laboratory 
was 33 hours (IQR 18, 42). Even more concerning is that the time from initial health care 
encounter to positive culture was 120 (IQR 86, 205) hours. By collecting rectal swabs at 
the point of care we will initiate specimen processing an average of 30 hours earlier. By 
employing the BioFire FilmArray we will reduce stool processing time by over 80 hours.  
 
2. Children with STEC infection will have dehydration reversed rapidly and they will 
be closely monitored: Following the rapid identification of children with STEC 
infection, all such children will be managed in accordance with a protocol developed by 
the APPETITE team for Alberta Health Services 
(http://www.albertahealthservices.ca/assets/info/hp/diseases/if-hp-dis-ecoli-stec.pdf), 
which includes the performance and monitoring of serum biochemistry and hematologic 
profiles and the prevention of dehydration. It is believed that the early administration of 
intravascular volume expansion has the potential to reduce the morbidity of HUS in 
affected children.  6,7  
 

Ethics ID: REB17-1916 
Title: Improvements Through the Use of a Rapid Multiplex PCR Enteric Pathogen Detection Kit in Children with 
Hematochezia 
PI: Dr. Stephen Freedman 
Version 5.0 – May 16, 2018 
Previous REB Approved Version Date: January 12, 2018 
 
 
 
 
Page 7 of 19 
 
3. Children who do not have STEC infection will avoid unnecessary blood tests, 
intravenous fluids and repeat visits: As ~10% of eligible children will have STEC 
infection, 25,26 the routine performance of blood tests, administration of intravascular 
hydration, and frequent monitoring is unnecessary in the vast majority of children with 
hematochezia. As such, knowledge that a result will be rapidly available will enable 
clinicians to confidently discharge children without performing unnecessary procedures. 
Those who test positive can be recalled to the ED for the appropriate management.  
 
4. Children will have alternative etiologies identified rapidly: The BioFire FilmArray 
will also enable the rapid identification of other pathogens, some of which will require 
immediate therapy such as Shigella sp., Clostridium difficile, and others that have public 
health implications such as Salmonella sp., and norovirus.  
 
5. Children who do not have infectious gastroenteritis will be rapidly identified: 
Children with a negative BioFire FilmArray will also be identified. This would raise the 
suspicion for alternative etiologies.  
 
6. Transmission will be reduced: The rapid identification of infected children has the 
potential to reduce spread, which is common and occurs early in the course of disease. 
Transmission might be reduced by separating infected index cases from their at risk 
siblings. 27  
3 Study Objectives 
The rapid identification of STEC-infected children will enable the provision of appropriate and 
timely care to such children. The rapid identification of other enteric pathogens (bacteria, virus, 
parasite) will enable the provision of targeted therapies as appropriate and the withholding of 
avoidable interventions. It will also provide information for clinicians to consider alternate, non-
infectious etiologies as appropriate. We hypothesize that the BioFire FilmArray can be used 
selectively in children with hematochezia to identify children with STEC infection and 
therefore will expeditiously identify children in need of therapeutic interventions and those 
for whom blood testing, intravenous fluids, and follow-up visits are unnecessary. 
Knowledge of the etiology early in the course of disease will enhance the provision of a patient-
level precision medicine approach to what is otherwise an ill-defined symptom.  
 
Research Questions  
1. Do individuals randomized to testing with the BioFire FilmArray undergo fewer 
procedures (i.e. blood tests, intravenous fluids) and repeat healthcare visits compared 
with children in the standard care arm?  
 
2. Are patient and caregiver satisfaction higher in individuals randomized to testing with 
the BioFire FilmArray arm compared with children in the standard care arm?  
 

Ethics ID: REB17-1916 
Title: Improvements Through the Use of a Rapid Multiplex PCR Enteric Pathogen Detection Kit in Children with 
Hematochezia 
PI: Dr. Stephen Freedman 
Version 5.0 – May 16, 2018 
Previous REB Approved Version Date: January 12, 2018 
 
 
 
 
Page 8 of 19 
 
Primary Objective  
1. To determine if use of the BioFire FilmArray in children with hematochezia 
results in a reduction in resource utilization. Primary outcome – any blood tests 
(dichotomous with sub-analysis by specific blood tests); secondary – intravenous fluid 
administration, ED encounters.  
 
