PrePhage - Faecal Bacteriophage Transfer for Enhanced Gastrointestinal Tract Maturation in Preterm Infants - Clinical Trial
Phase: EARLY_PHASE1
Conditions: Necrotizing Enterocolitis, Microbial Substitution
Sponsor: Rigshospitalet, Denmark

PrePhage – Faecal bacteriophage transfer for enhanced gastrointestinal tract maturation 
in preterm infants  
Ethical Registration Nr: 78259 
 
 
    
 
 
 
PrePhage 
1 
 
A Research Protocol:  
PrePhage – Faecal bacteriophage 
transfer for enhanced gastrointestinal 
tract maturation in preterm infants 
 
 
1. 
General information 
4 
1.1. 
Protocol title, version number, date, list of abbreviations 
4 
1.2. 
Good clinical practice 
6 
1.3. 
Time plan 
6 
1.4. 
Signature 
7 
1.5. 
Study Location 
7 
2. 
Summary 
8 
3. 
Background 
9 
4. 
Objectives, hypothesis and feasibility 
11 
5. 
Donor study 
14 
5.1. 
Design and recruitment 
14 
5.2. 
Inclusion criteria for donors 
15 
5.3. 
Exclusion criteria for donors 
15 
5.4. 
Inclusion criteria for mothers of donors 
15 
5.5. 
Exclusion criteria for mothers of donors 
16 
5.6. 
Primary endpoints 
16 
5.7. 
Secondary endpoints 
16 
5.8. 
Overview screening, data collection and fecal sample collection, donor study 
16 
5.8.1. 
Donor infant screening and data collection 
17 
5.8.2. 
Maternal screening and data collection 
20 
6. 
Clinical Trial 
22 

PrePhage – Faecal bacteriophage transfer for enhanced gastrointestinal tract maturation 
in preterm infants  
Ethical Registration Nr: 78259 
 
 
    
 
 
 
PrePhage 
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6.1. 
Design 
22 
6.2. 
Inclusion criteria for participant preterm infants 
23 
6.3. 
Exclusion criteria for participant preterm infants 
23 
6.4. 
Inclusion criteria for mothers of participants 
23 
6.5. 
Exclusion criteria for participant mothers 
23 
6.6. 
Endpoints, clinical trial 
23 
6.6.1. 
Primary endpoints 
23 
6.6.2. 
Secondary endpoints 
23 
6.7. 
Overview of data collection from participants 
24 
6.8. 
FFT treatment 
27 
6.8.1. 
Faecal viral transplant – Risk assessment 
27 
6.9. 
Preparation of faecal filtrate transplantation 
27 
6.10. 
Clinical observation and safety during admission 
28 
6.11. 
Long term follow-up after discharge 
28 
6.12. 
Randomization code 
28 
7. 
Analysis of microbiome and virome in faeces 
28 
8. 
Biobank and storage of samples 
29 
9. 
Recruitment, consent, and participant information 
30 
9.1. 
Recruitment strategy 
30 
9.1.1. 
Recruitment for Donor Study 
30 
9.1.2. 
Recruitment for Clinical Trial 
30 
9.2. 
Consent 
31 
9.3. 
Principles for informing participants and obtaining consent 
31 
10. 
Discontinuation and withdrawal 
32 
11. 
Adverse events 
33 
11.1. 
Definition of adverse events 
33 
11.2. 
Definition of serious adverse events 
33 
11.3. 
Baseline incidence of Serious Adverse Events 
33 
11.4. 
Follow-up of adverse events 
33 
11.5. 
Recording and reporting of the adverse events 
33 
11.6. 
Classification of causality 
34 
12. 
Side-effects and risks 
34 

PrePhage – Faecal bacteriophage transfer for enhanced gastrointestinal tract maturation 
in preterm infants  
Ethical Registration Nr: 78259 
 
 
    
 
 
 
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12.1. 
Faecal and urine samples 
34 
12.2. 
Blood samples 
34 
12.3. 
Safety during trial 
35 
13. 
Data Safety Monitoring Board (DSMB) 
35 
14. 
Termination of the study 
35 
15. 
Statistical analysis 
36 
15.1. 
General statistical considerations 
36 
15.2. 
Effect-modifying factors 
36 
15.2.1. Primary outcome 
36 
15.2.2. Secondary outcomes 
36 
15.3. 
Definition of datasets 
37 
15.4. 
Handling missing values 
37 
16. 
Data handling and storage 
37 
17. 
Ethical aspects 
38 
17.1. 
Administering FFT to the preterm infant: potential benefit and harm 
38 
18. 
Considerations on research with neonates 
38 
18.1.1. 
Study group 
38 
18.1.2. 
Considerations for ensuring minimal physical and emotional distress for 
participants 
39 
18.2. 
Investigator expertise 
39 
19. 
Patient record access and handling of personal data 
39 
20. 
Insurance 
40 
21. 
Reimbursement of participants 
40 
22. 
SARS-Cov2 
40 
23. 
Economy 
40 
24. 
Publication plan 
40 
25. 
Bibliography 
42 
 

PrePhage – Faecal bacteriophage transfer for enhanced gastrointestinal tract maturation 
in preterm infants  
Ethical Registration Nr: 78259 
 
 
    
 
 
 
PrePhage 
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1. 
General information 
1.1. 
Protocol title, version number, date, list of abbreviations 
Title: PrePhage - Faecal bacteriophage transfer for enhanced gastrointestinal tract maturation in 
preterm infants 
VEK submission number: 78259 
Version: Version 02 
Date: 27/07/2021 
 
List of abbreviations: 
AE: adverse events 
CMV: Cytomegalovirus 
DBS: dried blood spot 
DSMB: data safety monitoring board 
FFT: faecal filtrate transfer 
FMK: det fælles medicinkort 
FMT: Faecal microbiota transplantation  
GA: gestational age 
GI: gastrointestinal 
GM: gut microbiota 
HAV, HBV, HCV: Respectively Hepatitis A, B, and C virus 
HIV: Human immunodeficiency viruses 
IBD: inflammatory bowel disease 
ITT: intention-to-treat 
NEC: necrotizing enterocolitis 
NICU: neonatal intensive care unit 
VEK: De Nationale Videnskabsetiske Komitéer 
rCDI: recurrent clostridoides difficile infection 
RCT: randomized control trial 
SAE: serious adverse events 
SSI: Statens Serum Institut 
UCPH: university of Copenhagen 
VEGF: vascular endothelial growth factor 
 

PrePhage – Faecal bacteriophage transfer for enhanced gastrointestinal tract maturation 
in preterm infants  
Ethical Registration Nr: 78259 
 
 
    
 
 
 
PrePhage 
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Research team and responsibility 
Lise Aunsholt 
MD, Ph.D. Assoc Professor 
Department of Intensive Care for Infants and Toddlers 
Copenhagen University Hospital, Rigshospitalet 
Blegdams vej 9 
2100 Copenhagen Ø, DK 
+45 35451325/ +45 61991137 
lise.aunsholt@regionh.dk 
 
Gustav Riemer Jakobsen 
MD, PhD student 
Department of Intensive Care for Infants and Toddlers 
Copenhagen University Hospital, Rigshospitalet 
Blegdamsvej 9 
2100 Copenhagen Ø, DK 
+45 50 56 95 36 
gustav.riemer.jakobsen.01@regionh.dk 
 
Dennis Sandris Nielsen 
Professor, M.Sc., PhD 
Microbiology and Fermentation 
Department of Food Science 
University of Copenhagen 
+45 35 33 32 22 
dn@food.dk 
 
Anders Brunse 
Post doc, cand.scient., PhD 
Department of Veterinary and Animal Sciences 
Comparative Pediatrics and Nutrition 
Faculty of Health and Medical Sciences 
University of Copenhagen 

PrePhage – Faecal bacteriophage transfer for enhanced gastrointestinal tract maturation 
in preterm infants  
Ethical Registration Nr: 78259 
 
 
    
 
 
 
PrePhage 
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+45 61 46 02 11 
anderss@sund.ku.dk 
 
Susanne Soendergaard Kappel 
CN, MSc in Nursing, Ph.D. 
Clinical nurse specialist - Department of Intensive Care for Infants and Toddlers 
Copenhagen University Hospital, Rigshospitalet 
Blegdamsvej 9 
2100 Copenhagen 
Denmark 
Tlf.: + 45 20822378 
susanne.soendergaard.kappel@regionh.dk 
 
 
1.2. 
Good clinical practice 
We will consult the good clinical practice board of University Hospital of Copenhagen before 
initiation of the study. 
 
