Fecal Microbiota Transplantation in SAP（Severe Acute Pancreatitis）Patients With Intestinal Barrier Dysfunction
Phase: PHASE2
Conditions: Acute Pancreatitis, Intestinal Bacteria Flora Disturbance, Intestinal Dysfunction, Fecal Microbiota Transplantation
Sponsor: The First Affiliated Hospital of Nanchang University

Study Protocol and Statistical Analysis Plan 
 
Official title: Fecal Microbiota Transplantation in SAP (Severe Acute Pancreatitis) 
Patients With Intestinal Barrier Dysfunction 
 
NCT number: NCT02318134 
 
Document date: November 15, 2017 
 
Introduction 
Acute pancreatitis (AP) is a acute inflammatory disease of the pancreas. The 
incidence of AP ranges from 5 to 30 cases per 100,000, with an overall case fatality 
rate of 5%1. Approximately 80% cases of AP are mild and self-limited, and about 20% 
of patients suffer a severe disease course with persistent organ failure and/or 
infected pancreatic necrosis (IPN), with a mortality risk as high as 20~30%1. 
Gastrointestinal tract is considered not only as a target organ during systemin 
inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome 
(MODS), but also a “motor organ” of gut-derived infection2. Gastrointestinal 
dysfunction has been proven to be associated with adverse outcomes in AP3. 
Although the mechanisms underlying gastrointestinal dysfunction in the early phase 
of AP is complicated, early gastrointestinal barrier dysfunction was considered to be 
the main cause4,5. Gastrointestinal microbiota dysbiosis induces the injury of biologic 
barrier, which plays a key role in the pathogenic of gut-driven infection. Therefore, the 
maintenance of gastrointestinal microecology balance may be an effective method in 
treating gastrointestinal dysfunction, preventing gut-driven infection and improve 
clinical outcomes in AP. 
 Probiotics, as an adjunct to enteral nutrition, has long been studied as a measure to 
improve intestinal barrier dysfunction and prevent secondary infection in AP. Several 
clinical studies have assessed the effect of probiotics prophylaxis with contradictory 
results, and significant heterogeneity was noted between the trials with regard to the 

type, dose and treatment duration of probiotics6-11. A multicenter, double-blind, 
placebo-controlled trial have failed to show a beneficial effect of probiotic prophylaxis 
on the occurrence of infectious complications, and mortality in the probiotics group 
was about twice as high as in the placebo group8. The current data are not sufficient 
to draw a conclusion regarding the effects of probiotics on patients with predicted 
severe acute pancreatitis (SAP). Consequently, there is a clear need for other 
innovative strategies. 
Worldwide, interest in fecal microbiota transplantation (FMT) as an ‘ecological’ 
therapy for several diseases is growing rapidly. FMT presents another and more 
comprehensive approach to microbiota restoration. FMT consists of administering 
fecal material from a healthy donor into the intestinal tract of a patient. Unlike the few 
bacterial strains included in probiotics, FMT stool includes practically all the bacteria, 
viruses, eukaryotes, and metabolites from the healthy donor. In recurrent Clostridium 
difficile infections, FMT has shown excellent effects by restoring intestinal microbiota 
balance12. Several cases reported that patients with SIRS and severe diarrhea of 
unclear etiology persistently unresponsive to broad-spectrum antibiotics rapidly 
improved with FMT in the Intensive Care Unit (ICU). The improvements in clinical 
parameters were also associated with shifts in the recipients’ microbiota patterns 
toward that of the donors’, further attributing the patients’ recovery to FMT13-15. FMT 
could therefore theoretically be a possible treatment for AP patients with 
gastrointestinal dysfunction. No randomized controlled study evaluating FMT in AP 
patients has been published to date. 
Here, we performed the randomized, single-blind, parallel-group, placebo-controlled 
study to clarify the effects of FMT on gastrointestinal dysfunction and infectious 
complications, as well as gut microbiota in patients with AP. 
Methods 
This is a randomized, single-blind, parallel-group, placebo-control, single-center 
study in consecutive adults diagnosed with acute pancreatitis complicated with 
gastrointestinal failure in the setting of ICU. Eligible participants were randomized 1:1 
to receive fecal transplant or normal saline via a nasoduodenal tube for twice (once 