Secondary Objectives  
1. To quantify clinical outcomes – development of HUS, acute kidney injury, need for 
renal replacement therapy, turnaround time from BioFire result relative to stool 
culture result. 
2. To determine if use of the BioFire FilmArray is associated with greater family 
satisfaction with care.  
3. To determine testing criteria to optimize the clinical improvement emerging from use 
of the BioFire FilmArray (i.e. predictors of bacterial etiology).  
4 Patients and Methods 
4.1 Study Design 
We will prospectively identify children who present with hematochezia. Potentially eligible 
subjects will be identified by our Pediatric Emergency Medicine Research Associate Program 
(PEMRAP) and the Pediatric Emergency Research Team (PERT) nurses (ED coverage 14 
hours per day). Eligible children will have ≥3 episodes of diarrhea within the preceding 24 
hours and will have had blood identified in the stool (by physician, nurse or parent). Eligible 
subjects will be approached by a PERT team member to obtain consent (and assent when 
appropriate). Once consent has been obtained, study participants will be randomized by a 
REDCap randomization tool to ensure allocation concealment (i.e. research team, ED 
physicians will be unaware until after enrolled into the study and randomized). In order to 
optimize participants who have consented/assented to be contacted in the future regarding 
additional research studies as well as follow up, we will collect the following information at the 
initial point of contact: home, cell, work and alternate telephone numbers as well as e-mail 
address.  
 
Children randomized to the standard of care arm will have demographic and clinical 
information collected and the treating physician will be informed to proceed as per their usual 
practice and treatment patterns. If stool is unavailable a rectal swab will be collected and sent to 
Calgary Laboratory Services (CLS) for routine culture. A routine stool specimen for back-up 
culture will still be requested as per standard of care. Home stool collection will be performed 
for those unable to provide a sample at enrolment and will be achieved by providing families 
with collection kits. Following the completion of the visit, data regarding all testing, procedures, 
and medications administered will be collected. The family will be contacted 14 days later to 
collect outcome information (clinical and health resource use) and satisfaction. The medical 

Ethics ID: REB17-1916 
Title: Improvements Through the Use of a Rapid Multiplex PCR Enteric Pathogen Detection Kit in Children with 
Hematochezia 
PI: Dr. Stephen Freedman 
Version 5.0 – May 16, 2018 
Previous REB Approved Version Date: January 12, 2018 
 
 
 
 
Page 9 of 19 
 
records and select administrative databases may be reviewed to ascertain and confirm outcome 
data on Day 28.  
 
Children randomized to the BioFire FilmArray arm will have the same data collected, but 
stool, if available, will be sent STAT to Calgary Laboratory Services (CLS) for the performance 
of the BioFire FilmArray test and routine culture. If stool is unavailable, a rectal swab will be 
performed and sent to CLS for the performance of the BioFire FilmArray test and routine 
culture. A routine stool specimen for back-up culture will still be requested as per standard of 
care once it is available. The result of the BioFire test will be printed and brought to the ED 
where it will be provided to the ED physician for immediate review and management as 
appropriate. The result will be included in the ED chart for documentation purposes. Education 
regarding the interpretation of BioFire FilmArray results will have been performed in advance to 
all faculty members and a research team member will be available to answer any questions 
should they arise. We have worked with leaders at CLS including Drs. Gregson, Berenger and 
Vanderkooi who believe we should be able to receive a result within 2-3 hours of receipt of the 
specimen at CLS. Treatment decisions will be at the sole discretion of the ED treating physician 
who receives the result. Following the completion of the visit, data regarding all testing, 
procedures, and medications administered will be collected. The family will be contacted 14 
days later to collect outcome information (clinical and health resource use) and satisfaction. The 
medical records and select administrative databases may be reviewed to ascertain and confirm 
outcome data on Day 28. 
 
All children 6 months – 18 years of age will be eligible for participation (STEC infection is 
exceedingly rare in children < 6 months of age). Children previously enrolled in the study will be 
ineligible to participate. 
 
For those who decline consent, we will request consent to access select administrative data, 
Netcare and/or access to the child’s medical records related to the illness to document 
interventions and outcomes in order to be able to assess bias by comparing participating and 
non-participating children in this study. 
 
Biofire FilmArray™: The Health Canada and Food and Drug Administration-approved, 
multiplex Biofire FilmArray™ Gastrointestinal Panel can detect 22 analytes: Campylobacter (C. 
jejuni, C. coli, and C. upsaliensis), Salmonella, Shigella/Enteroinvasive E. coli (EIEC), Vibrio 
(V. parahaemolyticus, V. vulnificus, and V. cholerae), Yersinia enterocolitica, Shiga-like toxin-
producing E. coli (STEC) stx1/stx2, Enterotoxigenic E. coli (ETEC) lt/st, E. coli O157, 
Clostridium difficile (Toxin A/B), Plesiomonas shigelloides, Enteroaggregative E. coli (EAEC), 
Enteropathogenic E. coli (EPEC), norovirus GI/GII, rotavirus A, adenovirus F40/41, astrovirus, 
sapovirus (I, II, IV and V), Cryptosporidium, Entamoeba histolytica, Giardia lamblia, and 
Cyclospora cayetanensis. A single instrument can test one stool sample in approximately 1 hour 
with 2 minutes of hands-on time. After adding 200 μL of stool specimen in Cary Blair and 
hydration solution to the FilmArray pouch, the system performs nucleic acid extraction, nested 
multiplex polymerase chain reaction and lastly, endpoint melting curve analysis with the 

