Conditions: Recurrent Clostridium Difficile Infection Primary Clostridium Difficile Infection
Keywords: Clostridium difficile FMT Fecal Microbiota Transfer
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 93
Healthy volunteers: no

Brief summary:
Patients with microbiota alterations developed after being exposed to antibiotics are especially susceptible to Clostridioides difficile infections (CDI). The incidence and severity of CDI has increased in recent years and CDI recurrences (r-CDI) due to the appearance of new episodes in patients with a previous cured CDI, represent a serious and complex clinical issue. Although antibiotics are the recommended therapy for the first episode of CDI, treatment with oral vancomycin and/or metronidazole often results in significant treatment failure. In addition, the treatment of r-CDI is not adequately standardized, and although the most widely used treatment is the administration of fidaxomicin and bezlotoxumab, its efficacy in patients who already have r-CDI is not proven. In the late years, Fecal Microbiota Transfer (FMT) has emerged as the preferred non-pharmacological treatment to manage CDI with multiple recurrences and recent clinical trials have evaluated its potential efficacy and safety in the treatment of patients with primary CD infection.

The objective of this study is to assess the efficacy and safety of the MBK-01 medication, consisting of heterologous lyophilized fecal microbiota capsules coming from healthy donors in comparison to the treatment with fidaxomicin, in 92 patients with primary or r-CDI.

Detailed description:
This is a Phase III, multicenter, controlled and open label clinical trial in which patients who suffered an episode of Clostridioides difficile infection (either the first episode or subsequent recurrences) will be randomly assigned (1:1) to one of the following arms:

* Fidaxomicin
* MBK-01 (heterologous lyophilized fecal microbiota)

Objective: To assess the efficacy of FMT with capsules of lyophilized fecal microbiota (MBK-01), compared to the control (fidaxomicin) at 8 weeks after the start of the treatment. Also, assess the safety of MBK-01 and the quality of life of patients participating in the study.

Follow up: participants will return for clinic visits at 72 hours, week 3 and week 8 after the start of the treatment, and will receive follow-up phone calls at month 3 and month 6 after the start of the treatment. Stool samples will be collected from participants for further studies at time 0 and week 8 after the start of the treatment. Study Outcomes are detailed in the specific section of this website.

Rationale: The transferred microbiota restores the recipient's intestinal microbiota by reintroducing bacterial taxa that were absent or in low proportion in the recipient before the FMT. This supports the expansion of the recipient's own commensal microbiota and re-establishing a microbiota community with a high biodiversity.

Donors: All donors are screened to ensure they meet the strict requirements necessary to maintain the safety of the MBK-01.

Justification: The treatment of Clostridioides difficile infections (CDI) with antibiotics is usually effective for acute symptoms, but after the initial treatment, the probability of recurrence at 8 weeks ranges from 10-20 % of cases, and once a patient has a recurrence, the probability of further recurrences increases up to 40-65 %. In recent years, Fecal Microbiota Transfer (FMT) has emerged as the preferred non-pharmacological treatment to manage CDI with multiple recurrences and recent clinical trials have evaluated its potential efficacy and safety in the treatment of patients with primary CD infection. Although antibiotics are the recommended therapy for the first episode of CDI, treatment with oral vancomycin and/or metronidazole often results in significant treatment failure, with recurrences occurring in up to 30-40% of patients. Furthermore, antibiotic treatment does not correct deficiencies in the intestinal microbiota that facilitate CD infection and is associated with the risk of the emergence of antibiotic-resistant bacteria. Moreover, the treatment of recurrences is not adequately standardized. In recent years, although the most widely used alternatives have been fidaxomicin and bezlotoxumab, their efficacy in patients who already suffer from r-CDI is not proven. The administration of the FMT through oral capsules, although it is not standardized, has proven to be effective in the restoration of intestinal microbiota of patients with r-CDI. In addition, the use of lyophilized formulas facilitates the concentration of bacteria and further optimizes the donors' sample and reduces the amount of capsules that the patient has to ingest.

Arms:
arm 1: Participants will receive MBK-01 capsules of fecal microbiota coming from healthy donors (45 patients). arm 2: Participants will receive Fidaxomicin (47 patients).

Interventions: intervention 1: MBK-01 intervention 2: Fidaxomicin
Intervention descriptions: intervention 1: A single dose of 4 capsules of MBK-01 (heterologous lyophilized fecal microbiota coming from healthy donors) orally. intervention 2: Oral administration of 200mg/12 hours of fidaxomicin for 10 days.

Protocol document:
Prot_000.pdf: 
 
 
 
 
 
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A Randomised, Controlled, Open-label Phase III Clinical 
Trial in Patients With Primary or Recurrent Clostridioides 
Difficile (CD) Infection, to Evaluate the Efficacy and 
Safety of Capsules of Lyophilised Faecal Microbiota vs 
Fidaxomicin. 
Protocol code (for publication): ICD-01 
EudraCT: 2020-004591-17 
August 4, 2023, version 7.0 
MIKROBIOMIK 
BIC Bizkaia – Astondo Bidea, 
building no. 612 
48160, Derio – Vizcaya 
Spain 
 
 
 


 
 
 
 
 
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Table of Contents 
Version History ................................................................................................................................................... 5 
List of Acronyms ................................................................................................................................................. 6 
00. OVERVIEW ...................................................................................................................................................... 8 
00. 01. Study Title, Protocol ID ............................................................................................... 8 
00. 02. Sponsor ID...................................................................................................................... 8 
00. 03. Investigational Product ............................................................................................... 8 
00. 04. Planned Study Sites ...................................................................................................... 8 
00.05. Coordinating Investigator ........................................................................................... 9 
00.06. Clinical Research Ethics Committee .......................................................................... 9 
00.07. Study Design ................................................................................................................... 9 
00.08. Study Objectives and Endpoints ................................................................................. 9 
00.09. Study population ........................................................................................................... 9 
00.10. Calendar ........................................................................................................................ 10 
01. INTRODUCTION AND RATIONALE FOR THE STUDY .............................................................. 11 
01.01. Disease or disorder under study .............................................................................. 11 
01.02. Current Therapies ....................................................................................................... 13 
01.02.01. Metronidazole and vancomycin ......................................................................... 13 
01.02.02. Fidaxomicin ............................................................................................................ 14 
01.02.03. Bezlotoxumab ......................................................................................................... 15 
01.02.04. Fecal Microbiota Transplantation ...................................................................... 15 
01.03. Nonclinical Studies ...................................................................................................... 16 
01.04. Previous Clinical Experience .................................................................................... 17 
02. STUDY OBJECTIVES ................................................................................................................................. 26 
02.01. Primary Objective ....................................................................................................... 26 
02.02. Secondary Objectives ................................................................................................. 26 
03. TYPE OF CLINICAL STUDY AND DESIGN ....................................................................................... 26 
03.01. Type of study. Global Design ..................................................................................... 26 
03.02. Randomization Process ............................................................................................. 26 
03.03. Type of Control and Design ....................................................................................... 26 
03.04. Blinding Techniques ................................................................................................... 26 
03.05. Study Plan ..................................................................................................................... 26 
03.06 Early Trial Termination .............................................................................................. 27 
03.07. Treatment Efficacy Assessments .............................................................................. 27 
03.07.01. Definition of Study Endpoints ............................................................................. 27 
03.07.01.A. Efficacy Variables ............................................................................................... 27 
03.07.01.B. Safety Variables .................................................................................................. 28 
03.07.01.C. Quality of life variables ..................................................................................... 29 
03.07.02. Definitions............................................................................................................... 29 
04. STUDY POPULATION. PATIENT SELECTION .............................................................................. 30 
04.01. Inclusion Criteria ........................................................................................................ 30 
04.02. Exclusion Criteria ........................................................................................................ 30 
04.03. Expected number of patients .................................................................................... 31 
04.04. Withdrawal criteria and planned analysis of withdrawals ................................ 31 


 
 
 
 