every two days). The primary end point the recovery of gastrointestinal dysfunction 
assessed by gastrointestinal failure (GIF) score one week after intervention. 
Trial organization, committees and boards 
The designer of this trial is the First Affiliated Hospital of Nanchang University, China 
(http://www.cdyfy.com/). This study was investigator-initiated and investigator-driven 
and done in accordance with the principles of the Revised Declaration of Helsinki. 
The study protocol was approved by the institutional review boards of the First 
Affiliated Hospital of Nanchang University (approval No, 2014 [32]). Written informed 
consent was obtained from all participants or their legal representatives. All 
physicians involved in the study will repetitively be asked to report any potential 
adverse events. All possible adverse events will be listed and reported to the 
institutional review boards of the First Affiliated Hospital of Nanchang University. 
Study population 
Adult patients with AP were recruited from the ICU, Department of Gastroenterology, 
First Affiliated Hospital of Nanchang University. The diagnosis of AP requires two of 
the following three features: 1) upper abdominal pain; 2) serum lipase or amylase 
activity at least three times greater than the upper limit of normal; and 3) 
characteristic findings of AP on contrast-enhanced computed tomography (CECT), 
magnetic resonance imaging (MRI) or transabdominal ultrasonography16. 
Inclusion criteria 
1) Aged 18-70 year; 
2) Onset of pancreatitis within 2 weeks; 
3) Complicated with gastrointestinal failure, which was defined if the patients were 
complicated with obvious abdominal distention, abdominal rumbling sound 
weakening or disappearance, no self-defecation as well as intra-abdominal 
hypertension (IAH). 
Exclusion criteria 
1) Complicated by gastrointestinal bleeding or intestinal fistula; 
2) Pregnancy and lactation women; 
3) Not signed the informed consent; 

4) Diabetes and autoimmune disease; 
5) Multiple organ failure, which was defined as two or more organ failure including 
respiratory failure, renal failure or circulatory failure, which was defined as a 
score of 2 or more using the modified Marshall scoring system17. 
Sample size 
To detect a minimal clinically relevant difference of 35% between FMT group and 
control group in the primary outcome, and estimating that FMT would achieve 85% 
power and control would achieve 50% power and using a type I error of 0.05 and a 
type II error of 0.20 (80% power), we calculated that a minimal sample size of 48 with 
24 patients in each group was required. To allow for dropouts, we included 60 
participants with 30 in FMT group and 30 in control group. 
Randomization and masking 
A randomization sequence for 60 participants with an allocation ratio of 1:1 was 
generated using SPSS by an independent statistician who not involved in the clinical 
performance of the trial. The method of allocation concealment was sequentially 
numbered sealed opaque envelopes technique. Each participant got a study number 
at enrolment. An allocator (non-study personnel) then allocated participants to FMT 
or control group by the randomization sequence. This was done in a closed room and 
the allocation sequence were immediately disposed of. Investigators, patients and 
outcome assessors were kept masked to the allocation. The randomization key was 
revealed to researchers when all participants completed the 6-month follow-up and 
data analysis was completed. 
Study duration 
The planned starting data of the study is December 2017, and the planned finishing 
data is December 2018. 
Primary Endpoint 
The primary end point was the recovery of gastrointestinal dysfunction assessed by 
GIF score one week after intervention18. The normal gastrointestinal function 
assessed by GIF score means enteral feeding >50% of calculated needs, without FI 
and IAH. FI was defined when applied enteral feeding appeared to be unsuccessful 

and had to be discontinued because of repeated or profuse vomiting, high gastric 
residuals, ileus, severe diarrhea, abdominal pain, or distension. FI should not be 
considered as present if enteral feeding is electively not prescribed or is 
withheld/interrupted due to procedures. IAP was measured by the standardized 
methodology (preferably the transvesical method, with a maximal instillation of 25 cc 
of saline in the supine position, measured at the end of expiration, with zeroing done 
at the level of the midaxillary line). IAH was present if the IAP was found to be 12 
mmHg or higher, as confirmed by at least two measurements taken 1-6 h apart19. 
Secondary endpoints 
The secondary end points were any infectious complication, OF, functional 
assessments of gut barrier and inflammatory indicators, hospital stay and mortality. 
Infectious complications included IPN, infected ascites, bacteremia, pneumonia, 
urinary tract infection. Microbiological data for each of the infectious complications 
were collected. OF was defined as a score of 2 or more using the modified Marshall 
scoring system, which was recommended by 2012 Atlanta Classification Creation17. 
Persistent organ failure, that is, organ failure >48 hours16. Blood samples were 
collected from all included patients before and one week after intervention. Gut 
barrier function was assessed by detecting serum D-Amino Acid Oxidase (DAO) and 
endotoxin using commercial kits (Cloud-Clone Corp, TX, USA for DAO and ELX800 
(Bio Tek) for endotoxin). C-reactive protein (CRP) and procalcitonin (PCT) were 
measured by IMMAGE 800 (Beckman Coulter) and AFIAS-50 (JOINSTAR) 
separately. Stools were requested for microbiota analysis at baseline, one week and 
two week after intervention.  
Microbiomics analysis 
Fecal samples were longitudinally collected from patients at baseline before first 
FMT, one weeks and two weeks after first FMT. The fresh feces were collected in 
sterile cryopreservation tube by the nurses and kept at -20 ℃ immediately. Then, 
the feces were transferred within 6 hour after a bowel movement, and were stored at 
−80 °C until DNA extraction. 