Ethics ID: REB17-1916 
Title: Improvements Through the Use of a Rapid Multiplex PCR Enteric Pathogen Detection Kit in Children with 
Hematochezia 
PI: Dr. Stephen Freedman 
Version 5.0 – May 16, 2018 
Previous REB Approved Version Date: January 12, 2018 
 
 
 
 
Page 10 of 19 
 
FilmArray software. For this study, the BioFire FilmArray will be run as a research test at CLS 
and it will not go through full verification and implementation procedures at CLS. The product 
insert recommendations will be followed for verification purposes. 
 
Rectal Swabs: The APPETITE team has been evaluating the use of rectal swabs for over two 
years now and we have demonstrated that rectal swabs are a suitable alternative specimen when 
stool is unavailable. 28 In brief, we insert a sterile flocked rectal swab (FLOQSwab, Copan Italia, 
Brescia, Italy) into the rectum. It is then rotated a full circle once and then transported in 
modified Cary-Blair media. The current APPETITE protocol employs the Luminex xTAG 
Gastrointestinal Panel Platform and requires a full day for extraction (hence the switch to the 
Biofire FilmArray for the current study proposal). When compared with paired stool samples on 
1142 children, overall agreement is 90% (95% CI: 88, 92) with positive test result agreement of 
98% (95% CI: 96, 99). As it relates to bacterial pathogens the overall agreement is 93% (95% 
CI: 92, 95) with positive bacteria test result agreement of 81% (95% CI: 74, 86). These results 
were achieved through the use of a specimen procurement approach that would be replicated in 
the current proposal. 
 
In a 3-centre project currently underway in the U.S. employing rectal swab analysis on the 
Biofire FilmArray platform they have found performance of the rectal swab to be comparable to 
stool for detection of pathogens.29 Importantly, there were no discrepancies for bacterial or STEC 
targets. They concluded that rectal swab collection at the time of the patient visit enables rapid 
testing and the generation of actionable results in the acute care setting when a stool specimen 
cannot be provided. 
4.1.1 Outcome Variables 
We will track the following on all participants: 
Primary Outcome Measure: 
1. Blood test performance, any blood testing performed within 72 hours of 
randomization; 
Secondary Outcome Measures: 
2. Intravenous fluid administration, children administered IV fluids identified by 
chart review; 
3. Total physician visits (ED and non-ED), children visiting additional health-care 
practitioners identified by chart review; 
4. ED length of stay, ED length of stay during enrollment visit determined by chart 
review; 
5. Antibiotic use, antibiotic use identified by chart review; 
6. Hospital and intensive care unit admission, hospitalization identified by chart 
review; 
7. Diagnostic imaging performed, diagnostic imaging performed identified by 
chart review; 
8. HUS, children with HUS identified by chart review; 

Ethics ID: REB17-1916 
Title: Improvements Through the Use of a Rapid Multiplex PCR Enteric Pathogen Detection Kit in Children with 
Hematochezia 
PI: Dr. Stephen Freedman 
Version 5.0 – May 16, 2018 
Previous REB Approved Version Date: January 12, 2018 
 
 
 
 
Page 11 of 19 
 
9. Acute kidney injury, based on chart review in accordance with KDIGO 
guidelines; 
10. Need for renal replacement therapy, renal replacement therapy identified by 
chart review; and 
11. Caregiver and patient satisfaction, satisfaction of care received during ED visit 
answered in Day 14 follow-up form on a Likert scale 
 
4.2 Subject Selection and Withdrawal 
4.2.1 Inclusion Criteria 
Eligible children will: 
1. Be aged 6 months – 17.99 years of age 
2. Have ≥3 episodes of diarrhea within the preceding 24 hours and have blood identified in 
the stool (by physician, nurse or parent) 
 