1.3. 
Time plan 
  
ASAP 2022 2023 2024 2025 2026 
Start invitations 
  
  
  
  
  
  
Donor screening 
  
  
  
  
  
  
Clinical study 
  
  
  
  
  
  
Followup 
  
  
  
  
  
  
 
 

PrePhage – Faecal bacteriophage transfer for enhanced gastrointestinal tract maturation 
in preterm infants  
Ethical Registration Nr: 78259 
 
 
    
 
 
 
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1.4. 
Signature 
1.5. 
Study Location 
The clinical trial takes place at RH NICU. If the participants wish, some of the data collection 
can be performed at their homes.  
Biochemical and microbiological analysis is performed at the Department of Clinical 
Biochemistry at both RH and Odense University Hospital, as well as Statens Serum Institute 
(SSI).  
Analysis of the fecal microbiota will take place at Microbiology and Fermentation, at UCPH. A 
portion of the donated feces will be used in a piglet study at Comparative Pediatrics and 
Nutrition at UCPH.  
 
 

PrePhage – Faecal bacteriophage transfer for enhanced gastrointestinal tract maturation 
in preterm infants  
Ethical Registration Nr: 78259 
 
 
    
 
 
 
PrePhage 
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2. 
Summary 
This pilot study aims to investigate if faecal filtrate transfers (FFT) to preterm infants is safe and 
tolerable. To investigate this, we will recruit 10-20 donor infants and their mothers from time of 
delivery, and both will be subjected to a novel screening program including blood, urine, 
breastmilk, faecal screening and standard clinical investigation. Donor faecal samples will be 
collected from time of birth and with variating intervals for consecutive 3 years for 3 purposes: 1) 
to conduct safety studies in preterm piglets before transfer to preterm recipient infants, 2) to 
conduct FFT to preterm infants, and 3) to map normal microbiota development in healthy 
infants.  
The faeces used for donation will be collected between 2-4 weeks after birth. After one year, 
donated feces will be released for FFT to preterm, but only if the donor infant at this time has 
been healthy and normally developed. Donors are followed up for consecutive 3 years after 
birth. Maternal faecal samples will be compared to infant samples, to investigate maternal to 
infant transfer of microbiome. 20 preterm infants with gestational age (GA) between 26+0 – 
30+6 weeks + days, are block randomized to either FFT or saline placebo within 24 hours after 
birth and the following 3 days, in total 4 donations. The recipients are clinically and 
biochemically close monitored by attending staff and the group of research team according to 
best clinical practice and predefined clinical observation. The recipients are followed up for 
consecutive 3 years to evaluate potential late side-effects and to monitor change in faecal 
microbiome after transplant or placebo.  
The primary endpoint is to assess safety of FFT to preterm infants with expected no increase in 
necrotizing enterocolitis (NEC), sepsis and death in the intervention group. The secondary 
endpoint is to assess if, FFT treatment will reduce incidence of feeding tolerance and improve 
healthy gut development in recipient preterm infants. We expect to find FFT safe and with less 
cases of NEC and sepsis, though not powered to show the latter. We aim to follow up with a 
double-blinded multicenter randomized control trial, powered to document our hypothesis, that 
when colonizing with a healthy microbiome, we can decrease incidence of NEC in premature 
infants.  
 
 

PrePhage – Faecal bacteriophage transfer for enhanced gastrointestinal tract maturation 
in preterm infants  
Ethical Registration Nr: 78259 
 
 
    
 
 
 
PrePhage 
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3. 
Background 
Preterm birth 
Preterm births below gestational week 37 occur in 11% of all pregnancies worldwide and in 6% 
of Danish, with an incidence of very preterm pre-term births (< 32 weeks of gestation) of 1% in 
Denmark 1. Survival rates have improved over the years, due to improved obstetric and 
neonatal treatment, but a substantial fraction very preterm infants still experience subsequent 
comorbidities. 
 
Necrotizing enterocolitis 
Serious infections are common in preterm newborns due to aberrant microbial exposure and 
impaired host defense mechanisms. While the majority of these patients survive, many are left 
with chronic handicaps due to inflammation-related injuries including their developing brain 2. 
Necrotizing enterocolitis (NEC) is among the most prominent life-threatening infection of 
preterm newborns. NEC is a severe bowel inflammation associated with imbalanced gut 
microbiota (GM) development and impaired gut barrier function. Mortality is high (~30%), and 
treatment often involves surgical removal of the necrotized intestine with life-long consequences 
3. The incidence of NEC in the Copenhagen region is at present 9% (for infants born <1500g), 
with a maximum risk of 20.5% for infants with birth weight 500-599g 4,5. 
 
NEC and the gut microbiome 
Microbial colonization of the gut begins immediately after birth. Gestational age at birth, is the 
major determinant of the gut microbiome (GM) colonization pattern, exceeding the effects of 
both delivery mode, nutrition and antibiotic treatment 6,7. The GM of very preterm infants is 
dominated by Bacilli, Clostridia and Gamma-proteobacteria, with lowered bifido-bacteria 
presence, but with substantial intra-individual variability and significant fluctuations over time 8–
10. NEC has been linked to GM dysbiosis and NEC related GM patterns have been identified 
8,10,11. 
 
Bacteriophages and the early life gut virome 
Viruses are as abundant as bacteria in the human gastrointestinal (GI) tract. Of these, only few 
infect human cells, whereas the vast majority attack and kill bacteria. Such viruses are called 
bacteriophages (phages) and are specialized in targeting bacteria in a highly host specific 
manner, shaping microbial community structure, and maintaining bacterial diversity 12. 

PrePhage – Faecal bacteriophage transfer for enhanced gastrointestinal tract maturation 
in preterm infants  
Ethical Registration Nr: 78259 
 
 
    
 
 
 
PrePhage 
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The role of phages during early life microbiome establishment is poorly understood, but recent 
findings support that they play important roles in shaping the GM 12,13. Already during the first 
month of life the term infant gut is colonized by eukaryotic viruses such as Adenoviridae, and 
Annelloviridae as well as vast amounts of phages, with bacteria and phages being present in a 
ratio of 1:1 in the infant gut 13,14. In adults virome dysbiosis plays a pivotal role in the 
development of inflammatory bowel disease (IBD) 15, but the role of virome dysbiosis for 
diseases manifesting in early life is unknown. 
 
Microbial therapies for NEC prevention  
Probiotics are administered to preterm infants at many (but far from all) hospitals to prevent 
NEC and Meta-analysis supports implementation of probiotics as NEC prophylaxis in routine 
care of preterm infants 16. However, the NEC incidence reduction is rather modest and the large 
English PiPS trial did not find any beneficial effect of probiotic administration to preterm infants 
with respect to NEC 17,18. 
 
Faecal microbiota transplantation (FMT) has recently gained much interest due to efficient 
correction of GM dysbiosis and clinical improvement in patients suffering from recurrent 
Clostridioides difficile infection (rCDI) 19. In a preterm pig NEC model we recently showed 
beneficial effects of FMT on gut dysbiosis and NEC incidence 20. However, FMT also had 
adverse effects, increasing the risk of bacterial translocation, inner organ colonization and 
sepsis. Hence, FMT based on crude donor feces should not be tested in preterm infants. 
Despite of that, it should be noted that in a study in Helsinki, Finland, mother-to-infant low-dose 
FMT for term infants delivered by Caesarean section showed no adverse events and a 
significant improvement in gut microbiome maturation 21.  
Interestingly, it has been shown that removal of all intact bacteria from a donor faecal slurry by 
sterile filtration (leaving only viruses and small-size molecules) before administration to rCDI 
patients has similar efficacy as regular FMT 22. Inspired by this, we tested donor faecal filtrate 
transfer (FFT) in comparison to regular FMT in the preterm pig NEC model. Orally administered 
FFT was superior to conventional FMT, consistently preventing NEC and reducing gut 
permeability with no obvious adverse effects 23. Our data indicate that the donor-derived phages 
specifically inhabit the viscous mucus layer, where they eliminate NEC-related pathogens.  

PrePhage – Faecal bacteriophage transfer for enhanced gastrointestinal tract maturation 
in preterm infants  
Ethical Registration Nr: 78259 
 
 
    
 
 
 
PrePhage 
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The term infant gut bacterial microbiome undergoes significant fluctuations during the first years 
of life 24, and the optimal age of a faecal donor for FFT to preterm infants is unknown. However, 
a few weeks into life the GM of term infants’ transits from a very dynamic, unstable phase into a 
more stable. Colonization of the GI-tract by bacteria begins in the first few weeks and a primitive 
flora is formed at 1 month of age 13. Further, in piglets we have observed that 10-day old piglets’ 
function well as donors for FFT to preterm piglets 23. Based on our findings from the preterm pig 
model, we hypothesize that 2-4 weeks after birth is optimal time for donation. 
 