Bacterial DNA was extracted from fecal samples using the E.Z.N.A. Soil DNA Kit 
(Omega Bio-Tek, Norcross, GA, USA) according to the manufacturer’s instructions. 
The final DNA concentration and purification were determined by NanoDrop 2000 
UV-vis spectrophotometer (Thermo Scientific, Wilmington, USA), and DNA quality 
was checked by 1% agarose gel electrophoresis. The V3-V4 hypervariable regions of 
the bacteria 16S rRNA gene were amplified with primers 338F (5’-
ACTCCTACGGGAGGCAGCAG-3’) and 806R (5’-GGACTACHVGGGTWTCTAAT-3’) 
by thermocycler PCR system (GeneAmp 9700, ABI, USA). The PCR reactions were 
conducted using the following program: 3 min of denaturation at 95℃, 27 cycles of 
30 s at 95℃, 30 s for annealing at 55℃, and 45 s for elongation at 72℃, and a final 
extension at 72℃ for 10 min. PCR reactions were performed in triplicate 20 μL 
mixture containing 4 μL of 5 × FastPfu Buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of each 
primer (5 μM), 0.4 μL of FastPfu Polymerase and 10 ng of template DNA. The 
resulted PCR products were extracted from a 2% agarose gel and further purified 
using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, 
USA) and qualified using QuantiFluro-ST (Promega, USA). 
Purified amplicons were pooled in equimolar and paired-end sequenced (2×300) on 
an Illumina MiSeq platform (Illumina, San Diego, USA). The raw reads were 
deposited into the NCBI Sequence Read Archive (SRA) database (Accession 
Number: SRP). The raw reads were demultiplexed and quality-filtered by 
Trimmonmatic and merged by FLASH software. The high-quality sequences were 
assigned to samples according to barcodes. Operational taxonomic units (OTUs) 
were clustered with 97% similarity cutoff using UPAPSE (version 7.1, 
http://drive5.com/uparse/) and chimeric sequences were identified and removed 
using UCHIME. The taxonomy of each 16S rRNA gene sequencing was analyzed by 
RDP Classifier algorithm (http://rdp.cme.msu.edu/) against the Siva (SSU123) 16S 
rRNA database using confidence threshold of 70%. 
Intervention 
All participants underwent interventions one day after allocation. In the FMT group, 

participants received 200 mL fresh donor feces for twice (once every two days). In 
the control group, participants received 200 mL normal saline. The fecal transplant or 
normal saline were drawn on 50 mL sterile sealed syringe and infused into each 
patients via a nasoduodenal tube, which was inserted into distal duodenum by 
endoscopy. The procedure was performed within 5 min at the bedside of pancreatic 
intensive care unit by a researcher who were not involved in the clinical performance 
of the trial. The patients were blinded to the identity of the intervention that they were 
receiving because either FMT or placebo were stored on a sealed syringe. Before 
FMT or normal saline, Laxative was stopped for at least one hour to retain stool. To 
assure maximum delivery and colonization of these fecal transplant, first we assured 
a at least 4-hour gap between study medication and antibiotics (if prescribed) or 
laxative. No probiotics or lactulose was used during hospitalization. Considering the 
feasibility, we altered the protocol (after registration at ClinicalTrials.gov, but before 
enrolment of participants) to implement FMT or normal saline via a nasoduodenal 
tube instead of via retention enema. 
General treatment regimen 
All participants received routine treatment at admission according to the AP 
guidelines, including goal-oriented fluid resuscitation, enteral nutrition as early as 
possible, organ support as needed. Antibiotic prophylaxis was not given routinely. 
The use of antibiotics was recorded, irrespective of indication. As all the patients 
enrolled was complicated with gastrointestinal failure, we used traditional Chinese 
medicine-rhubarb (and/or mirabilite) for all patients. Some other purge measures 
including gastrointestinal decompression or mannitol via nasoduodenal tube were 
conducted according to patients’ conditions. Abdominal puncture and drainage were 
for patients with abdominal effusion. 
Data collection 
Data collection was prospectively entered by a researcher, who completed 
standardized case report forms. During the study an independent data monitor 
checked the individual patients’ data against the primary source data. After double 
check of key variables by two researchers, data were exported unedited for statistical 

analyses. 
Follow up 
Patients are followed during their hospital stay. There is one follow-up visit, 6 months 
after discharge by clinical visit or telephone visit, to assess readmission, mortality 
and adverse events. 
Statistical analysis 
Quantitative variables are presented as medians (ranges) or means ± standard 
deviations and were analysed using the Mann-Whitney U test or t-test as appropriate. 
Categorical variables are reported as absolute numbers and proportions and were 
tested by the chi-square test or Fisher’s exact test as appropriate. The differences 
between outcomes for matching data were tested with the paired t test or Wilcoxon 
signed-rank test as appropriate. We estimated relative risks (RRs) and 95% 
confidence intervals (CIs) for primary outcomes and secondary outcomes. A two-
tailed P value <0.05 was considered statistically significant. Data were analyzed 
using SPSS software (v17.0; SPSS Inc., Chicago, IL, USA). 
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