4.2.2 Exclusion Criteria 
Children with any of the following will not be eligible: 
1. Previously enrolled in the study 
2. Unavailable for Day 14 follow-up  
3. Currently (most recent complete blood count) known to be neutropenic (Neutrophils 
<1000), or at high-risk of being neutropenic (receiving chemotherapy) at present 
4. Known to be STEC positive (stool culture, PCT, or toxin) 
5. Language barrier that prevents the ability to obtain informed consent and assent (when 
appropriate) 
 
4.2.3 Study Withdrawal and Discontinuation 
Subjects will be withdrawn if: 
1. If it is deemed by the investigator or treating physician that the child’s health may be 
jeopardized by continued participation in the study (prior to sample collection) 
2. The patient’s caregivers wish to no longer participate and withdraws their child for 
whatever reason 
3. The patient wishes to no longer participate and withdraws themselves for whatever 
reason 
4. The assented child wishes to no longer participate and withdraws for whatever reason 
 
If the patient, patient’s caregiver or the assented child respectively chooses to withdraw the 
participant from the study, they will be provided with a choice regarding their exit from the 
study: 
A. The patient, caregiver or assented child may choose to withdraw the participant from the 
study, as well as all data collected from their participation in the study. In this case, all 

Ethics ID: REB17-1916 
Title: Improvements Through the Use of a Rapid Multiplex PCR Enteric Pathogen Detection Kit in Children with 
Hematochezia 
PI: Dr. Stephen Freedman 
Version 5.0 – May 16, 2018 
Previous REB Approved Version Date: January 12, 2018 
 
 
 
 
Page 12 of 19 
 
data will be excluded from analysis and not incorporated into the study. The data will 
also be destroyed in accordance with local data destruction policy and TCPS2 guidelines. 
B. The patient, caregiver or assented child may choose to withdraw the participant from the 
study; however they will allow continued use of study data collected from their 
participation in the study. 
 
Subjects will discontinue the study if: 
1. If it is deemed by the investigator or treating physician that the child’s health may be 
jeopardized by continued participation in the study (following sample collection) 
4.3 Study Procedures 
Children randomized to the standard of care arm and the BioFire FilmArray arm will have 
clinical data collected:  
 
1) Day 0 – ED enrollment visit (In person): At the initial screening visit, the research 
assistant or research nurse will explain the nature of the research project to the family. 
Informed consent, and assent if applicable, will be obtained. A detailed medical history 
will be documented to ensure the subject’s eligibility for inclusion in the study. Parents 
will be asked to participate in a standardized 10 minute survey administered by study 
personnel. The survey will collect descriptive data regarding current medical history (i.e. 
diarrhea – frequency/duration/blood; vomiting – frequency/duration; fever; pain; health 
care use; treatment required/procedures performed), previous medical history (i.e. 
investigations), household pets, and childcare arrangements. The information will be 
entered in our electronic data capture system (REDCap), and all charts will be reviewed 
in the ED.  
a. Specimen Collection: Subjects who qualify for the study will have a stool sample 
collected, or a rectal swab specimen will be collected if stool is not available for 
either cohort (Standard Care and BioFire). Children who have a rectal swab 
collected at this visit will be requested to submit a stool specimen for culture after 
the ED visit.  
b. Letter to parents: Only subjects randomized to the BioFire FilmArray arm will be 
given a letter. The letter outlines their assignment to the BioFire arm and what 
that means. 
2) Day 14 Follow-Up: Data regarding ongoing symptoms, any hospital visits/admissions 
and locations of the respective hospital visits/admissions, testing and procedures 
conducted as well as parental satisfaction of the experience at the ED will be collected. 
Parental reports will act as source documentation for data that is related to any additional 
healthcare visits as well as care in relation to the respective visits not associated with the 
Alberta Children’s Hospital. At the initial point of contact, we will collect the following 
data: home, cell, work and alternate telephone numbers.  For phone follow-up, we will 
call all numbers provided at least once daily (maximum 5 attempts). Study participants 
will not be identified by name on study document except the Contact Sheet.   
 

Ethics ID: REB17-1916 
Title: Improvements Through the Use of a Rapid Multiplex PCR Enteric Pathogen Detection Kit in Children with 
Hematochezia 
PI: Dr. Stephen Freedman 
Version 5.0 – May 16, 2018 
Previous REB Approved Version Date: January 12, 2018 
 
 
 
 
Page 13 of 19 
 
All standardized surveys employed in this study will consist of a series of single-and 
multiple-select closed-ended items.  We will also allow for the selection of a residual 
“other” category.  In cases in which the respondent feels that his or her response does not 
correspond to one of those already listed (ie. he/she selects “other”), the respondent will 
be asked to elaborate on his or her answer in full text. 
 