 
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04.05. Withdrawal criteria for lack of efficacy .................................................................. 31 
04.05.01. Discontinuation from treatment ........................................................................ 31 
04.06. Duration of participation ........................................................................................... 32 
05. DESCRIPTION OF STUDY TREATMENT ......................................................................................... 32 
05.01. Definition of treatment .............................................................................................. 32 
05.02. Investigational Product ............................................................................................. 32 
05.02.01. Description of the manufacturing technique(s) of the proposed 
therapy(ies) .............................................................................................................................. 32 
05.02.02. Pharmacokinetics .................................................................................................. 35 
05.02.02.A. Bioavailability of the investigational medication........................................ 35 
05.02.03. Adverse effects ....................................................................................................... 35 
05.02.04. Presentation and instructions for proper administration ............................ 36 
05.02.05. Storage and receipt of study product ................................................................ 36 
05.02.06. Administration and Dosing Schedule ................................................................ 36 
05.02.07. Supportive measures during treatment ........................................................... 36 
05.02.08. Dose modification .................................................................................................. 36 
05.03. Fidaxomicin .................................................................................................................. 37 
05.04. Permitted and Prohibited Concomitant Treatments ........................................... 37 
06. STUDY ASSESSMENTS ............................................................................................................................. 37 
06.01. Medical History ............................................................................................................ 37 
06.02. Physical examination ................................................................................................. 37 
06.03. Laboratory tests .......................................................................................................... 38 
06.03.01. Stool samples .......................................................................................................... 38 
06.03.02. Other laboratory tests .......................................................................................... 38 
06.04. Follow-up Assessments: Study Visits ...................................................................... 38 
Visit 1. Baseline Visit (Randomization and Treatment) .................................................. 38 
Visit 2. Clinical course assessment (f2f): 72 hours ±1 day after treatment initiation.
 
40 
Visit 3. Post-Treatment Assessment (F2F): three weeks ±4 days after treatment 
initiation. ................................................................................................................................... 40 
Visit 4. Post-Treatment Assessment (F2F): eight weeks ±4 days after treatment 
initiation. ................................................................................................................................... 40 
Visit 5. Early follow-up (telephone call): three months ±6 days after treatment start.
 
41 
Visit 6 (end of study). Late follow-up (phone call): six months ±6 days after 
treatment initiation ................................................................................................................ 41 
Unscheduled Visits .................................................................................................................. 41 
07. MONITORING .............................................................................................................................................. 41 
08. ADVERSE EVENTS ..................................................................................................................................... 42 
08.01. General Considerations .............................................................................................. 42 
08.02. Adverse Event (AE) ..................................................................................................... 42 
08.03. Adverse Drug Reaction (ADR) .................................................................................. 42 
08.04. Serious Adverse Event (SAE) ..................................................................................... 42 
08.05. Suspected Unexpected Serious Adverse Reaction (SUSAR) ................................ 42 
08.06. Causality assessment .................................................................................................. 43 
09. ETHICAL ASPECTS .................................................................................................................................... 43 


 
 
 
 
 
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09.01. General Considerations. Approvals ......................................................................... 43 
09.02. Informed Consent Form (ICF) ................................................................................... 44 
09.03. Confidentiality ............................................................................................................. 44 
09.04. Data Integrity and Quality Assurance ..................................................................... 44 
10. STATISTICAL ANALYSIS ........................................................................................................................ 44 
10.01. General Statistical Considerations ........................................................................... 44 
10.02. Determination of sample size ................................................................................... 45 
10.03. Randomization ............................................................................................................ 45 
10.04. Study Populations ....................................................................................................... 46 
10.05. Efficacy Analyses ......................................................................................................... 46 
10.06. Safety Analyses ............................................................................................................ 47 
10.07. Variables and Subgroups ........................................................................................... 47 
10.08. Interim Statistical Analysis ....................................................................................... 49 
11. REFERENCES ................................................................................................................................................ 50 
ANNEX 1: SUMMARY OF ICD-01 PROTOCOL AMENDMENTS ..................................................... 59 
Summary and justification of Substantial Modification No.01 ....................................... 59 
Summary and justification of Substantial Modification No.02 ....................................... 59 
Summary and justification of Substantial Modification No.03 ....................................... 60 
Summary and justification of Substantial Modification No.04 ....................................... 61 
Summary and justification of Substantial Modification No.05 ....................................... 61 
Summary and justification of Substantial Modification No.06 ....................................... 62 
Summary and justification of Substantial Modification No.07 ....................................... 63 
 
Table List 
Table 1. Donor Inclusion Criteria. ........................................................................................... 32 
Table 2. Donor exclusion criteria. ........................................................................................... 32 
Table 3. Study variables and subgroups to be considered in the statistical analysis. ......... 48 
 
 
 


 
 
 
 
 
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Version History 
 
VERSION 
DATE 
CHANGES 
Version 3.0 
30-July-2021 
Modification of exclusion criteria 
Version 4.0 
07-December-2021 
Modification of inclusion and exclusion 
criteria 
Expanding Sites 
Modified definition of poor evolution 
Modification of interim analysis 
Version 5.0 
20-July-2022 
Modification of study population 
Modification of inclusion criteria 
Change in sample size 
Expanding Sites 
Version 6.0 
17-May-2023 
Modification of donor exclusion criteria 
Sample Size Modification  
Version 7.0 
04-August-2023 
Sample size calculation 
Definition of study variables 
Premature Termination of Study 
Safety follow-up (withdrawn patients) 
 
 
 


 
 
 
 
 
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List of Acronyms 
ADR: Adverse Drug Reaction 
AE: Adverse Event 
AEMPS: Spanish Agency for Medicinal Products and Medical Devices (Agencia Española de 
Medicamentos y Productos Sanitarios) 
ALT: Alanine transaminase 
AR: Adverse Reaction 
AST: Aspartate aminotransferase 
BMI: Body Mass Index 
CD: Clostridioides difficile 
CDI: Clostridioides difficile Infection 
CEIm: Clinical Research Ethics Committee (Comité de Ética de la Investigación con 
medicamentos) 
CI: Confidence Interval 
CRA: Clinical Research Associate 
CRP: C-reactive protein 
eCRF: electronic Case Report Form 
EPEC: Enteropathogenic Escherichia coli  
ESCMID: European Society of Clinical Microbiology and Infectious Diseases 
FDA: US Food and Drug Administration 
FMT: Fecal Microbiota Transplantation 
GCP: Good Clinical Practice 
GDH: Glutamate dehydrogenase 
GDPs: Good Distributing Practices 
GGTP: Gamma-glutamyltransferase 
GMP: Good Manufacturing Practices  


 
 
 
 
 
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HDL: High-Density Lipoprotein 
HDPE: High-Density Polyethylene 
ICF: Informed Consent Form 
ICU: Intensive Care Unit 
IDSA: Infectious Diseases Society of America 
ITT: Intention-to-treat 
LDH: Lactate dehydrogenase 
LDL: Low-Density Lipoprotein 
OS: Overall Survival 
PaLoc: Pathogenicity region 
PCR: Polymerase chain reaction 
PI: Principal Investigator 
PP: per Protocol Population 
PPI: Proton-Pump inhibitors 
QP: Query Plan 
r-CDI: Recurrent Clostridioides difficile Infection 
SAE: Serious Adverse Event 
SAP: Statistical Analysis Plan 
SDU: Standard Drink Unit 
SEIMC: Spanish Society of Infectious Diseases and Clinical Microbiology (Sociedad Española 
de Enfermedades Infecciosas y Microbiología Clínica) 
SOP: Standard Operating Procedure 
SP: Safety Population 
STEC: Shiga Toxin-producing Escherichia coli 
SUSAR: Suspected Unexpected Serious Adverse Reaction 
 
 


 
 
 
 