Given these encouraging preliminary findings, PrePhage aims to develop a phage-based 
prophylactic treatment, assess its safety and NEC-preventive efficacy in preclinical piglet 
experiments, and ultimately test its safety and feasibility in a phase 1 clinical trial on preterm 
infants in a danish neonatal care unit.  
 
4. 
Objectives, hypothesis and feasibility 
The primary purpose of this study is to demonstrate the safety and investigate the feasibility of 
administering FFT to preterm infants.  
 
At first, a carefully designed donor screening program is developed, selected donors are 
screened, and faecal samples collected. Donors and the mothers of donors are followed for 3 
years to ensure that the donor is and remains healthy. 
Faecal samples are thoroughly screened for known pathogens, filtrated and manipulated to 
remove known eukaryotic vira associated with risk of infection. The product is then transplanted 
to preterm piglets. If no adverse effects are observed, validated FFT will be transplanted to 
preterm infants in a progressive clinical pilot study.  
The study will be blinded. Statistical and laboratory analyses will be performed blindly. 
 
Stop/go decision 
The decision to continue to the clinical pilot trial depends on the results of an in-vivo evaluation 
of safety in preterm piglets. Pre-term piglets are given human FFT, followed for 2 weeks and 
euthanized. The safety in piglets is evaluated in terms of growth impact, feeding tolerance, stool 
patterns, gut histopathology, complete blood count, hepatic and renal function tests as well as 
blood culture and arterial blood gas analysis in case of suspected infection. If the study gives 

PrePhage – Faecal bacteriophage transfer for enhanced gastrointestinal tract maturation 
in preterm infants  
Ethical Registration Nr: 78259 
 
 
    
 
 
 
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any indication that it would be unsafe to continue the trial, the study will be either discontinued 
or a contingency plan will be developed and submitted to “De Videnskabsetiske Komitéer” 
(VEK) before continuation. 
 
Our primary aim is to demonstrate that administering FFT from donor term infants is safe and 
tolerable when used from the first 24 hours of life in preterm infants born between 26+0 and 
31+6 weeks of gestational age, hence the pilot trial. 
 
Our secondary aim is to examine if FFT can improve gastrointestinal outcome for preterm 
infants in terms of:  
● Reduced time to development of balanced gut microbiome and virome 
● Increased feeding tolerance 
● Reduced time to full enteral feeding 
● Improved infant thriving 
● Reduced incidence of NEC 
 
The tertiary purpose of this trial is to map development of faecal virome over time and to 
determine donor characteristics for optimal outcome of FFT, hence the donor study. 
We will map the development of the infant virome in conjunction with child feeding patterns, GI-
development and thriving as well as in relation to maternal microbiome.  
 
Finally, we wish to examine a broad range of explorative outcomes to study mechanism-of-
action of FFT as well as effects of differences in gut microbiome and virome on normal 
gastrointestinal development and lastly to identify effect modifying factors. 
 
● Faecal calprotectin 
● Key inflammatory markers 
● Total DNA and virus-like particles will be extracted from faecal samples 
● Absolute bacterial abundance of faecal samples 
● Determination of gut metagenome in faecal samples using shotgun high-
throughput sequencing 
 

PrePhage – Faecal bacteriophage transfer for enhanced gastrointestinal tract maturation 
in preterm infants  
Ethical Registration Nr: 78259 
 
 
    
 
 
 
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As the clinical study is a feasibility study, with only 10 infants receiving FFT and 10 infants 
receiving placebo, sample size is not likely to be large enough to demonstrate above effects. 
Therefore, it is our intention to follow up with a larger randomized clinical trial, as well as 
develop a biobank for use in further studies if safety and tolerability is confirmed in this study.  
 
Feasibility 
Motivations to participate in clinical trials are altruism along with the hope of personal gain 25. 
For infants, the informed consent is given by the parents, who can be in a conflict of interest 
since their role is to protect and care for the infant.  
To gain more knowledge about motivation to participate in a clinical trial like PrePhage a nurse-
run feasibility study of FFT to preterm infants will be carried out. Using ethnographic fieldwork in 
the NICU and semi-structured interviews with parents and the NICU staff, their response and 
reactions toward the study will be investigated.  
The aim is to generate qualitative and quantitative data to identify factors of importance for later 
larger clinical trials. 
Feasibility outcomes: 
1) Consent rate (willingness to participate) 
2) Adherence sample collection 
a. Proportion of missing blood and fecal sample 
b. Proportion of incomplete questioners 
3) Qualitative evaluation of source documentation and procedures 
a. Informed consent procedures 
b. Data collection procedures and tools 
c. Sample collection of biological material (sample collection, freezing) 
 
Based on data material, combined with ethnographic fieldwork in the NICU, the group of 
coordinating investigators will create an overview (see below) to help the clinically responsible 
and the project leader (in correspondence with the DSMB) to decide whether it is feasible to go 
ahead with the planning of a larger RCT based on results and practical experience from the pilot 
study. 
To generate knowledge about informed consent we plan to do interviews of parents both given 
consent and declined consent (informed consent will be collected before). To generate 
knowledge about data collection procedures, handling of the FVT product, procedures for 

PrePhage – Faecal bacteriophage transfer for enhanced gastrointestinal tract maturation 
in preterm infants  
Ethical Registration Nr: 78259 
 
 
    
 
 
 
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reporting adverse events and sample collections we plan to do an ethnographic fieldwork of the 
staff members involved in the project. In case of concerns corrections will be made before a 
larger RCT is initiated.    
 
 
 
 
 
 
Consent rate 
>70% 
50-70% 
<50% 
Proportion of completed blood and faecal 
samples  
>50% 
20-50% 
<20% 
Proportion of incomplete questioners 
<20% 
20-50% 
>50% 
 
Pre-defined criteria used to decide whether, or how, to proceed with a later, larger RCT. Green 
color: fully acceptable and feasible. Yellow color: feasibility concern, changes to be decided. 
Red color: serious feasibility concerns. Clear actions must be taken before a larger RCT is 
planned. 
5. 
Donor study 
5.1. 
Design and recruitment 
 
10-20 eligible donor infants will be recruited while intrauterine for 3 purposes: 1) to donate feces 
for a safety trial in piglets, 2) to donate feces for the pilot FFT trial 3) to study development of 
gut virome and microbiome over time. The piglet safety trial is described in a separate protocol, 
and thus not described in detail in this protocol.   
Infants included will be followed up one year before donation can be executed and consecutive 
for three years (2022 – 2024). Faecal samples and questionnaires will be collected throughout 
the period. A novel donor screening program, including blood, faecal and urine samples from up 
to 20 term infant donors is applied. 
 
The mothers of infants will be recruited from in-patient maternity clinics in The Capital Region of 
Denmark (Region Hovedstaden) and will be included in the study, for three purposes. 1) To 
screen for preventable transmittable disease. 2) To compare development of the infant faecal 
microbiome to the microbiome of their mothers. 3) To identify external factors that might lead to 

PrePhage – Faecal bacteriophage transfer for enhanced gastrointestinal tract maturation 
in preterm infants  
Ethical Registration Nr: 78259 
 
 
    
 
 
 
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unforeseen changes in donor microbiome development. They will be followed up for 1 year or 
until the child is no longer breastfed, whichever comes first. They will be asked to answer 
questionnaires, provide blood- milk- urine- and faecal samples as well as provide access to their 
obstetric and medical record (Fælles Medicin Kort (FMK)). 
 