3) Day 28: The medical records, Netcare and select administrative databases may be 
reviewed to ascertain and confirm outcome data on Day 28. This chart review will collect 
descriptive data regarding subsequent hospital visits and/or subsequent hospital 
admissions. Variables such as date and time of visit/admission, discharge diagnosis, 
investigations completed, complications or procedures performed and medications 
administered will be collected. Chart reviews will serve as source documentation for all 
data that is related to the Alberta Children’s Hospital. 
4.4 Statistical Plan 
4.4.1 Sample Size Determination 
The desired sample size – 54 subjects – was calculated employing a baseline blood testing rate of 
60% in the control group, a power of 80%, type I error of 5%, and a minimally clinically 
important difference of 40% in the proportion of children who experience the primary outcome 
(i.e. have any blood testing performed within 72 hours). Thus, we hypothesize that 20% of 
children in the intervention arm will have blood testing performed. The estimation of children 
having any blood testing performed within 72 hours (i.e. meeting our primary outcome measure) 
is based upon data from the APPETITE study (currently 62% among children with 
hematochezia). Based on previous experience we estimate that 10% of subjects will be lost to 
follow-up; hence the final sample size is 60 in total. Sample size requirements will be 
recalculated after the enrollment of 30 children. Based on the enrollment into APPETITE of 935 
children at ACH since April 2015 and the presence of hematochezia in approximately 5% of 
children (23/506 on whom these data were collected), we anticipate that through the 
identification of potential participants outside of research coverage hours, we should be able to 
recruit our cohort over the span of 12 months. 
4.4.2 Statistical Methods 
All analyses will be undertaken by the intention-to-treat (ITT) principle. Patients who drop out or 
crossover will be followed and included in the ITT analysis. All statistical tests will be two-
sided. Baseline characteristics will be analyzed and compared. Sensitivity analyses will be 
performed to assess the possibility and consequences of non-random loss to follow-up. The 
proportion of children who have any blood testing performed within 72 hours of randomization, 
the primary outcome, will be analyzed by comparing proportions utilizing a Fisher’s Exact test. 
We will use an alpha level of 0.05 for statistical testing.  
 
The overall significance level for statistical tests on the secondary outcomes will be set at 0.05 
with adjustment for multiple comparisons performed using a Bonferroni correction. 

Ethics ID: REB17-1916 
Title: Improvements Through the Use of a Rapid Multiplex PCR Enteric Pathogen Detection Kit in Children with 
Hematochezia 
PI: Dr. Stephen Freedman 
Version 5.0 – May 16, 2018 
Previous REB Approved Version Date: January 12, 2018 
 
 
 
 
Page 14 of 19 
 
Dichotomous outcomes to be evaluated include 1) intravenous rehydration, 2) ED revisits, 3) any 
healthcare revisits and 4) hospitalization within the subsequent 14 days. Clinical outcomes will 
also include development of 5) HUS, 6) Kidney Disease Improving Global Outcomes Stage 2 or 
3 creatinine based acute kidney injury,31 7) antibiotic use, and 8) diagnostic imaging. 
 
Recursive partitioning analysis will be employed to identify clinical features that select 
candidates for BioFire testing that maximize specificity and sensitivity to identify STEC patients. 
Analyses will be performed blinded to allocation assignment. 
5 Adverse Event Reporting 
Anticipated disease outcomes (listed below) are sought to be a natural continuation of the disease 
and as a result, the following will not be reported to REB: 
1. IV insertion 
2. Blood tests 
3. Abdominal pain 
4. Fever 
5. Vomiting 
6. Diarrhea 
7. Bloody stool 
8. Dialysis 
9. Renal abnormalities 
10. Hematological abnormalities 
11. Biochemical abnormalities 
12. Transfusions 
13. Seizures 
14. Hospital admissions 
15. Other known complications of STEC infection 
6 Data Handling and Record Keeping 
Patients will be assessed and treated by ED staff without any regard to this study.  Stool 
specimen testing will continue as per standard of care.  Once the inclusion criteria are confirmed 
and parental consent is obtained (and child assent when appropriate) an enrollment ID number 
will be assigned.  Recruitment and specimen collection will commence after ethics approval is 
obtained. 
 
We may need to contact subjects in the future regarding research questions which that may be 
discovered from the data collected in this study. Select administrative data may also be accessed 
to allow linkages beyond study collected outcomes. 
6.1 Confidentiality 
Data collected on paper will be stored in a locked cabinet in a locked office of a securely 
accessed building. 