 
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00. OVERVIEW 
00. 01. Study Title, Protocol ID 
Title: A Randomised, Controlled, Open-label Phase III Clinical Trial in Patients With 
Primary or Recurrent Clostridioides Difficile (CD) Infection, to Evaluate the Efficacy and 
Safety of Capsules of Lyophilised Faecal Microbiota vs Fidaxomicin. 
No. Protocol (for publication): ICD-01, Version: 7.0 
Date: August 4, 2023 
EudraCT No.: 2020-004591-17 
00. 02. Sponsor ID 
Mikrobiomik 
BIC Bizkaia – Astondo Bidea, edificio nº612. 48160 Derio – Vizcaya 
E-mail: ensayosclinicos@mikrobiomik.net 
Phone number: (+34)944 540 166 
00. 03. Investigational Product 
Drug product consisting of heterologous lyophilized fecal microbiota capsules from healthy 
donors.  
00. 04. Planned Study Sites 
Hospital Ramón y Cajal [Ramón y Cajal Hospital] (Madrid) 
Hospital Universitario 12 de Octubre [12 de Octubre University Hospital] (Madrid) 
Hospital Universitario Puerta de Hierro [Puerta de Hierro University Hospital] (Madrid) 
Hospital Universitario de Bellvitge [Bellvitge University Hospital] (Barcelona) 
Hospital Clínic [Clinical Hospital] (Barcelona) 
Hospital Universitario Reina Sofia [Reina Sofia University Hospital] (Cordoba) 
Hospital Universitario Son Espases [Son Espases University Hospital] (Mallorca)  
Hospital Universitario Marqués de Valdecilla [Marqués de Valdecilla University Hospital] 
(Santander) 
Hospital Universitario de Cruces [Cruces University Hospital] (Bilbao) 
Hospital Universitario de Basurto [University Hospital of Basurto] (Bilbao) 
Hospital Universitario de Araba [University Hospital of Araba] (Alava) 
Hospital Universitario de Donostia [University Hospital of Donostia] (San Sebastian) 
Hospital Clínico Universitario Lozano Blesa [Lozano Blesa University Clinical Hospital] 
(Zaragoza) 
Hospital San Pedro [San Pedro Hospital] (Logroño) 


 
 
 
 
 
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Hospital Universitario y Politécnico La Fe [La Fe University and Polytecnic Hospital] 
(Valencia) 
Hospital General Universitario Gregorio Marañón [Gregorio Marañón University General 
Hospital] (Madrid) 
La Paz University Hospital [Hospital Universitario La Paz] (Madrid) 
Hospital General Universitario de Alicante [University General Hospital of Alicante] 
(Alicante) 
Hospital Universitario de Gerona Doctor Josep Trueta [University Hospital of Gerona Doctor 
Josep Trueta] (Gerona) 
Hospital Universitario Quironsalud Madrid [Quironsalud University Hospital Madrid] 
(Madrid) 
Quironsalud Barcelona Hospital [Quironsalud Hospital of Barcelona] (Barcelona) 
00.05. Coordinating Investigator 
Javier Cobo, MD 
Hospital Universitario Ramón y Cajal [Ramón y Cajal University Hospital] 
00.06. Clinical Research Ethics Committee 
Ethics Committee for Investigation with Medicinal Products of Euskadi 
00.07. Study Design 
A multi-center, open-label, controlled phase III prospective follow-up clinical trial in a 
cohort of patients with a diagnosis of CDI (a first episode or subsequent recurrences). The 
proposed treatment is a single-dose orally administered drug consisting of lyophilized fecal 
microbiota capsules from healthy donors.  
00.08. Study Objectives and Endpoints 
Primary objective: to evaluate the efficacy of the investigational medicinal product (MBK-
01) compared to the control (fidaxomicin). 
Primary variable: no CD diarrhea without need for retreatment (or no recurrence as defined 
in Section 03.07.02), as assessed by subject interview and physical examination. The 
primary variable is operated by the probability of recurrence. 
Secondary Objectives: Evaluate the safety of the investigational medicinal product and 
quality 
of patients participating in the study.  
00.09. Study population 


 
 
 
 
 
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Patients who experience an episode of CD infection (either a first episode or subsequent 
recurrences). 
00.10. Calendar  
Run-In Period: August 2021 - December 2023 
Estimated follow-up: six months 
Estimated End of Study Date: June 2024 
 


 
 
 
 
 
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01. INTRODUCTION AND RATIONALE FOR THE STUDY 
01.01. Disease or disorder under study  
Clostridioides difficile (CD) bacteria is the leading pathogen responsible for diarrhea 
associated with previous antibiotic use in healthcare facilities in developed countries and 
an increasingly common cause of community diarrhea. Although it usually presents with 
mild diarrhea, it can also cause severe conditions such as pseudomembranous colitis, toxic 
megacolon, septic shock and, in extreme cases, death. The epidemiology of Clostridioides 
difficile infection (CDI) has changed dramatically in recent decades and has become a major 
global health problem. CDI has experienced significant epidemiological changes in the past 
15 years, with increased incidence, mortality, and recurrence observed (Dieterle et al., 
2019). This is due in part to the appearance of increasingly virulent strains but also to the 
improvement of available diagnostic methods and the inappropriate use of antibiotics, as 
well as the aging of a population affected by a larger number of chronic and debilitating 
diseases (Abreu et al., 2019). 
Over the last decade, serious interhospital outbreaks have occurred in North America and 
Europe, reaching CDI at an incidence of up to 92 per 100,000 population. Data from the 
EUCLID study estimate that in Europe, the mean number of CDI episodes is 7 per 10,000 
patients admitted and an estimated 23% of cases are not detected (Davies et al., 2014). 
Although there is no national CDI registry in Spain, the incidence of nosocomial-origin CDI 
is estimated to be 10 episodes per 10,000 hospital stays, while limited descriptive CDI 
studies show recurrence percentages of 12-18% (Rodriguez-Pardo et al., 2013; Reigadas et 
al., 2015; Larrainzar-Coghen 2016).  
The recurrence of CDI poses a serious problem at both the health and economic levels. 
Recurrence aggravates the patient’s general condition and promotes disease spread to 
other patients. On the other hand, recurrence increases healthcare expenditure to a greater 
extent than initial episodes (€4,900 vs. €3,900) (Asensio et al., 2013). CD is a gram-positive, 
strict anaerobic bacillus of oral-fecal transmission capable of producing spores whose 
primary pathogenic mechanism are two exotoxins (toxin A and B), which are responsible 
for the clinical manifestations. Toxins are the major virulence factors of CD; toxin A or 
enterotoxin (308 kDa) and toxin B or cytotoxin (270 kDa) that are encoded, respectively, by 
the tcdA and tcdB genes located in a region of pathogenicity (PaLoc) of the CD chromosome 
(Oka et al., 2012). Toxins A and B cause inflammation at the level of the large intestine and 
lead to increased epithelial permeability, cytokine production, neutrophil infiltration, 
oxygen reactive intermediate production, mast cell activation, substance P production, and 
direct intestinal mucosal damage (Perez et al., 2013). 
Other virulence factors that have been described are binary toxin and surface proteins that 
mediate CD adherence to host epithelial cells. The presence of these varies in different 
strains of CD and could influence the pathogen’s ability to bind to the colon epithelium, 
acting synergistically with toxins A and B (Voth et al. 2005; McDonald et al., 2006; Tenover 
et al., 2011; Penichea et al., 2013). CDI may present with different clinical manifestations 


 
 
 
 
 
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ranging from mild or moderate diarrhea to fulminant pseudomembranous colitis, toxic 
megacolon, or death (SEIMC, Alcalá-Hernández et al., 2016). 
The type of disease and its severity will depend on factors such as the virulence of the 
infective strain and the host’s immune response. Contact with spores of a toxin-producing 
strain of CD in combination with disruption of the host microbiota allows colonization by 
CD. 
The main risks of CD infection are exposure to antibiotics (especially to clindamycin, 
cephalosporins, beta-lactams and fluoroquinolones), prolonged hospitalization, admission 
to an intensive care unit, the physical proximity of infected individuals, advanced age (over 
65 years old), the severity of another underlying disease, immunosuppression, a poor 
immune response to CD toxins, the performance of non-surgical gastrointestinal 
procedures and the use of antacids (SEIMC, Alcala-Hernández et al. 2016). 
Elderly, hospitalized and antibiotic-treated patients are the main risk group for CDI; 
however, in recent years, an increase of cases – around 25% of total CDI cases – has been 
observed in patients without traditional risk factors: young subjects who have not had 
contact with hospital institutions or antibiotic treatment (SEIMC, Alcalá-Hernández et al., 
2016).  
Patients with clinical symptoms of the disease can be classified according to different 
severity criteria. Severity classification is important when assessing the need for treatment, 
type of treatment, or even the need for surgery or admission to an intensive-care unit to 
control infection (SEIMC, Alcalá-Hernández et al., 2016). 
Mild disease is characterized by only diarrhea, with an absence of symptoms and signs of 
colitis, while patients with moderate disease typically present with diarrhea and evidence 
of colitis characterized by fever and abdominal colic, usually in the lower quadrants. 
Laboratory signs of mild-moderate CDI include leukocytosis <15,000 cells/μL and serum 
creatinine levels below 1.5 times the pre-morbidity levels (SEIMC, Alcalá-Hernández et al., 
2016). 
Systemic manifestations typically occur in moderate or severe disease. Evidence of severe 
colitis includes fever (up to 40ºC), abdominal colic, leukocytosis ≥15,000 cells/μL, 
hypoalbuminemia (with serum albumin levels <2.5 mg/dL), presence of leukocytes in feces, 
and colon inflammation visualized by endoscopy (for pseudomembranes), or computed 
tomography (to observe colon wall thickening) (BSC, Alkalah-Hernán et al. 2016).  
Fulminant CD disease occurs in less than 5% of patients and is characterized by severe 
abdominal pain, profound diarrhea (although diarrhea can sometimes be absent), while the 
patient rapidly progresses to the development of ileus or toxic megacolon (SEIMC, Alcalá-
Hernández et al., 2016). 
The risk of recurrence ranges from 20% after initial infection to 60% after multiple 
recurrences, with the recurrence rate being similar in nosocomial or community-acquired 
infection (SEIMC, Alcalá-Hernández et al., 2016). 
Diarrhea is the most common clinical manifestation of CDI and is mediated by toxins A and 
B. Diarrhea is defined as the presence of loose stools (taken into the shape of the container 