Mother and child cannot be enrolled separately. If either mother or child does not fulfill 
inclusion/exclusion criteria, neither can participate. 
Some maternal samples may be collected before birth. If complications arise during or after birth 
so they no longer fulfill inclusion criteria, mother and child are excluded from the study and all 
data and biological material collected up till this exclusion will be destroyed. 
5.2. 
Inclusion criteria for donors 
• 
The donor must be of term (>37+0 weeks GA, < 41+0 weeks GA),  
• 
Be born vaginally with no maternal pre-birth infection,  
• 
Be exclusively breastfed un till fulfilled donation at 4 weeks of age,  
• 
Have no known predisposition for disease.  
5.3. 
Exclusion criteria for donors 
• 
Antibiotic exposure before collection of faecal material for donation,  
• 
Disease between time of birth and collection of feces for donation,  
• 
Major congenital anomalies or birth defects, perinatal asphyxia, need for mechanical 
ventilation or cardiovascular support before time of inclusion.  
• 
Positive stool sample for C. difficile toxin, parasites or other pathogens 
• 
Positive HIV, HBV, or HCV or CMV 
• 
Parents who do not want to know the HIV, HBV or HCV status of the child 
5.4. 
Inclusion criteria for mothers of donors 
• 
Women aged 18-45 and currently healthy 
• 
No continuous medical consumption with effects on microbiome 
• 
Non-smoking 
• 
Ability to give informed consent 

PrePhage – Faecal bacteriophage transfer for enhanced gastrointestinal tract maturation 
in preterm infants  
Ethical Registration Nr: 78259 
 
 
    
 
 
 
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5.5. 
Exclusion criteria for mothers of donors 
• 
Known or high risk of infectious disease such as HIV, HBV, or HCV 
• 
Positive CMV IgM during pregnancy 
• 
Positive stool sample for C. difficile toxin, parasites or other pathogens 
• 
Systemic antibiotic treatment < 1 months prior to study 
• 
New tattoo < 1 month prior to study 
• 
Risky sexual behavior    
• 
Gestational diabetes 
• 
Family history of inflammatory bowel disease 
 
5.6. 
Primary endpoints 
Characterization of faecal virome and microbiome over time in the term infant to map early gut 
development and relation to feeding patterns, growth, normal infant development and 
association to maternal faecal microbiome. 
Further we find it important to identify if any donors develop serious gastrointestinal disease (not 
including infectious diarrhea) during the study period, to ensure their feces is not used for 
human FFT. 
5.7. 
Secondary endpoints 
Clinical evaluation of normal development, anthropometrics from birth to end of observation (3 
years of age), time to establish breastfeeding, length of hospital stay related to birth, days to 
regain birth weight, stool characteristics and frequency (combining Amsterdam Stool Scale 26 
and Diapered Infant Stool Scale 27 at first, switching to a combination of Bristol stool scale and 
Diapered Infant Stool Scale after full solid nutrition), feeding pattern (feeding intolerance, 
frequency, type of nutrition,) and time switch from fluid to full solid food as well as frequency of 
infection from birth to end of observation. 
5.8. 
Overview screening, data collection and fecal sample collection, donor 
study 
Potential donors are screened at Rigshospitalet according to age-adjusted international 
standards for FMT treatment 28,29 as well as clinical guidelines 30. Mothers of potential donors 

PrePhage – Faecal bacteriophage transfer for enhanced gastrointestinal tract maturation 
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are likewise screened at Rigshospitalet according to adjusted international standards for FMT 
treatment 28,29. 
Donors are screened before sampling for donation, then donor stool is quarantined for one year 
till successful first year follow-up. Following a flawless screening and normal development of the 
donor the collected stool is released for use in the human safety study. Our preclinical 
experiments suggest that the NEC-reducing efficacy of FFT is maintained despite 2-3 years 
storage of donor stool at -80°C. 
 
 
Figure 1 Overview of data collection, donor study 
 
5.8.1. 
Donor infant screening and data collection 
• 
Blood sample for screening is taken at between birth and 14 days:  

PrePhage – Faecal bacteriophage transfer for enhanced gastrointestinal tract maturation 
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o Serological test for hepatitis A (HAV-IgM), B (HbsAg), and C (anti -HCV); HIV-1 
and HIV-2 (anti-HIV-1,2); syphilis (screening: WR and RPR), CMV (CMV-IgM 
and CMV-IgG) and EBV (VCA IgM, VCA-IgG and EBNA IgG), as well as NAT 
screening for HIV, HBV, and HCV. 
▪ 
HbsAg, Anti-HCV, HIV-1 and 2, NAT-Screening for HIV, HBV and HCV 
will be performed on dried blood spots (DBS) on the PKU at Department 
of Clinical Biochemistry at Odense University Hospital 
▪ 
CMV will be analyzed as DBS from the PKU at Statens Serum Institut 
(SSI) 
▪ 
EBV antibodies as well as syphilis reactions analyses are currently 
neither available as capillary blood samples nor analyses on dried blood 
spots.  
• 
We are in correspondence with collaborators to develop a 
possibility for this before initiation of the trial. If this proves 
impossible to do, we will omit these samples from the infant. The 
probability of the infant having syphilis while the mother does not 
(as she is screened at inclusion) is extremely low. In terms of 
screening for EBV, it is improbable, that the donor infant will 
contract and become infected the virus without displaying 
symptoms of it in the 2-4 weeks after birth, where feces for 
donation is collected. Therefore, we do not believe the benefit of 
this screening outweighs the discomfort of taking a venous blood 
sample, nor can we justify drawing the large blood volume 
currently needed to perform the analysis. 
o Blood samples will be collected as capillary samples by trained personnel at RH 
NICU.  
o Donor infants are expected have 80 ml of blood/kg and to weigh at least 3000 g, 
thus having a total blood volume of at least 240 ml. Current regional guidelines 
advise that blood volume taken at a given time is not to exceed 1% of total blood 
volume and 3% over a week 31.  
o The total volume will be 2.45 ml, 1 % of minimally expected blood volume in total. 
• 
Nasal swab: if donor infants are symptomatic of an upper respiratory tract infection at 
sample collection or clinical examination, a nasal swab for RS virus as well as influenza 

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A and B virus will be performed 
 
• 
Urine sample taken at inclusion and week 2, 3, 4, 6, 8, 12 and 24 to screen for CMV 
o Urine samples are collected using a urine collection bag, as they are exclusively 
used to screen for CMV 
• 
Faecal samples, growth measurement and: weekly during the first month, every 14 days 
from month 1-3, every 3 months until the age of 2 years, once at the age of 3 years.  
o Faecal samples are collected from diapers. If donor is potty-trained, they will be 
provided with standard collecting kits. Feces is removed from the diaper/potty, as 
soon as possible after defecation. As soon as removed, feces are stored in a 
container and kept until picked up by research team at home. During week 2-4, 
feces are divided in to 3 at the hospital, two small proportion and one larger. 
Feces is then stored in containers marked with each donor’s ID, until used for 1) 
faecal sample screening as described beneath, 2) testing in preterm piglets and 
2) donation for preterm infants. 
o Faecal samples collected from week 2-4 after birth are potentially used for 
transplantation and are screened for:  
▪ 
Clostridioides difficile, Salmonella spp., Shigella spp., Campylobacter coli 
/ jejuni, Yersinia enterocolitica, Aeromonas spp. 
▪ 
Diarrhea-causing Escherichia coli (E. coli): (Verocytotoxin producing E. 
coli (VTEC), Enteropathogenic E. coli). Enterotoxigenic E. coli (ETEC), 
Enteroinvasive E. coli (EIEC), and Attaching and Effacing E. coli (A / 
EEC)  
▪ 
Intestinal pathogenic viruses: Adenovirus, Atrovirus, Sapovirus, 
Enterovirus, Parechovirus, Rotavirus and Norovirus; intestinal pathogenic 
parasites: Entamoeba histolytica, Cryptosporidium parvum/hominis, 
Giardia lamblia and worms  
▪ 
Multiresistant bacteria: Extended-spectrum beta-lactamase (ESBL) 
producing E. coli, Klebsiella pneumoniae and Proteus mirabilis, 
Carbapenemase-producing organism (CPO), vancomycin-resistant 
enterococci (VRE) 
▪ 
In addition, they are tested for Helicobacter pylori antigen, SARS-CoV-2 
RNA, as well as RS-virus and Influenza A and B virus 