Ethics ID: REB17-1916 
Title: Improvements Through the Use of a Rapid Multiplex PCR Enteric Pathogen Detection Kit in Children with 
Hematochezia 
PI: Dr. Stephen Freedman 
Version 5.0 – May 16, 2018 
Previous REB Approved Version Date: January 12, 2018 
 
 
 
 
Page 15 of 19 
 
 
Data will be entered from paper to REDCap, or entered directly into REDCap, which is a data 
capture system that has been validated by Health Canada. REDCap access will be determined 
based on project involvement. Only the investigators and research staff listed on the ethics 
submission will have access to data. Additionally the data management team will have 
administrative access to the database and the servers on which it resides. The data management 
team will only access study data as required to perform maintenance and support duties. 
 
The database contains sensitive patient information as well as personal identifiers however, each 
patient is assigned a study ID that is used to communicate about the patient. Information from 
the database will be coded and stored into the secure server. The database will have robust and 
modern security principles enforced in an auditable fashion as outlined in the Health Information 
Act and in most cases in line with the guidelines set out in the FDA's CFR Part 11 Guidance 
document. 
 
Deidentified data may also be stored on secure network drives including Alberta Health Services, 
University’s hydrogen drive and computers on the ProvLab Laboratory Information System. 
Authorized access will be granted to relevant research team members and requires a specific user 
ID and password. 
 
Because this study will recruit participants in an ED setting, the likelihood of one study 
participant speaking with another is very low. These participants will likely only have one visit, 
at which time they will be enrolled. All follow up will be done confidentially over the phone or 
by secure email.  
6.2 Records Retention 
Name of patient is collected and kept for future contact after data collection has been completed 
only if the patient consents to contact for a future research study. 
 
6.3 Regulatory Binder 
A regulatory binder containing all information pertinent to the study will be kept. 
 
 
 
 
 
 
7 Budget 
 
PEMRAP 
$2,500 – coordination, training, supplies, data collection 
CLS 
$100/test x 30 = $3,000 

Ethics ID: REB17-1916 
Title: Improvements Through the Use of a Rapid Multiplex PCR Enteric Pathogen Detection Kit in Children with 
Hematochezia 
PI: Dr. Stephen Freedman 
Version 5.0 – May 16, 2018 
Previous REB Approved Version Date: January 12, 2018 
 
 
 
 
Page 16 of 19 
 
Flocked Rectal Swabs 
$0.75 x 30 = $23 
Study Coordinator 
Start-up: $40/hr x 37.5 hrs/wk/FTE x 0.5 FTE x 8 weeks x 1.23 (benefits) = $7,380 
Ongoing: $40/hr x 37.5 hrs/wk/FTE x 0.1 FTE x 52 weeks x 1.23 (benefits)  $9,594 
Database/randomization 
$2,500 – database creation, design, logic, limits, and computer based randomization 
Statistical Analysis 
To be done by Dr. Stephen Freedman (with consultation requested as needed) 
Biofire FilmArray 
$29,000 (in-kind for device, training, service, kits) 
TOTAL 
$24,997 
 
We have been working with bioMérieux Canada, the distributor of the Biofire FilmArray on 
approaches to integrate rapid diagnostics to improve the care of children with STEC infection. 
For the current study proposal they have agreed to provide an in-kind device ($6,000/year), 
service ($5,000/year), and 100 test kits ($18,000) for total in-kind support of $29,000. 
8 Publication Plan 
We will interpret our findings in the context of value added. We will compare the outcomes in 
children randomized to testing with the Biofire FilmArray and those randomized to standard of 
care. We will evaluate the role the Biofire FilmArray might play in rapidly identifying the 
etiology of the hematochezia compared with standard testing. We will explore the role the 
Biofire FilmArray might play in selectively identifying children who require blood tests, 
intravascular volume expansion and close monitoring. 
 
If this study yields findings of clinical relevance, we would seek subsequent funding to 
conduct an economic evaluation to determine if the cost of care provided to individuals 
randomized to testing with the BioFire FilmArray is less than that provided to children in the 
standard care group. We will employ data collected regarding utilization of testing (BioFire 
FilmArray, blood, urine, stool), diagnostic imaging, ED care (i.e. visit cost), physician 
billing, and medication administration to evaluate the relative cost of the two approaches and 
we will determine the cost per case of STEC infection identified. 
 