 
 
 
 
 
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or graded between types 5 and 7 on the Bristol scale) accompanied by at least 3 stools in 24 
hours or less or the patient’s perception of a higher than normal number of stools. These 
are usually watery, greenish stools with a characteristically bad odor, although they can 
sometimes contain mucous and are soft. The presence of blood is rare (SEIMC, Alcalá-
Hernández et al., 2016). 
For the CDI diagnosis to be made, at least two criteria must be met: on the one hand, there 
must be microbiological detection of the toxin and/or isolation of toxin-producing CD in the 
absence of another cause for diarrhea, or colonoscopic or histopathological evidence of 
pseudomembranous colitis, and, on the other hand, the presence of diarrhea (≥3 non-
formed stools in 24 hours) or, evidence of ileus or toxic megacolon through imaging tests. 
Laboratory diagnosis should be made only in symptomatic patients (diarrhea and/or 
abdominal pain, frequently accompanied by leukocytosis and fever) and in non-formed 
feces (Bristol scale levels 5-7), with the important exception of cases in which paralytic ileus 
or toxic megacolon is suspected, diarrhea may not be present and feces may be formed 
(SEIMC, Alcalá-Hern 2016 et al).  
Commercial immunological techniques are available for the detection of toxin A and/or B, 
the Glutamate dehydrogenase (GDH) enzyme and, also, a combination of both. In addition, 
nucleic acid amplification testing on the same day as sample receipt allows detection of gene 
encoding for toxins A and/or B (SEIMC, Alcalá-Hernández et al., 2016) to be performed.  
 
01.02. Current Therapies 
Current treatment recommendations vary by country and clinical definition and are 
primarily linked to the number of episodes and severity of CDI that each patient presents 
(Bouza et al., 2020). The optimal treatment of patients with recurrence and/or severe 
complications is not entirely clear and is based on limited clinical evidence (Bouza et al., 
2013; McDonald et al., 2018). 
 
01.02.01. Metronidazole and vancomycin 
First-line drugs for the treatment of CDI are metronidazole and vancomycin. They have a 
similar degree of efficacy, with response rates of around 90-97%, and oral administration 
is recommended.  
Oral treatment with metronidazole is generally well tolerated, although systemic effects 
may occur. In one study conducted over a ten-year period, more than 600 patients with IDC 
were treated with metronidazole, and only 1% of them had significant adverse effects. 
These may include nausea, a metallic taste, disulfiram-like reactions with alcohol, and the 
effect of anticoagulants may be potentiated (Gonzalez-García et al., 2005). In addition, when 
metronidazole is administered for more than four weeks, it may generate neurotoxicity 
(McDonald et al., 2018). Systemic effects of oral vancomycin are rare. The main drawbacks 


 
 
 
 
 
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are the risk of developing resistant enterococci and the lack of therapeutic activity for CD 
when administered intravenously (Gonzalez-García et al., 2005).  
In cases of severe infection, the use of vancomycin as first-line drug is recommended. This 
recommendation is based on clinical experiences in which patients appear to respond more 
quickly to vancomycin than to metronidazole.  
The recommended duration of antibiotic treatment (metronidazole or vancomycin) in CD 
diarrhea is 10-14 days, both in initial treatment and in relapses (McDonald et al., 2018).  
Currently, and according to the American Society for Infectious Diseases (IDSA) (McDonald 
et al., 2018), the use of vancomycin and fidaxomicin over metronidazole is recommended 
for initial episodes of CDI unless access to them is limiting, in which case metronidazole is 
recommended only for non-severe cases. 
 
01.02.02. Fidaxomicin  
Fidaxomicin is a macrocyclic antibiotic approved for the treatment of CDI in Europe and 
USA (Johnson et al., 2012; Debast et al., 2014), more respectful of gut microbiota than 
vancomycin and metronidazole (Louie et al., 2012). There are two completed prospective, 
randomized, double-blind clinical trials demonstrating that the clinical efficacy observed 
following oral treatment with fidaxomicin is not inferior to that observed following oral 
treatment with vancomycin. However, differences in the number of recurrences were 
observed.  
In one of the studies, the percentage of recurrences observed in vancomycin-treated 
patients was 31%, whereas in those receiving fidaxomicin it was 21% (p=0.14) (Louie et al., 
2011). In another study, in which patients from the previous study were compared together 
with the de novo study, the rate of CDI recurrence was evaluated in a total of 1,164 patients 
treated with vancomycin vs. fidaxomicin in two phase III clinical trials. Treatment efficacy 
was similar (>90% cure) in both groups (fidaxomicin-treated patients vs. vancomycin-
treated patients) (Cornely et al., 2012).  
However, 28-day recurrence reached 35.5% for vancomycin-treated patients and 19.7% for 
fidaxomicin-treated patients (-15.8% difference; 95% confidence interval, -30.4% to -0.3%; 
P=0.045). Early recurrence (at 14 days) occurred in 27% of vancomycin-treated patients 
and 8% of fidaxomicin-treated patients (p=0.003). In patients with a first recurrence of CDI, 
vancomycin and fidaxomicin were similar in terms of clinical cure at the end of therapy, but 
fidaxomicin was superior when preventing a second recurrence within the 28-day interval 
(Cornely et al., 2012). Both trials also showed an overall increase in sustained response over 
time (Louie et al., 2011; Johnson et al., 2012; Cornely et al., 2012). Oral fidaxomicin is as well 
tolerated as oral vancomycin is, and both treatments have been shown to have a similar 
safety profile (Louie et al., 2011; Cornely et al., 2012). 
In 2014, the European Society for Clinical Microbiology and Infectious Diseases (ESCMID) 
recommended fidaxomicin for the first episodes of CDI, for the treatment of subjects with 
first recurrences, in patients at high risk of recurrence, and in patients with multiple 


 
 
 
 
 
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recurrences of CDI (Debast et al., 2014). Currently, the IDSA-published guideline 
recommends both vancomycin and fidaxomicin as the best initial treatment for CDI 
(McDonald et al., 2018); fidaxomicin is the recommended treatment for CDI patients at a 
very high risk of recurrence (Cornely et al., 2014; Bouza et al., 2020). Fidaxomicin is 
recommended by groups of Spanish and European experts for critically ill, 
immunocompromised, chronic renal failure, transplant patients and cancer patients 
(Aguado et al., 2015; Bouza et al., 2019). 
Fidaxomicin is a very high-cost treatment and is, therefore, limited in use in many European 
hospitals (Cornely et al., 2012; Bouza et al., 2020).  
 