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o Faecal samples collected during week 2-4 are prepared for FFT to preterm 
piglets and preterm infants, as described in section 6.9 
o Faecal samples collected outside week 2-4 are only used for microbial 
characterization.    
o Gut microbiome and virome are analyzed as described in section 7. 
o At the first donation, approx. 10 mL of faeces will be stored. These are stored at -
80 ° C in case a recipient is later diagnosed with known or currently unknown 
infectious disease, where it is desired to investigate whether the donor material 
contained the microbiological agent. 
o Screening of faecal samples will be performed at RH Department of Microbiology 
• 
Questionnaires: are performed at the same time intervals as faecal samples. Questions 
are arranged in 5 domains:  stool characteristics, feeding pattern, infection and, and 
behavior (Questionnaire included in appendix 7) 
• 
Clinical examination: at inclusion, at 2 weeks, at 4 weeks, 6 months, 1, 2 and 3 years 
o Clinical examination is performed according to best clinical practice to ensure 
normal development of the infant. The clinical examinations will be documented 
in the patient record.  
• 
Access to patient record and FMK 
o FMK access is required throughout the data-collection period to adjust for any 
antibiotic use, when analyzing composition of faecal mikrobiota. Further, time 
since last antibiotic treatment is required, to adjust for impact on faecal 
microbiota. All medication that potentially could affect the flora of faeces must be 
identified via FMK.    
o Patient record access is required throughout the data-collection period to identify 
any donors who develop significant gastrointestinal, autoimmune or acute 
diseases that would prohibit the infant from being a donor. (i.e., inflammatory 
bowel disease or major congenital disease requiring surgical treatment, 
diabetes). Records will be accessed in conjunction with the clinical examinations.   
5.8.2. 
Maternal screening and data collection 
• 
History at inclusion: ethnicity, number of siblings in the household, socioeconomic status 
(level of education and current income), smoking 

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• 
Blood samples: taken at inclusion for screening purposes, lipid profile, Hba1c as well as 
complete blood count (i.e. leukocytes, differential count, platelets, hemoglobin), 
immunoglobulin A (IgA) and serological test for hepatitis A (HAV-IgM), B (HbsAg and 
Anti-Hbc), and C (anti -HCV); HIV-1 and HIV-2 (anti-HIV-1,2); syphilis (screening: WR 
and RPR), CMV (CMV-IgM and CMV-IgG) and EBV (VCA IgM, VCA-IgG and EBNA 
IgG), as well as NAT screening for HIV, HBV and HCV. 
o All maternal serological screening will be analyzed as venous blood samples at 
RH Department of Microbiology 
• 
Urine samples at weeks 1, 2, 3, 4 to screen for CMV. Milk samples at weeks 1, 2, 3, 4, 6, 
8, 12 and 24 to screen for CMV. Milk samples are collected until cessation of 
breastfeeding.  
o Both urine and milk samples are collected according to standard procedure 
• 
Faecal samples: before birth, weekly during the first month after birth, at 6 months and 
12 months.  
o After birth faecal samples are used for microbial characterization. Gut 
microbiome and virome are analyzed as described in section 7 
o Faecal samples before birth are testet for Clostridioides difficile, Salmonella spp., 
Shigella spp., Campylobacter coli / jejuni, Yersinia enterocolitica, Aeromonas 
spp., diarrhea-causing Escherichia coli (E. coli): (Verocytotoxin producing E. coli 
(VTEC), Enteropathogenic E. coli). Enterotoxigenic E. coli (ETEC), 
Enteroinvasive E. coli (EIEC), and Attaching and Effacing E. coli (A / EEC); 
intestinal pathogenic viruses: Adenovirus, Atrovirus, Sapovirus, Enterovirus, 
Parechovirus, Rotavirus and Norovirus; intestinal pathogenic parasites: 
Entamoeba histolytica, Cryptosporidium parvum/hominis, Giardia lamblia and 
worms; multiresistant bacteria: Extended-spectrum beta-lactamase (ESBL) 
producing E. coli, Klebsiella pneumoniae and Proteus mirabilis, Carbapenemase-
producing organisms (CPO), vancomycin-resistant enterococci (VRE). In 
addition, the samples will be tested for Helicobacter pylori antigen and SARS-
CoV-2 RNA.  
 
• 
Questionnaire: Monthly during study period until the child is primarily eating solid foods. 
Questions are asked regarding diet, smoking, alcohol intake, gastrointestinal symptoms, 
stool characteristics, and use of medicine (appendix 7) 

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• 
Obstetric record and FMK:  
o Before  inclusion: Access to obstetrical information and FMK, is a allowed to 
ensure the participant is eligible for inclusion,  according  listed inclusion criteria..  
o At inclusion and until the mother stop breastfeeding the child: Access to FMK to 
adjust for any antibiotic use or other drugs that could affect the faecal microbiota. 
Knowledge is used when analyzing data of microbiota and especially in comparison 
between mother and infant microbiota 
• 
Throat swab:  to test for SARS-CoV-2 and Respiratory Syncytial Virus (RSV) . Testing 
will follow current local guidelines at Rigshospitalet NICU.  
 
6. 
Clinical Trial 
In a double-blinded, block randomized study, 20 preterm infants (GA 26+0 to 30+6) will be 
randomized to either FFT or saline solution.  
6.1. 
Design 
 
Block-Randomization 
The block randomization will be performed before treatment is initiated. Infants who meet the 
inclusion criteria will have FFT or saline solution administered within the first 24 hours after birth 
and treatment will be double-blinded. We aim to give first treatment during daytime. 
Treatment will be stepwise, and the Data Safety Monitoring Board will receive information about 
acceptance of treatment, suspected adverse events or other observations, after each step. 
Each step will be monitored for 4 weeks before moving onwards to the next. Steps will be as: 
 
1) 1+1 
2) 1+1 
3) 1+1 
4) 7+7 
 
All cases will be followed-up for at least 4-weeks or until discharge, whichever comes first. 
Long-term follow-up is performed till 3 years after study completion. 

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6.2. 
Inclusion criteria for participant preterm infants 
● Preterm infants born between GA 26+0 and 30+6 
● Delivery at RH or transferred to RH NICU within 24 hours of delivery 
● Signed parental consent 
6.3. 
Exclusion criteria for participant preterm infants 
● Major congenital anomalies or birth defects 
● Congenital infection 
● Extremely SGA infant (weight SD score < -3 SD) 
● Need for mechanical ventilation or cardiovascular support before first FFT 
treatment 
6.4. 
Inclusion criteria for mothers of participants 
● Women aged 18-45 
● Ability to give informed consent 
6.5. 
Exclusion criteria for participant mothers 
 
● Mothers who have severe infection, defined by need for other treatment 
to support infection-related comorbidities, besides from antibiotics (e.g. 
inotropic treatment, iv fluid resuscitation) 
6.6. 
Endpoints, clinical trial 
The trial is designed as a feasibility study. We are aware, that it is not powered to investigate 
any of the endpoints with statistical significance.  
6.6.1. 
Primary endpoints 
To demonstrate the safety and tolerability to FFT in terms of no serious adverse events: 
specifically, no increased incidence of serious infections including NEC, sepsis, and death. 
6.6.2. 
Secondary endpoints 
We wish to examine the potential benefits of FFT in terms of 

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• 
Characterization of faecal virome and microbiome over time in the term infant to 
map early gut development determine gut microbiome maturation relative to the 
known progression in term infants 
• 
feeding intolerance 
• 
time to full enteral feeding 
• 
stool characteristics 
• 
paraclinical data (e.g. CRP, leukocytes, and capillary acid/base status) 
• 
anthropometric data 
• 
days of hospitalization 
6.7. 
Overview of data collection from participants 
 
Figure 2 Overview of data collection, clinical trial 
• 
Blood samples in the neonatal period: routine blood samples taken for clinical purposes 
are used in the study (capillary acid/base, thrombocytes, CRP, total leucocytes and 
coagulation factors). These are standard blood tests taken at birth, after 12 hours and 
daily for the first week for the vast majority of infants admitted to Rigshospitalet NICU 
o After the first week we ask for permission to continue the use of daily routine 
samples (capillary acid/base, CRP and in case CRP increase, then thrombocytes 
and total leucocytes) for one more week and in case of suspicion of adverse 
events.  
▪ 
Recipient infants are expected to have 80 ml of blood/kg and expected 
weights between 600 and 1500 g at inclusion, leading to a total blood 

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volume of 48-120 ml of blood. 32,33 Current regional guidelines on clinical 
samples advise that blood volume taken at a given time does not exceed 
3% of total blood volume, and that total volume collected during a 30-day 
period does not exceed 10% of total blood volume 31. 
▪ 
Required volumes for samples:  
• 
Acid/base-status: 0.1 ml 
• 
CRP: 0.01 ml 
• 
White blood cell count and platelets: 0.25 ml 
▪ 
Daily samples are maximally 0.36 ml of blood, 0.8% of expected minimum 
blood volume. Planned samples comprise 0.77 ml, 1.6 % of expected 
minimum blood volume. If it is deemed necessary to collect white blood 
cell count and platelets for clinical reasons (suspected infection), this is 
considered to be of clinical need and is not counted in total blood volume 
for research purposes.    
 