Funding will be sought from a variety of sources to support the hiring of graduate students. 
Potential sources include philanthropic (IPH, ACHRI, U of C, AIHS, CIHR), industry 
(bioMerieux), or stakeholders (CLS, ProvLab, AHS, MNCY, kidney or emergency SCN).  If 
safe and effective, we will subsequently work with CLS to integrate this technology into 
standard of care. We will also use the data obtained to justify the use of this technology in a 
CIHR RCT proposal that is currently under review that proposes to use rapid diagnostic 
approaches to enroll STEC-infected children in a volume expansion trial. We will also be 
submitting an NIH over-the-cap R01 multinational proposal that will include this technology 
(to be submitted in 2019; NIH R34 in May 2017). 
9 References 
1. 
Sockett P, Goebel SE, Varela NP, et al. Verotoxigenic Escherichia coli: costs of 
illness in Canada, including long- term health outcomes. J Food Prot 2014;77:216-26. 
2. 
Mody RK, Gu W, Griffin PM, et al. Postdiarrheal hemolytic uremic syndrome in 

Ethics ID: REB17-1916 
Title: Improvements Through the Use of a Rapid Multiplex PCR Enteric Pathogen Detection Kit in Children with 
Hematochezia 
PI: Dr. Stephen Freedman 
Version 5.0 – May 16, 2018 
Previous REB Approved Version Date: January 12, 2018 
 
 
 
 
Page 17 of 19 
 
United States children: clinical spectrum and predictors of in-hospital death. J Pediatr 
2015;166:1022-9. 
3. 
Garg AX, Suri RS, Barrowman N, et al. Long-term renal prognosis of diarrhea-
associated hemolytic uremic syndrome: a systematic review, meta-analysis, and meta-
regression. JAMA 2003;290:1360-70. 
4. 
Oakes RS, Kirkham JK, Nelson RD, Siegler RL. Duration of oliguria and anuria 
as predictors of chronic renal- related sequelae in post-diarrheal hemolytic uremic 
syndrome. Pediatr Nephrol 2008;23:1303-8. 
5. 
Freedman SB, Eltorki M, Chui L, et al. Province-Wide Review of Pediatric Shiga 
Toxin-Producing Escherichia coli Case Management. J Pediatr 2016. 
6. 
Ake JA, Jelacic S, Ciol MA, et al. Relative nephroprotection during 
Escherichia coli O157:H7 infections: association with intravenous volume 
expansion. Pediatrics 2005;115:e673-80. 
7. 
Hickey CA, Beattie TJ, Cowieson J, et al. Early volume expansion during diarrhea 
and relative nephroprotection during subsequent hemolytic uremic syndrome. Arch Pediatr 
Adolesc Med 2011;165:884-9. 
8. 
Ardissino G, Dacco V, Testa S, et al. Hemoconcentration: a major risk factor for 
neurological involvement in hemolytic uremic syndrome. Pediatr Nephrol 2015;30:345-
52. 
9. 
Ardissino G, Tel F, Possenti I, et al. Early Volume Expansion and Outcomes of 
Hemolytic Uremic Syndrome. Pediatrics 2016;137:e20152153. 
10. 
Goldwater PN, Bettelheim KA. Treatment of enterohemorrhagic Escherichia coli 
(EHEC) infection and hemolytic uremic syndrome (HUS). BMC Med 2012;10:12. 
11. 
Tarr PI, Gordon CA, Chandler WL. Shiga-toxin-producing Escherichia coli and 
haemolytic uraemic syndrome. Lancet 2005;365:1073-86. 
12. 
Holtz LR, Neill MA, Tarr PI. Acute bloody diarrhea: a medical emergency for patients 
of all ages. Gastroenterology 2009;136:1887-98. 
13. 
Balestracci A, Martin SM, Toledo I, Alvarado C, Wainsztein RE. Dehydration at 
admission increased the need for dialysis in hemolytic uremic syndrome children. Pediatr 
Nephrol 2012;27:1407-10. 
14. 
Ojeda JM, Kohout I, Cuestas E. Dehydration upon admission is a risk factor for 
incomplete recovery of renal function in children with haemolytic uremic syndrome. 
Nefrologia 2013;33:372-6. 
15. 
Oakes RS, Siegler RL, McReynolds MA, Pysher T, Pavia AT. Predictors of 
fatality in postdiarrheal hemolytic uremic syndrome. Pediatrics 2006;117:1656-62. 
16. 
Grisaru S, Xie J, Samuel S, et al. Associations Between Hydration Status, 
Intravenous Fluid Administration, and Outcomes of Patients Infected With Shiga Toxin-
Producing Escherichia coli: A Systematic Review and Meta-analysis. JAMA Pediatr 2016. 
17. 
FilmArray Gastrointestinal Panel. Available at: http://www.biofiredx.com/wp-
content/uploads/2016/08/MRKT- PRT-0234-
08_FilmArray_Info_Sheet_GI_Panel_STD.pdf. Accessed September 29, 2016. 
18. 
De Rauw K, Detemmerman L, Breynaert J, Pierard D. Detection of Shiga toxin-
producing and other diarrheagenic Escherichia coli by the BioFire FilmArray(R) 