01.02.03. Bezlotoxumab 
Bezlotoxumab is a recombinant human monoclonal antibody that prevents the binding of 
toxin B to intestinal cell receptors. This blocks the first step of the cellular intoxication 
cascade. Currently, the approved indication for bezlotoxumab is prevention of CDI 
recurrence in adult patients (≥18 years) receiving treatment for CDI and at a high risk for 
recurrence (IPT 31, 2018).  
The efficacy of bezlotoxumab has been evaluated in two randomized, double-blind, placebo-
controlled, multicenter (320 centers in 30 countries) phase III studies called MODIFY I and 
MODIFY II (Wilcox et al., 2017). The results of the comparison show that, among adult 
patients receiving antimicrobial therapy versus a primary or recurrent episode of CDI, the 
use of this antimicrobial therapy in combination with bezlotoxumab reduces the number of 
patients who will have a recurrence of CDI over the next 12 weeks by 37.5%; in other words: 
a 10% reduction in absolute relapse values in a population with an estimated prevalence of 
relapse of 26.6%.  
Following the good results, in Spain, the Spanish Agency of Medicines and Medical Devices 
(AEMPS, Agencia Española de Medicamentos y Productos Sanitarios) (IPT, 31. 2018) 
authorizes the use of bezlotoxumab as adjunctive treatment for the treatment of CDI (to 
date, it is the only antibody-based product approved for the treatment of recurrences) in 
patients at high risk of recurrence, including patients ≥65 years of age with prior episodes 
of CDI in the previous six months, in immunosuppressed patients, in patients with CDI 
produced by hypervirulent strains (such as 027 and 244), patients with severe CDI (Zar ≥2), 
and patients with a high probability of recurrence based on externally validated predictor 
models. 
 
01.02.04. Fecal Microbiota Transplantation 
Fecal Microbiota Transplantation (FMT) consists of the infusion of stools from a healthy 
donor into a patient’s intestinal tract to treat a specific disease associated with alteration of 
the intestinal microbiota (Cammarota et al., 2017), as is the case for CDI. Within this context, 
FMT has emerged as a clinical procedure demonstrating high efficacy and safety in the 
treatment of recurrent CD infection (r-CDI) by restoring the gut microbiota and preventing 
successive recurrences (van Nood et al., 2013). Because of the beneficial effects obtained in 


 
 
 
 
 
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patients with r-CDI, studies have also been conducted to propose FMT as a treatment for 
the first CD infection and have also yielded favorable efficacy and safety results within this 
population (Camacho-Ortiz et al., 2017; Ng SCC et al., 2017; Juul et al., 2018). FMT can be 
administered by colonoscopy, enema, naso-duodenal or naso-jejunal tubes and oral 
capsules; and is, even after the onset of bezlotoxumab, the preferred non-antimicrobial 
treatment for treating CDI recurrences (Reigadas et al., 2019). The European Society for 
Clinical Microbiology and Infectious Diseases (ESCMID) Guidance in 2014 strongly 
recommended the use of FMT in cases of multiple CDI recurrences (Debast et al., 2014). 
Moreover, a panel of 28 experts from 10 countries across Europe recently published a 
European Consensus in which they recommended FMT as a treatment option for refractory 
CDI and moderate-to-severe CDI recurrences, as well as implementation in clinical practice, 
based on the quality of evidence (Cammarota et al., 2017). 
01.03. Nonclinical Studies 
CDI has been extensively studied in different animal models. Distinct aspects of the disease 
(colonization, pathophysiology, transmission, recurrence, mechanism of action of toxins, 
etc.) have been investigated in small animal models (hamster, mouse, rabbit, hare, guinea 
pig, prairie dog, partridge, or zebrafish embryos) as well as in a limited number of large 
animal studies (foal, pig, and monkey) (Hamm et al., 2006; Lanis et al., 2010; Best et al., 
2012; Hutton et al., 2014).  
The primary therapy for the treatment of CDI is antibiotics against the pathogen. Once the 
antibiotic treatment is underway, normal intestinal function is restored. To prevent disease 
recurrence, the indigenous microbiota must also be restored. Current antibiotic therapy 
does not favor this. Thus, FMT is an alternative treatment that contributes to the definitive 
restoration of the indigenous microbiota through the inoculation of a full ecosystem from a 
healthy donor (Khoruts et al., 2010, Greham et al., 2010). 
Different studies in mouse models of r-CDI have evaluated the efficacy of FMT treatment. In 
a study comparing vancomycin treatment with FMT, vancomycin-only mice were shown to 
have a recurrence of CDI when treatment was stopped. However, recurrence was prevented 
in mice treated with FMT (Seekatz et al., 2015). Another study demonstrated that FMT is an 
effective method for palliating the inflammatory effects of mice with antibiotic-induced CDI, 
as well as for eliminating the infectious agent and recovery of the gut microbiota (Litchman 
et al., 2016). Similar results were obtained in other animal (Buffie et al., 2015, Lawley et al., 
2012) and human (Seekatz et al., 2014, Hamilton et al., 2013, Song et al., 2013) models, 
suggesting that FMT is an effective treatment for the restoration of microbiota and may be 
necessary to prevent CD recurrence in CDI patients.  
In a study in mice with antibiotic-induced CDI, the efficacy of fresh, frozen, and lyophilized 
FMT was evaluated for 2 to 15 months, and so was the stability. The transferred microbiota 
was of human origin, and the number of days that the mice had diarrhea after being infected 
with CD strain VPI 10463, toxin A and B was evaluated. The results showed that both frozen 
and lyophilized products can be stored for up to seven months and that, during that time, 
they maintained the same clinical effectiveness as the fresh product. However, after seven 


 
 
 
 
 
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months, the products began to lose efficacy in vivo. These results demonstrate that FMT 
products have a limited storage time, even if they are stored at very low temperatures (Jiang 
et al., 2017b). 
Animal FMT studies have shown certain limitations, such as lack of inter-individual 
variability. In addition, in the case of preclinical studies, the statistic for being able to 
determine differences is mainly based on the potential of the treatment. When treatment is 
less effective, a larger study sample size is required. On the other hand, it is difficult to 
extrapolate the results of FMT in animals to humans, adverse effects and safety are not 
always adequately assessable in animal models; therefore, the safety profiles of FMT in 
previous human studies should be analyzed (Staley et al., 2017, Arrieta et al., 2016, Best et 
al., 2012). 
01.04. Previous Clinical Experience 
As described above, oral vancomycin and fidaxomicin are the antibiotics of choice for the 
treatment of CDI, with FMT treatment being reserved for recurrences (Bouza et al., 2020, 
McDonald et al., 2018). It has been shown that the use of FMT not only protects against 
infection but also prevents future recurrences (Khoruts et al., 2010, Greham et al., 2010). 
On the other hand, there are also clinical trials that have evaluated its potential efficacy and 
safety for the treatment of patients with primary CD infection. Although antibiotic therapy 
is recommended for the first episode of CDI, treatment with oral vancomycin and/or 
metronidazole often leads to significant treatment failure, with recurrences in up to 30-40% 
of patients experiencing a first episode. In addition, antibiotic treatment does not correct 
deficiencies in the intestinal microbiota that facilitate CD infection and is associated with 
the risk of antibiotic-resistant bacteria emerging (Camacho-Ortiz et al., 2017). Because 
administration of FMT results in faster reconstitution of the underlying intestinal 
microbiota alterations, as well as eradication of infection, and has been used for the 
treatment of patients with recurrent CDI with high efficacy and safety even in 
immunocompromised patients, the following trials have studied FMT administration as a 
potential treatment for primary CD infection.  
Camacho-Ortiz et al. conducted an open-label trial with two treatment arms that patients 
were randomized to in a 1:1 ratio: oral vancomycin (250 mg every 6 hours for 10-14 days) 
or FMT (consisting of a mixture of fecal donors). Patients in either arm who did not clinically 
improve by 72 hours after the first treatment received a second FMT treatment. The study 
was conducted in adults hospitalized with the first episode of CDI. None of the patients had 
had a prior CDI or had received prior treatment for their current CDI. Patients with toxic 
megacolon, documented or suspected intestinal perforation, pregnancy, or concurrent 
colon neoplasms were excluded.  
FMT doses were prepared by collecting three stool samples from each donor, mixing the 
stools from all donors, resuspending in 0.9% saline and filtering. Finally, glycerol, at a final 
concentration of 15% (v/v), was added before storing the mixture in 45 mL aliquots and 
freezing at -80ºC. A 45-mL aliquot was considered a dose. In total, 881.62 g of feces were 
collected. The final suspension had a concentration of 0.19 g/mL. FMT could be 


 
 
 
 