• 
Urine samples: 1 sample each week for 4 weeks, the first urine is sampled after 1 week 
o Urine samples are collected using a collecting bag 
o Urine samples are used only to detect CMV 
• 
Faecal samples:  
o Feces is removed from the diaper as soon as possible after defecation. Feces is 
removed from the diaper, as soon as possible after defecation. As soon as 
removed, feces are stored in a container and is given to either RH NICU staff or 
research team while admitted to the NICU or collected by research team if 
discharged. 
o Sample collection: Daily until 7 samples are collected, then weekly during the first 
month, every 14 days from month 1-3, every 3 months until the age of 2 years, 
and once at the age of 3 years. 
o Gut microbiome and virome are analyzed as described in section 7 
o Faecal calprotectin is analyzed in all samples during the first month as a marker 
of gut inflammation. 
 
• 
Growth: Daily measurement of weight till regained birth weight then weekly along with 
length and head circumference while admitted to the NICU. After discharge, 

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anthropometrics will be measured at the same times as faecal sample collection. 
 
• 
Evaluation of feeding tolerance: Clinical evaluation every second hour according to 
standardized procedure. Parameters include ventricle content from nasogastric tube, 
abdominal appearance, and quantity and characteristics of stool (appendix 8) 
 
• 
Questionnaire on feeding, faecal pattern, and well-being: Once the infant is on full 
enteral feeds and discharged from the NICU, participants will fill out the same 
questionnaires as the donors (Questionnaire included in appendix 7). 
 
• 
Clinical examination by research team in addition to routine examinations performed at 
RH NICU: daily for the first week, at 2 weeks, 3 weeks, 4 weeks, 6 months, 1, 2 and 3 
years 
o Clinical examination is done according to best clinical practice for examining 
normal health and development 
 
• 
Access to patient record and FMK 
o Before inclusion: patient record of mothers, who are at risk for preterm birth, will 
be accessed to ensure mothers fulfil inclusion criteria and that infants are not 
intrauterine diagnosed with known congenital diseases that will led to exclusion. 
If mother and infant before birth are eligible for inclusion, this information will be 
passed on to the research team.   
o After birth patient records of both mother and infant is accessed to ensure all 
inclusion criteria is still fulfilled 
o Infant FMK access is required throughout the data-collection period to adjust for 
any use of antibiotics or other drugs with potential effects on analysis  of faecal 
microbiota composition ..  
o Infant patient record access is required throughout the data-collection period to 
identify positive effects and potential side effects to intervention during admission 
and till termination of the study. This is part of the safety follow-up, to ensure that 
treatment does not cause long-term side-effects. Patient records will be 
accessed in conjunction with the clinical examinations.   

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6.8. 
FFT treatment 
FFT will be administered in a suspension via a nasogastric tube, which the preterm infants will 
have as part of their treatment in all cases. The first dose will be given within 24 hours and 
repeated every 24 hours for 3 days for a total of 4 doses. The volume of each dose will be 1 ml. 
The small volume ensures that the process of administering the transplant will not disturb 
enteral feeding of the infant. 
6.8.1. 
Faecal viral transplant – Risk assessment 
The viral transplant will be screened for known pathogens as described in the donor study. 
Furthermore, the risk of translocation of bacteria to the blood stream seen with full faecal 
transplants will be avoided, since the filtrate will contain virtually no bacteria.  
 
As a nasogastric tube is already necessary for nutritional purposes in the premature infant, this 
will provide no additional discomfort to the infant. Administration of faecal microbiome 
transplantation by nasogastric tube has been seen to be well-tolerated and safe 21. It should be 
noted that the dosage administered was relatively low (0.7–16 3 10^6 live bacterial cells). 
 
6.9. 
Preparation of faecal filtrate transplantation 
The faecal virome used for transplantation will be isolated from approved donors (10-20 donors) 
after the thorough screening process. Donor faeces are processed without mixing faeces from 
several donors, i.e. each recipient will receive faecal virome transplantation from the same 
donor. This is done to ensure traceability between donor and recipient. 
The faecal donor material is collected in anaerobic sterile zip-bag. The faecal samples will be 
dissolved in buffer (stabilizing phages). Bacteria and other larger fragments are removed by 
centrifugation followed by sterile filtration (0.45 µm filter) after which the virome will be 
concentrated using Centrisart® tubes to the same volume, as the original sample 34–36. The final 
mixture will be stored at -80 °C until use. The faecal virome will be administered to the infants 
mixed in their daily feed. A total volume of 1 ml of faecal virome will be administered to the 
infants on day 0, 1, 2 and 3. The faecal virome will contain the same number of bacteriophage 
particles, as the original faecal (approximately 10(9) bacteriophages/ml). 
 

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Figure 3 Illustration of the FFT preparation 
6.10. 
Clinical observation and safety during admission 
Participants in the study will be admitted to RH NICU. They will be monitored and treated 
according to best clinical practice. Participation in the study will not influence other treatment 
given while admitted to the NICU. NICU personnel will have access to routine samples taken 
during the study as well as study evaluation of feeding tolerance (appendix 8).  
Management of any potential complications will be done in collaboration with NICU doctors. 
6.11. 
Long term follow-up after discharge 
Participants are followed clinically until the age of 3 as described in overview of data collection. 
Parents have direct access to project staff throughout the study period and are encouraged to 
contact research team with any potential questions or unforeseen complications.  
6.12. 
Randomization code 
Randomization code is kept securely. Access to the code is done if serious adverse events are 
suspected.  
7. 
Analysis of microbiome and virome in faeces 
 
Faecal samples will be collected according to standard operation procedures for subsequent 
standardized total DNA extraction as well as total virome DNA/RNA extraction. All analysis will 

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be performed at University of Copenhagen, Food Science, Department of Microbiology and 
Fermentation.  
 
Total genomic DNA will be subjected to deep metagenome sequencing and related to the study 
outcomes. When extracting faecal DNA as well as viral DNA/RNA, physical fractionation or 
selective lysis will be employed to ensure host DNA is kept to a minimum. Remaining host DNA 
material will be removed during bioinformatics filtering and mapping of the shotgun 
metagenomics data. These procedures will ensure that no human genome data end up in the 
microbiome data. The content of metabolites in faeces will be analysed based on metabolomics 
at Department of Food Science, UCPH using a combination of proton NMR spectroscopy and 
LC-MS. 
8. 
Biobank and storage of samples 
Biological material that is not immediately sent for analysis (feces and milk samples) is stored in 
a research-biobank created for this study and registered centrally at NEXS, UCPH, until it is 
analyzed. In this, biological material is stores until analyzed. Further mothers and parents of 
participants are asked, for permission to donate excess biological material, in case of any. This 
excessed material will be stored in a registered biobank at NEXS, UCPH in accordance to 
Datatilsynets regulations as decribed in section 5.3 
(https://www.nvk.dk/emner/biobanker/vejledning-om-bio-mat). If “Datatilsynet” does not give its 
consent to create the new biobank, all excess material will be destroyed at the conclusion of the 
study in 31/12/2030. 
Consent for donation of material to the biobank is part of the signed consent form (appendix 4, 5 
and 6), and patients are informed about this in the participant information (appendix 2 and 3). 
The material will only be used in new and related research projects after these have been 
approved by the VEK, unless otherwise specified by the guidelines of VEK. If a participant 
agrees to donate possible excess material to the biobank and later regret it, they can contact 
either the research team or NEXS and have their material destroyed, unless the samples have 
been anonymized. This means that any ID-log that connects study participants and biologic 
material is destroyed. 

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Capillary blood samples and dried blood spots from the clinical study are not stored, as we 
expect to have no excess material. Blood samples are sent for analyses directly after collection.  
Urine samples are analyzed for CMV directly after collection. Excess urine is not stored.  
Part of the milk samples are sent directly to analysis for CMV after collection. Excess material, if 
any, is stored as described above.  
The PI is aware of the fact, that collection of biologic material for a biobank is not covered by the 
committee law, but by the GDPR/Databeskyttelsesloven. All biological samples in the Biobank 
are handled confidentially and stored in accordance with applicable law, GDPR and 
“Databeskyttelsesloven”. 
9. 
Recruitment, consent, and participant information 
9.1. 
Recruitment strategy 
9.1.1. 
Recruitment for Donor Study 
Participants for the donor study are recruited in 2 different ways. The first is using posters 
placed at strategic locations at the campuses for medicine and veterinary medicine at UCPH, as 
well as uploading digital versions of the posters to relevant online study platforms. The second 
is through the obstetric out-patient clinics in Region Hovedstaden (RH, Herlev and Hvidovre, but 
not Nordsjælland), where clinical staff will inform briefly about the study when the family is there 
for routine checkups. They will be given a small pamphlet with information regarding the study 
and contact information of the research team. When families contact the research team, an 
information meeting will be planned directly.  
9.1.2. 
Recruitment for Clinical Trial 
Parents admitted to the Department of Obstetrics at Rigshospitalet will be informed briefly 
verbally and in writing about the study when they are given standard information regarding 
preterm birth. If they are interested, they will be given the written participant information and will 
be contacted by the research team to be fully informed before potential inclusion in the study.  
 