Ethics ID: REB17-1916 
Title: Improvements Through the Use of a Rapid Multiplex PCR Enteric Pathogen Detection Kit in Children with 
Hematochezia 
PI: Dr. Stephen Freedman 
Version 5.0 – May 16, 2018 
Previous REB Approved Version Date: January 12, 2018 
 
 
 
 
Page 18 of 19 
 
Gastrointestinal Panel in human fecal samples. Eur J Clin Microbiol Infect Dis 2016;35:1479-
86. 
19. 
Stockmann C, Pavia AT, Graham B, et al. Detection of 23 Gastrointestinal Pathogens 
Among Children Who Present With Diarrhea. J Pediatric Infect Dis Soc 2016. 
20. 
Spina A, Kerr KG, Cormican M, et al. Spectrum of enteropathogens detected by 
the FilmArray GI Panel in a multicentre study of community-acquired gastroenteritis. 
Clin Microbiol Infect 2015;21:719-28. 
21. 
Buss SN, Leber A, Chapin K, et al. Multicenter evaluation of the BioFire 
FilmArray gastrointestinal panel for etiologic diagnosis of infectious gastroenteritis. J 
Clin Microbiol 2015;53:915-25. 
22. 
Scallan E, Hoekstra RM, Angulo FJ, et al. Foodborne illness acquired in the United 
States--major pathogens. Emerg Infect Dis 2011;17:7-15. 
23. 
Slutsker L, Ries AA, Greene KD, Wells JG, Hutwagner L, Griffin PM. Escherichia 
coli O157:H7 diarrhea in the United States: clinical and epidemiologic features. Ann Intern 
Med 1997;126:505-13. 
24. 
Grisaru S, Midgley JP, Hamiwka LA, Wade AW, Samuel SM. Diarrhea-associated 
hemolytic uremic syndrome in southern Alberta: A long-term single-centre experience. 
Paediatr Child Health 2011;16:337-40. 
25. 
Klein EJ, Boster DR, Stapp JR, et al. Diarrhea etiology in a Children's 
Hospital Emergency Department: a prospective cohort study. Clin Infect Dis 
2006;43:807-13. 
26. 
Klein EJ, Stapp JR, Clausen CR, et al. Shiga toxin-producing Escherichia coli 
in children with diarrhea: a prospective point-of-care study. J Pediatr 2002;141:172-
7. 
27. 
Werber D, Mason BW, Evans MR, Salmon RL. Preventing household 
transmission of Shiga toxin-producing Escherichia coli O157 infection: promptly 
separating siblings might be the key. Clin Infect Dis 2008;46:1189-96. 
28. 
Freedman SB, Xie J, Nettel-Aguirre A, Lee B, Chui L, Pang XL, et al. 
Enteropathogen detection in children with diarrhoea, or vomiting, or both, comparing 
rectal flocked swabs with stool specimens: an outpatient cohort study. Lancet 
Gastroenterol Hepatol. 2017;2(9):662-9 
29. 
Chapin K, Duffy S, LeBlanc L, et al. Comparison of Results Obtained with the 
FilmArray GI Panel using Rectal Swabs and Cary-Blair stool from Patients with 
Gastroenteritis in the Pediatric Emergency Dept. 2016 Clinical Virology Symposium; 
Daytona, FL. Available at: nhttp://www.copanusa.com/education/scientific-
studies/comparison-results- obtained-filmarray-gi-panel-using-rectal-swabs-and-cary-blair-
stool-patients-gastroenteritis-pediatric-emerge/. Accessed October 7, 2016. 
30. 
Freedman SB, Xie J, Neufeld MS, et al. Shiga Toxin-Producing Escherichia coli 
Infection, Antibiotics, and Risk of Developing Hemolytic Uremic Syndrome: A Meta-
analysis. Clin Infect Dis 2016;62:1251-8. 
31. 
Section 2: AKI Definition. Kidney Int Suppl (2011) 2012;2:19-36. 
  
 

Ethics ID: REB17-1916 
Title: Improvements Through the Use of a Rapid Multiplex PCR Enteric Pathogen Detection Kit in Children with 
Hematochezia 
PI: Dr. Stephen Freedman 
Version 5.0 – May 16, 2018 
Previous REB Approved Version Date: January 12, 2018 
 
 
 
 
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10 Attachments 
 
Appendix 1: Quantified time lag to diagnosis in Alberta’s children.