 
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administered by different routes at the discretion of the physician. FMT was administered 
by upper endoscopy in all but two cases: one patient received FMT by a nasojejunal tube 
already in place, and another, by colonoscopy. 
A total of 16 patients out of the 19 recruited completed the study. Out of the 9 patients in 
the vancomycin treatment group, 8 patients experienced resolution of their infectious 
symptoms (88.9%); in the FMT treatment group, 4 out of 7 patients (57.1%) experienced 
resolution after the first dose of FMT (P=0.26), and 5 out of 7 patients, after the second dose 
of FMT (P=0.55). The microbial composition of the participants over time in the vancomycin 
group was different from the microbiome in the FMT group, confirming the impact oral 
antibiotics have on the gut microbiota. Regarding treatment safety, no adverse effects 
related to FMT were observed throughout the study (Camacho-Ortiz et al., 2017). 
Juul et al conducted a randomized, blinded Phase 2 clinical trial in patients with primary CD 
infection (NCT02301000). Twenty-one adult patients with acute C. difficile infection who 
had not had a previous C. difficile infection were randomly assigned to receive oral 
metronidazole (a dose of 400 mg three times daily for 10 days) or FMT (an enema 
administered by a renal catheter of 60 mL of anaerobically cultured human intestinal 
microbiota). The primary objective of the study was to evaluate disease resolution without 
evidence of recurrence of CD infection at Day 70. One patient was excluded, and, out of the 
20 eligible patients, 9 were randomly assigned to fecal microbiota transplantation and 11, 
to metronidazole.  
A primary complete response was observed in five patients (56%; 95% CI: 21-86) in the 
FMT group and in 5 in the metronidazole arm (45%; 95% CI: 17-77) (exact P=1.00). Three 
of the remaining four patients in the FMT group received antibiotics on day 4 after 
treatment initiation, and two of them had a secondary complete response. In the 
metronidazole group, of the remaining six patients, none had a complete secondary 
response because the infection was refractory or recurrent. Thus, overall treatment 
response (primary or secondary complete response) was achieved in seven patients in the 
FMT-treated group (78%; 95% CI: 40-97), compared to five in the metronidazole group 
(45%; 95% CI: 17-77) (P=0.20). Serious treatment-related adverse events were not 
observed in either study group (Juul et al, 2018).  
Due to the beneficial results obtained in this study, patients are currently being recruited 
for a multi-center, randomized, controlled, parallel-group, two-arm (FMT and antibiotic) 
phase III clinical trial (NCT03796650), which is a continuation of the phase II trial for 
primary Clostridium difficile infection. In this trial, patients with CDI and no prior CDI within 
12 months prior to enrollment will be randomized in a 1:1 ratio to receive FMT or be treated 
with vancomycin (10 days, 125 mg, four times daily). The trial is still active, so no results 
are available as yet.  
Another clinical trial (Ng SCC et al., 2019) has been conducted in patients with a primary 
episode of CDI, which evaluated the efficacy of FMT compared to the standard regimen of 
vancomycin. This was an open-label, randomized study in which 30 patients were assigned 
to receive either oral vancomycin (500 mg, four times daily) followed by FMT (a single 
infusion of stool from donors administered via nasoduodenal tube), or a standard regimen 


 
 
 
 
 
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of oral vancomycin (500 mg four times daily for 10 days). The primary objective was to 
demonstrate resolution of CDI-associated diarrhea without relapse within 10 weeks of 
treatment initiation. In addition, 30-day and six-month mortality, 30-day colectomy rates, 
length of hospital stay, adverse effects, and fecal microbiota alteration after FMT were 
studied as secondary objectives. Sixty percent of subjects in the vancomycin arm and 47% 
of subjects in the FMT arm, respectively, had severe CDI. Resolution of C. difficile infection 
occurred in 10 of 15 patients (66.7%) who received vancomycin and in 11 of 15 patients 
(73.3%) who received FMT (p=1.00). Two deaths occurred in the vancomycin group and 
none occurred in the FMT group within 30 days. After six months of the study, there were 
nine (60%) deaths in the vancomycin arm and three (20%) deaths in the FMT arm. 
Therefore, mortality was higher in the vancomycin-treated group. None of the patients had 
to have a colectomy. The median length of stay was the same in the two groups (13 vs. 15 
days; p=0.95). In patients receiving FMT, a restoration of enriched bacteria from healthy 
donors was observed in recipients after FMT. Regarding treatment safety, no serious 
adverse events attributed to FMT were detected.  
Therefore, while results still must be proven in studies with a larger sample size, these trials 
reflect the potential efficacy and safety of FMT for the treatment of patients with primary 
CD infection. 
A meta-analysis published by Madoff and colleagues evaluated all the clinical trials 
conducted up to February 2018 that analyzed the efficacy and safety of different treatments 
for the prevention of CDI recurrence. Treatments used were antibiotics, FMT, monoclonal 
antibodies, probiotics, or other compounds (Madoff et al., 2020). Out of the clinical trials 
discussed in this review, eight evaluated the efficacy of FMT treatment for the prevention of 
CDI recurrence in a total of 582 patients. In all the studies evaluating FMT, only mild 
intervention-related adverse effects such as abdominal distension, cramping, nausea, and 
vomiting were reported. No serious adverse effects were reported in any case (Van Nood et 
al., 2013; Youngster et al., 2014a; Kelly et al., 2016; Lee et al. 2016; Hota et al., 2017; Jiang 
et al., 2017a; Kao et al., 2017). 
The first clinical trial demonstrating superiority of FMT treatment versus vancomycin 
treatment in patients with recurrent CDI was published in 2013. In this clinical trial, 
patients were randomly assigned to three treatment groups: the first group received four 
days of treatment with oral vancomycin (500 mg, 4 times daily), followed by bowel lavage 
and FMT via gavage; the second group was treated with oral vancomycin for 14 days, and a 
third group received oral vancomycin for 14 days, followed by bowel lavage. In patients 
receiving FMT, diarrhea resolved more effectively compared with the other two groups of 
patients who did not receive FMT (p <0.001). The frequency of adverse events was similar 
in all three groups. Of the 16 patients in the FMT group, 81% (n=13) showed resolution of 
CD-associated diarrhea after a single transfer. Of the remaining three patients who received 
a second microbiota transfer, the infection was resolved in two of them. In the vancomycin-
treated group, 31% (4/13 patients) resolved the diarrhea caused by CD, and 23% (3/13 
patients) in the third group (Van Nood et al., 2013). 


 
 
 
 
 
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Another subsequent study compared FMT treatment by colonoscopy to a standard cycle of 
oral vancomycin (125 mg 4 times daily, followed by 125-500 mg/day every 2-3 days for at 
least three weeks). This study demonstrated that FMT treatment is significantly more 
effective in preventing CDI recurrence than the standard vancomycin regimen (p <0.001). 
This study ended one year from the start, as 90% (18/20 patients) of those treated with 
FMT showed clinical resolution of the disease; whereas, in the vancomycin-treated group, 
only 26% (5/19 patients) experienced resolution; the cure rate was 87% for FMT-treated 
patients and 55% for vancomycin-treated patients (Cammarota et al., 2015). 
Following early studies showing the efficacy of FMT as a treatment for recurrent CDI, other 
studies emerged that evaluated the differences in efficacy and safety between the different 
routes of FMT administration, as well as the development of different preparations that 
could be stored to allow for greater availability and flexibility for treatment application. 
In a comparative study between FMT treatment administered by colonoscopy vs. oral liquid 
suspension capsules, no differences in efficacy were found between both routes of 
administration (RR 0.98 [95% CI, 0.14-6.71]) (Kao et al., 2017). Another study comparing 
colonoscopy vs. nasogastric tube administration also found no differences in efficacy (RR 
0.20 [95% CI, 0.01-3.70]) (Youngster et al., 2014a). 
Jiang et al compared the efficacy of frozen FMT suspension administered via enema versus 
lyophilized FMT administered orally via capsules in a total of 65 patients with three or more 
episodes of CDI recurrence. Patients treated with oral FMT received a dose of lyophilized 
microbiota obtained from 100 g feces (1.5 g microbiota powder/100 g fecal product), which 
was subsequently increased to 200 g to improve treatment efficacy. Patients treated with 
FMT via enema were administered a dose of microbiota obtained from 100 g of feces. The 
efficacy in preventing CDI recurrence 60 days after treatment was 84% for patients treated 
orally and 88% for patients treated with enema. Normalization of microbiota diversity was 
observed in all patients, although Bacteroides repopulation was lower in orally treated 
patients. No differences were observed in adverse effects (flatulence, nausea...) found after 
both administrations (Jiang et al., 2018). 
Studies evaluating the efficacy of different routes of administration and different FMT 
preparations conclude that administration of FMT with previously frozen, colonoscopy-
administered microbiota has an efficacy similar to that of fresh FMT suspension (RR 9.36 
[95% CI, 0.53-165.03]) (Jiang et al., 2017a). Treatment efficacy was not reduced in cases in 
which administration of previously frozen FMT was via enema (RR 0.84 [95% CI, 0.54-
1.30]) (Lee et al., 2016). 
Hecker and colleagues conducted a study comparing the efficacy of lyophilized microbiota 
oral capsules to frozen microbiota liquid suspension capsules. Twenty patients with 
recurrent CDI were treated and dosed with 20 to 40 capsules (sized 0 capsules containing 
60 mg lyophilized feces, including 00 capsules), obtained from approximately 40 g of feces 
(total anaerobic concentration/mL lyophilized vs. frozen microbiota was 8.5 ±0.5 and 
9.1 ±0.2 log10 colony forming units, respectively). One month after treatment, diarrhea 
resolved in 85% of patients, and overall resolution amounted to 90% after treatment with 
a second dose of FMT in patients who had relapsed after the first dose. No serious adverse 