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9.2. 
Consent 
Both parents (if two) must give consent to inclusion of the infant in both donor study and clinical 
trial for the infant to be enrolled in the study. In the donor study, the mother must also give 
consent to participating in the study. 
9.3. 
Principles for informing participants and obtaining consent 
Potential participants identified as described above receive an informative consultation. Prior to 
this, the investigators will consult the participants medical record and FMK in order to ensure, 
they fulfill the inclusion criteria.  
In both studies the informative consultation will take place in privacy, in a room where it is 
possible to ensure disturbance is prohibited and with possibility of a bystander. In the donor 
study, information could be given at the family’s homes or at the NICU at RH. In the clinical 
study, the consultation will include considerations to the condition of the mother. While admitted 
with risk of preterm birth, the consultation will take place in the room where the mother is 
admitted and bedridden and it is secured that it is given in privacy. 
 
If the family wishes, they can have access to a bystander of their own choice. If they feel the 
need for a bystander with neonatal competences, they can have access to one from among the 
personnel at RH NICU with no direct relation to the research project. In case of single parents, 
they will be asked to bring along an assessor. Besides written and oral information about the 
study (Information, appendix 2 and 3), the pamphlet “Før du beslutter dig: Om at være 
forsøgsperson i sundhedsvidenskabelige forsøg” from VEK, together with the pamphlet 
“Forsøgspersonens rettigheder i et sundhedsvidenskabeligt forskningsprojekt'' reviewed and 
published by the ethical committee system of 21st Sep 2019, and a copy of the informed consent 
forms (appendix 4, 5 and 6).  
 
 
In the clinical trial, we aim to obtain consent from both parents before  time of birth in order to 
allow the treatment to start within 24 hours. In case, this is not possible, we will aim to obtain 
consent as soon as possible after birth to be able to give treatment as fast as possible. In the 
donor study, we aim to have consent before the time of birth. If possible, a consideration period 
of 24 hours will be offered before signing the informed consent, in which case a new meeting 

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(the screening) will be scheduled. However, as FFT is to be administrated within 24 hours of 
birth, the consideration period might be shortened due to practical considerations. If participants 
do not require time for consideration, the declaration of informed consent may be signed 
immediately after the information meeting by the participant mother or the parents of the 
participant and the investigator (or research team who have had this responsibility assigned by 
the investigator). When the informed consent is signed, the participant will be offered a copy. 
Signed informed consent is a prerequisite for screening or any study-related procedures to start. 
 
 
They will be informed that the participation is completely voluntary, and that they have the right 
to withdraw their informed consent at any time without explanation or consequence. Potential 
participants and assessors will be invited to ask questions with trained research team members. 
If the research team or the RH NICU staff judge that a participant needs a second explanation 
of the study, this will be encouraged. Participants will also be informed that data collected before 
they withdraw their informed consent may be used by the researchers in the overall data 
analysis. In addition, they will be informed that by signing the informed consents they also give 
permission for the use of their personal data in accordance with the purpose of this project and 
controlling authorities to access their data and that a person that carries out these functions is 
participant to rules of confidentiality.  
10. 
Discontinuation and withdrawal 
The parents are free to withdraw the participants at any time during the intervention period as 
well as during the data collection period. The reason(s) will be recorded. When possible, the 
parents will be asked if they will allow that we use collected data before withdrawal for 
publication. The parents may ask to have biological material stored in the biobank destroyed at 
any time. The neonatologists and research staff can discontinue the participants from the study 
at any time, and the reason(s) will be recorded and reported. 
 

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11. 
Adverse events 
11.1. 
Definition of adverse events 
An adverse event (AE) is defined as any untoward occurrence in a participant in the clinical 
study, which does not necessarily have a causal relationship with the study intervention. AEs 
can be illnesses, signs, symptoms, or irregular results of routine laboratory tests occurring 
during the intervention period, and until discharge. 
11.2. 
Definition of serious adverse events 
A serious adverse event (SAE) is a fatal or life-threatening event, which causes or may cause 
death, prolonged hospitalization, persistent or significant disability, or requiring long-term 
intervention to prevent permanent impairment or damage. 
 
11.3. 
Baseline incidence of Serious Adverse Events 
In all cases, being born preterm infant is associated with significant risks. Therefore, we cannot 
expect zero adverse events during the study. However, it is our expectation that our rate of 
death, NEC and sepsis does not exceed the latest incidence rates from very low birth weight 
infants at Rigshospitalet NICU. The current rates are mortality 16.7 %, NEC 7.1% and positive 
blood cultures 17.1%. The current use of antibiotics at Rigshospitalet NICU, is 33% of all 
admitted infants. Though not reflecting numbers of infants with bacterial sepsis, we use the 
incidence of positive blood cultures in Copenhagen as a surrogate measure 4,5. 
11.4. 
Follow-up of adverse events 
All adverse events will be followed until they have abated, or until they have reached a stable 
situation. Depending on the event, follow up may require additional tests and/or medical 
procedures. 
11.5. 
Recording and reporting of the adverse events 
The very preterm infants in a NICU are often seriously ill and some die. Many of the adverse 
events will be observed with or without the FFT intervention, and therefore AEs and SAEs will 
likely occur in the study. Therefore, recording and reporting of all AEs is not realistic and 
relevant.  
 

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In the current study, sepsis, surgical NEC, and death will be recorded by the research team and 
reported to the chief investigator and the DSMB. The CI will re-evaluate the causal relation to 
the study. If it is probable, or certain, the CI will report this to the ethical committee. Severe 
unexpected suspected adverse reactions will be similarly reported. 
 
A safety report will be sent annually to the relevant standing ethical committees. This safety 
report will include the interim analysis of the DSMB (see below). 
11.6. 
Classification of causality 
The research team and attending neonatologists will evaluate the possibility of a connection 
between the FFT intervention and adverse events on the basis of the following criteria: 
1) Unrelated: There is an obvious other explanation for the AE, e.g.: 
The AE is clearly explained by the condition of the participant 
The AE is related to the side-effect of one or more medical treatments 
2) Unlikely relation: Possible connection with the intake of FFT, but there is another 
plausible explanation for the occurrence of the AE. Participant does not have a clear 
response to intervention withdrawal. 
3) Probable relation: Reasonable temporal relationship with the intake of FFT, and 
plausible reasons point to a causal relationship with the study product. 
4) Certain relation: Reasonable temporal relationship with the intake of FFT, and there is 
no other explanation for the AE. Descending and/or disappearing of the AE happen 
when withdrawing the intervention. 
 
12. 
Side-effects and risks 
12.1. 
Faecal and urine samples 
Collecting both urine samples and faecal samples is both safe and tolerable. There is a minor 
practical inconvenience associated with collecting the sample.  
12.2. 
Blood samples 
Blood samples will be drawn by trained personnel at Rigshospitalet NICU. When possible, 
project blood samples will be coordinated with blood samples drawn for clinical purposes. It is 

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anticipated that the needle used, will not cause any harm to the participants besides that 
associated with the insertion i.e. a small local pain. There is a risk that the skin around the entry 
point may swell slightly, become sore and blue/brown discoloration emerges on the day after 
blood sampling.  
12.3. 
Safety during trial 
To our knowledge, FFT in the preterm has not been performed before. A recent study of 
maternal faecal microbiota transplant in 10 cesarean-born infants has been shown to be safe 21. 
Administration of the FFT will be given while admitted to Rigshospitalet NICU, giving optimal 
circumstances for managing any adverse event. The families of both donors and participants 
are given contact information to the research team. They are encouraged to call, text, or write 
an email with any possible questions about potential side effects, fear or discomfort. 
Furthermore, clinical examination by trained professionals is planned to ensure that signs of 
potential disease not observed by family are found.  
 
13. 
Data Safety Monitoring Board (DSMB) 
There will be a DSMB instituted for this study. Persons with the following professions will take 
place in the board: 
● Neonatology 
● Infections 
● Methodology in clinical trials 
 
14. 
Termination of the study 
Safety and tolerability of FFT transplantation will be evaluated by the neonatologists in the 
neonatal departments of RH, as well as the research team. This will happen at least after each 
of block of randomization. This will be accessed by the clinical outcomes, the routine laboratory 
analysis, the plasma amino acid profiles, and SAEs. The study will be terminated if FFT is not 
tolerated or found unsafe. Before termination, the DSMB and VEK are asked for advice. 