 
 
 
 
 
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effects were reported during the 204-day mean follow-up (range 31-408 days). Therefore, 
the use of lyophilized microbiota oral capsules was shown to be effective and to allow for 
the delivery of a high concentration of viable bacteria, with a predominance of anaerobes 
(Hecker et al., 2016). 
Overall, no significant differences in clinical efficacy and safety outcomes were found for the 
different routes of FMT administration in patients with recurrent CDI, nor were there 
differences between the different sample preparations. 
Numerous studies have evaluated not only the efficacy but also the safety of FMT treatment 
by oral capsules in patients with CDI (Youngster et al., 2014b, Hirsch et al., 2015, Youngster 
et al., 2016, Hecker et al., 2016, Staley et al., 2017, Staley et al., 2019, Reigadas et al., 2018, 
Reigadas et al., 2019). 
One of the studies discussing the efficacy and safety of FMT administered as previously 
frozen oral capsules evaluated treatment in 20 patients with recurrent CDI. Each patient 
was administered 30 microbiota capsules (15 capsules daily, for two consecutive days); this 
microbiota was obtained from the processed 48 g of feces, with a mean dose equivalent to 
1.6 g of microbiota/capsule (range 1.0-2.05 g/capsule). Resolution of diarrhea was assessed 
at eight weeks post-treatment, and follow-up was prolonged for six months. Seventy 
percent of patients had no diarrhea after eight weeks of treatment, and the percentage 
amounted to 90% after a second dose of FMT. The efficacy obtained in this study 
corroborates the results of previous studies of this same group in which the treatment 
applied by the nasogastric route or colonoscopy was evaluated. During the six months of 
follow-up, 30% of patients reported mild adverse events such as abdominal cramps or 
bloating that resolved spontaneously at 72 hours. On the other hand, freezing of the 
microbiota was shown to not diminish treatment efficacy, which allowed for the detection 
of other types of possible infections in the donor, thus increasing safety in treated patients 
(Youngster et al., 2014b). Another study in the same group evaluated the efficacy and safety 
of FMT via frozen microbiota capsules in a total of 180 patients with at least three moderate 
CDI episodes and at least two severe CDI episodes (requiring hospitalization). Each patient 
was administered 30 capsules of FMT microbiota obtained from the 48 g of feces processed, 
15 per day, for two consecutive days. Again, resolution of diarrhea was assessed for eight 
weeks following FMT treatment at six months of follow-up. After eight weeks, the primary 
resolution of diarrhea in such patients was 82%. Following a re-transfer with the same dose 
of 30 capsules, the overall resolution increased to 91% and then to 93% after a third 
transfer (Youngster et al., 2016). The efficacy of administration of frozen microbiota 
capsules was shown to be the same as that observed in previous studies by the same team, 
in which FMT was administered by liquid preparations via gavage or colonoscopy 
(Youngster et al., 2014a, Youngster et al., 2016). Only three grade 2 or higher effects (one 
episode of transient high fever and two newly diagnosed cases of ulcerative colitis) 
considered to be related or possibly related to FMT were observed during the six months of 
follow-up. The other cases reported in 84% of patients were grade 1-adverse events, mostly 
mild intestinal discomfort. (Youngster et al, 2016). 


 
 
 
 
 
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Another study evaluated treatment with liquid microbiota oral capsules to treat patients 
(n=19) with two or more recurrence episodes (a mean of four recurrences per patient) of 
CDI. All patients had previously received treatment with metronidazole, vancomycin, 
and/or fidaxomicin. Each patient was administered 8-12 capsules, each one containing 0.4 
mL of liquid suspension at 0.5 g/mL (patients received 1.1 to 8.3 g of input material). A 90-
day follow-up was performed, after which 68% of patients had resolved; after a second 
administration of FMT, the percentage of diarrhea resolution was 89%. In this study, the 
minimum effective dose was lower than the minimum dose used in other studies, as 70% of 
patients showed a positive response with a dose less than 3 g of starting material, and with 
an estimate of 9.7 x1010 viable bacteria. No serious adverse effects were recorded during 
the entire follow-up period. In addition, the capsule presentation was shown to be an easy 
and safe type of administration for patients of advanced age and/or with comorbidities 
(Hirsch et al., 2015). 
The efficacy of FMT was evaluated in patients in whom treatment with antibiotics (including 
vancomycin) had failed. Forty-nine patients with 3-4 episodes of CDI (patients with 
inflammatory bowel disease excluded from the study) were treated with frozen and 
lyophilized oral capsules. Patients were initially treated with 24-27 capsules (~2.5 x1012 
bacteria) and, subsequently, due to a limitation on the number of FMT capsules obtained in 
the development phase, the dose was reduced to 2-3 capsules per patient (~2.1-2.5 x1011 
bacteria), which were administered as a single dose. Two months after transfer, overall 
recovery was 87.7%, with no significant differences observed between the highest (78.9% 
recovery; 15/19 patients) and lowest (93.3% recovery; 28/30 patients) doses. Following 
treatment, the fecal intestinal microbiota increased in time toward the normalization of the 
patient’s microbiota one month after FMT, with no differences in kinetics or in this increase 
observed between high and low doses. However, they recorded a delay in this normalization 
of the microbiota, especially Bacteroides, when compared to the team studies in which FMT 
administration was performed with colonoscopy, although it did not affect clinical response. 
Administration of these capsules was shown to be safe, as the adverse effects that one third 
of patients showed consisted of abdominal swelling, flatulence, etc., in the first weeks after 
FMT (Staley et al., 2017). 
This same group performed the assessment of colonization restoration in FMT-treated 
patients in order to account for the effect of this treatment on the recovery of the indigenous 
microbiota. The study was conducted in 18 patients with at least two episodes of CDI 
recurrence who had been previously treated with vancomycin. Treatment consisted of 
administration of 2 to 4 lyophilized FMT capsules, with the bacterial dose ratio of 2.1 x1011 
to 5 x1011 bacteria/capsule. All patients showed clinical recovery and were free of CDI, 
although with varying degrees of colonization restoration throughout the year of follow-up. 
Sixty-one percent of patients had a successful restoration of colonization after one week of 
FMT, and it was maintained throughout the year. Twenty-two percent of patients had 
decreased colonization after one month, and 17% of patients, of whom two were taking 
metformin, had poor colonization. Following these observations, it can be concluded that 
no more than one dose of FMT is required to achieve clinical recovery in patients. 
Furthermore, no difference in efficacy was observed between the high and low doses 


 
 
 
 