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15. 
Statistical analysis 
15.1. 
General statistical considerations 
The statistical analyses described in this protocol will be carried out by the research team. The 
level of significance is set at 0.05 and if necessary, data will be transformed to reach normality. 
The overall statistical modelling approach will the Mann–Whitney U test, as our sample size is 
likely too small to fit normal distribution. Results from fitted models will be reported with and 
without control for multiplicity. For control for multiplicity e.g. Bonferroni corrections will be 
performed.  
This study is a feasibility study. Therefore, initial small sample size is made in terms of practical 
and safety considerations. If this study goes according to plan, we will follow up with a larger 
randomized clinical trial.  
All statistical analysis will be performed using “R” (https://www.r-project.org/). 
15.2. 
Effect-modifying factors 
• 
Gender 
• 
Ethnicity 
• 
Parenteral nutrition 
• 
Time till meconium discharge 
• 
Antibiotic consumption 
• 
Infections 
• 
Time on respiratory support 
 
15.2.1. 
Primary outcome 
As the main outcome of the study is to have no serious adverse events causally attributed to 
FFT, no statistical analysis will be performed for the primary outcome. Difference between 
incidence of NEC and late-onset sepsis will be analyzed, but since the study is not powered 
enough to show differences, the findings will be descriptive.  
15.2.2. 
Secondary outcomes 
For testing preliminary effects of FFT, clinical and paraclinical outcomes will be compared 
between the two treatment groups. Statistical analysis will be performed blindly. Continuous 
outcomes will be checked for the normal distribution, and if not, they will be transformed to fulfill 

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the normal distribution. If no proper transformation method can be found, non-parametric 
analyses will be performed. For continuous outcomes, General Linear Model with or without 
covariates will be used. For binary outcomes, logistic regression will be used. For repeated 
measurement, mixed model with repeated measurement will be applied. 
15.3. 
Definition of datasets 
To allow evaluation of the biological effect of the FFT treatment, complete-case analysis and 
per-protocol analyses of the primary and secondary outcomes will be carried out. Available-case 
analysis, utilizing all available data will be used for the exploratory analyses as appropriate. 
Additionally, Intention-to-treat (ITT) analyses may be carried out for all outcomes. 
15.4. 
Handling missing values 
For ITT analyses, missing outcome values will be handled using last-observation-carried-
forward method. 
16. 
Data handling and storage 
All information obtained during the study will be handled according to local regulations and the 
GDPR. All data collected during the study including relevant information from the participants 
medical record (antibiotic use, infections, presence of gastrointestinal disease) will be stored 
using a project specific account at REDCap (https://www.project-redcap.org/). The account is 
protected by security code and is only accessible by registered users. The database has an in-
built audit-trail. The project database will be erased after 10 years.  
 
If necessary, in conjunction with mandatory monitoring by the appropriate regulatory authorities 
or internal quality control, the project ID will be retraced to the participant, who may then gain 
access to the research database as well as the patient’s medical record.  
 
Questionnaires answered at home by the participants (appendix 7) will be answered directly via 
REDCap, and consent forms will be signed digitally using Nem-ID and REDCap.  
The staff at RH NICU will be trained in the use of the feeding tolerance evaluation (appendix 8) 
before initiation of the study to minimize interrater variability. They will be entered digitally in a 
standardized manner and stored using REDCap. The physical copies will be stored safely at RH 
NICU by the research team. 

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Any biological material not analyzed directly in the clinical setting will be identified only using a 
specific project ID after collecting the material.  
 
The randomization code is stored by the primary investigator in a locked safe. The intervention 
code is broken if serious side-effects are suspected that are deemed unacceptable for the 
participant. 
17. 
Ethical aspects 
17.1. 
Administering FFT to the preterm infant: potential benefit and harm 
We believe that administration of FFT will support beneficial GI tract maturation and decrease 
the risk of NEC, which must be one of the most cruel diseases related to prematurity. 
Based on previous piglet studies of FFT and the steps taken to develop the filtrate with thorough 
screening for pathogenic vira and exclusion of bacteria, it is our belief that the risk of serious 
infection because of the procedure is minimal.  
 
Placing a nasogastric tube is a routine procedure all premature infants admitted to RH NICU is 
exposed to and causes minimal distress to the infant. Administration of FFT via NG-tube has 
been shown to be equally efficacious to administration using colonoscopy.  
Infant feeding pattern is not expected to be disturbed, as the FFT is administered using an 
already placed nasogastric tube, and the volume is not expected to exceed 1 ml per dosage and 
4 ml in total.  
18. 
Considerations on research with neonates 
18.1.1. 
Study group 
We believe that the study can only be performed using premature infants, as they are the 
primary group affected by NEC. Hence, results from feasibility studies in term infants will not 
generalize to preterm infants, and as such we believe it inadvisable to perform the study in 
another population.  

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18.1.2. 
Considerations for ensuring minimal physical and emotional 
distress for participants 
Screening samples from donor infants will be coordinated with standard sample screenings for 
metabolic disease (PKU). Routine blood samples are coordinated with routine clinical care and 
continued only in the extent we believe it necessary to ensure safety during study protocol.  
Collecting faecal samples from diapered children does not cause any significant emotional 
distress. We offer the parents the service of collecting samples at home and measuring growth 
at home if they so prefer.  
 
18.2. 
Investigator expertise 
The group of responsible research team collaborating in this study all have a broad experience 
within each of their competences (Dennis Sandris Nielsen, microbiome, Andres Brunse, animal 
studies, Susanne Soendergaard Kappel, ethnographic fieldwork in the neonatal setting, 
 and Lise Aunsholt neonatal clinical studies). Besides from that, people within the group have 
long-term collaboration with each other. The primary clinical investigator, Lise Aunsholt, has 
experience in translational studies, combining animal studies with clinical studies, in a classical 
bench to bedside set-up and has investigated novel interventions previously. Apart from that, 
she has experience in large-scale national and international (China) multicenter studies 
involving more neonatal intensive care units and collaborators.  
The Ph.D. student involved in this study will acquire competences within translational studies 
during his Ph.D. under close supervision be the senior team.  
 
19. 
Patient record access and handling of personal data 
 
The patient record will be accessed as described in the observations and measurements. The 
patient records will not be accessed after the study conclusion.  
When consent to participate in the project has been given, based on written and verbal 
information, then the research team have permission to access patient records including FMK, 
in order to perform mandatory quality control. Furthermore, consent will allow controlling 
authorities access to the patient record in order to perform legally mandated control of clinical 
research projects.  

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Personal information (including CPR-number) is used in the project. All personal data will be 
treated in accordance with “Databeskyttelsesforordningen” (EU forordninger 2016/679 af 27. 
april 2016) and “Databeskyttelsesloven” (lov nr 502 af 23/5/2018).  
 
20. 
Insurance 
Both donors and participants are covered by the public patient insurance of Denmark  
( https://www.nvk.dk/emner/forsikring-og-erstatning/vejledning-om-forsikring ) 
21. 
Reimbursement of participants 
The participants will not be reimbursed for participation. Treatment and medical advice given 
during the study will be free of charge. 
22. 
SARS-Cov2 
Testing of participants and personnel as well as social distancing and use of protective 
equipment will always follow the current guidelines at RH NICU. 
23. 
Economy 
This project was initiated by Dennis Sandris Nielsen, Anders Brunse, and Lise Aunsholt and 
funding for the study which include synergy between 3 institutions was applied as a synergy 
application. The project is funded by the Novo Nordisk Foundation with 15.000.000 DKr. (public 
at www.novonordiskfonden.dk). Each institution has a budget of 5.000.000 DKr., which include 
salary for involved staff, sample analysis and costs of publication as well as congress 
presentations. For the clinical study, Lise Aunsholt holds an account at Rigshospitalet. Lise 
Aunsholt has no relation to the Novo Nordisk Foundation. 
24. 
Publication plan 
The pilot study will be registered on ClinicalTrials.gov. Positive, negative, or inconclusive results 
are all planned to be published in peer-reviewed international journals. If the amount and quality 
of obtained results from this pilot study do not meet the normal criteria for publication in 
international journals, results will be published as a public report available online UCPH. 

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Authorship will be determined according to the International Committee of Medical Journal 
Editors Guideline. 
 
 

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