 
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administered. The results of this study describing the restoration of long-term colonization 
following FMT treatment also suggest that the numerical similarity of the donor is not 
strictly related to clinical outcome, and they identify a persistent donor-specific effect on 
fecal bacterial communities in the patient (Staley et al. 2019). 
In line with the studies described above, Hospital Universitario Gregorio Marañón has been 
conducting several cohort studies in 2014 in which patients with recurrent CDI are treated 
with FMT. One of the studies conducted by this group was in 13 patients with 2 or more 
episodes of CDI recurrence in which FMT is applied via three different routes of 
administration: liquid-containing oral capsules, colonoscopy, and nasojejunal tube. Some of 
the patients included in the study had other comorbidities such as hematological diseases 
or cirrhosis. Overall efficacy with a single dose of FMT was 83.3%; when patients who 
received more than one dose were included, efficacy reached 91.7%. Oral capsule use was 
found to be as effective as treatment given by colonoscopy or nasojejunal route. Only one 
adverse effect was reported in one of the patients who experienced bacteremia. This study 
demonstrated that the use of FMT is safe and effective, even in patients with different 
comorbidities (Reigadas et al., 2018). Another study conducted by the same group in which 
32 patients with recurrent CDI were treated (a mean of two relapses/patient) and supplied 
with 4-5 lyophilized fecal content capsules at a single dose (final bacterial dose range per 
patient was 2.1 to 2.5 x1011) has recently been published, with the minimum administered 
dose of 2.1 x1011. The primary efficacy was 81.3% and, after a second transfer, the overall 
efficacy amounted to 87.5%. During the follow-up time, no adverse effects were observed 
in patients, and the capsules were well tolerated (Reigadas et al., 2019). 
Overall, a high recovery rate is observed for patients with recurrent CDI treated with FMT 
with similar percentages across studies. It has been seen that in cases where the first FMT 
transfer is not effective, the administration of a second dose is effective. One of the most 
recent meta-analyses evaluating the efficacy of FMT in recurrent CDI is 92% for both 
controlled and uncontrolled studies (Quraishi et al., 2017). 
On the other hand, with respect to the optimal dose of FMT there is a disparity between the 
different studies. There is considerable heterogeneity among published studies with respect 
to the dose to be used, both in the treatment with liquid FMT (administered by colonoscopy, 
nasogastric tube or enema) and in the form of capsules. In addition, not all studies 
adequately detail the dose that has been used; most papers using liquid FMT with non-oral 
or oral gavage administration say they use a stool dose of ≥50g. There are some studies 
reaching 152 g ±32 g of donor stool (Cammarota et al. 2015) with 90% resolution, and 
another study or so using a mean dose of 62 g, not as high as the previous one, that shows a 
recurrence of 9.1% (Kelly et al. 2016, Madoff et al. 2020). Other studies use a dose of 100 g 
via enema, with a recurrence of CDI of 12%, somewhat higher than that reported with lower 
doses, although the difference is not significant (Jiang et al., 2018).  
In addition, when performing FMT via capsules across studies, the effectiveness and efficacy 
of FMT is similar from one study to another, even if different doses are applied. Using 
lyophilized formulations facilitates bacteria concentration, leverages donor samples better, 


 
 
 
 
 
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and decreases the number of capsules the patient needs to swallow (Hirsch et al., 2015, 
Hecker et al., 2016, Reigadas et al., 2019).  
The few studies that have evaluated the minimum bacterial doses indicate that FMT 
containing bacterial doses of 9.7 x1010 for liquid capsules and 2.1-2.5 x1011 for lyophilized 
capsules were effective in the treatment of CDI. Dosing in the range of 2.1-5.7 x1011 makes 
it possible to administer a low number of capsules and obtain an effective treatment 
outcome (Reigadas et al., 2019).  
In terms of assessing the safety of FMT in patients with CDI recurrence, treatment has been 
shown to have a high level of safety in all previous studies, with a low number of adverse 
effects reported, mostly moderate and self-limited. In a review of the safety of FMT, 50 
studies were analyzed and it was concluded that the safety profile of FMT is positive, given 
that patients have no serious adverse effects, with only mild or moderate adverse effects 
having been reported, such as nausea, vomiting, swelling, abdominal pain or cramps or 
flatulence, all self-limited episodes with a short duration occurring shortly after FMT 
(Youngster et al 2014a, Youngster et al. 2016, Staley et al. 2017, Cheng et al. 2019, Madoff 
et al. 2020). In addition, the type or severity of adverse effects was shown not to differ 
depending on the route of administration, although patient perception is more positive 
when capsules are administered by oral administration (Kao et al., 2017, Hirsch et al., 2015, 
Staley et al., 2017, Jiang et al., 2018, Cheng et al., 2019).  
The short-term safety of treatment has been demonstrated in all studies. Long-term safety 
was assessed in one of the studies, which follows patients treated with FMT for one year. 
This study reports that patients had no gastrointestinal adverse effects (Staley et al., 2019).  
The safety of FMT has been demonstrated even in immunocompromised patients who are 
at increased risk of CDI. The safety of FMT was evaluated in a study of 80 
immunocompromised patients (75 adults and 5 pediatric) with relapsed, refractory, or 
severe CDI treated with FMT who were followed up on during a mean of 11 months (3 to 46 
months of follow-up). Overall treatment efficacy was 89%. In addition, the safety of FMT 
was demonstrated in these patients, who had the mild adverse effects described above. 
None of the patients had infectious complications (Kelly C.R. et al., 2014). The safety of this 
treatment has been evaluated in another immunocompromised population: patients with a 
history of solid organ transplants and, therefore, also immunocompromised. In these 
patients the treatment was administered by colonoscopy and enema. After three months, 
58.7% of single-transfer patients recovered, and this increased to 91.3% after repeat 
transfer (repeating administration up to 2, 3, 4 or more times). The recovery ratio with a 
single transfer was lower for patients with severe or fulminant CDI, although it was also 
observed that the mean immunosuppressive medication was higher in the patients in whom 
the transfer failed. Twenty-two percent of patients showed the adverse effects described 
above, no cases of bacteremia (3.2%) were due to FMT, and only 14% of cases with 
underlying CMV (three patients) had reactivation. They concluded that treatment with FMT 
is safe for immunocompromised patients (Cheng et al., 2019).  
No severe adverse effects due to transmission of such organisms have been seen in more 
than 45,000 FMT treatments provided by OpenBiome, a not-for-profit stool bank (founded 


 
 
 
 
 
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in 2012 in Cambridge, MA, USA) or in randomized controlled trials for other indications 
(Kuijper 2019). This transmission is also absent in both the systematic review of more than 
50 articles (Wang et al. 2016) and the retrospective analysis of the 80 immunocompromised 
patients treated with FMT (Kelly C.R. et al. 2014). In conclusion, the safety of FMT is high, 
and no serious adverse effects resulting from this treatment have been reported. However, 
it should be noted that further analyses are needed to verify long-term safety (Tan et al. 
2019). 
However, despite the positive safety results reported in the FMT studies, in July 2019 the 
US Food and Drug Administration (FDA) issued a safety alert regarding serious adverse 
reactions caused by the transfer of multidrug-resistant organisms via FMT; concretely, in a 
case in which there was transmission of a β-lactamase-producing strain of Escherichia coli 
from the feces of one donor to two immunocompromised patients. Subsequently, six 
additional cases of CDI patients with enteropathogenic E. coli or CTE infection (two 
immunocompromised patients) and Shiga toxin-producing E. coli or STEC infection (four 
patients) were reported in March 2020 after receiving an FMT provided by OpenBiome. Out 
of these six cases, four were hospitalized and subsequently discharged, and two were 
treated as outpatients (important safety alert regarding use of fecal microbiota for 
transplantation and risk of serious adverse reactions due to transmission of multi-drug 
resistant organisms, FDA 2019, Safety Alert Regarding Use of Fecal Microbiota for 
Transplantation and Risk of Serious Adverse Events Likely Due to Transmission of 
Pathogenic Organisms, FDA 2020). Moreover, in August 2022, the FDA issued a safety alert 
on the potential risk of transmission of monkey smallpox via FMT. Although the risk of 
transmitting this virus via FMT is not yet determined, there are studies that have 
documented the presence of this DNA in rectal swabs and/or stool samples of infected 
individuals. In one study, virus DNA was detected in rectal swabs from three individuals 
who reported no symptoms of monkey smallpox disease, and in two of them the virus was 
isolated from the viable monkey smallpox in the rectal swabs. (“Safety Alert Regarding Use 
of Fecal Microbiota for Transplantation and Additional Safety Protections Pertaining to 
Monkeypox Virus”, FDA 2022). 
In relation to these potential risks, Mikrobiomik performs selective cultures to detect 
enteropathogenic E. coli and multi-resistant strains. All donors in which this type of bacteria 
is detected will be discarded, thus minimizing the risk of transmission of these organisms 
via FMT. With regard to monkey smallpox, the questionnaire that is administered to donors 
raises the question as to whether the patient is infected with this virus or has been in contact 
with someone with this disease. In addition, the presence of the monkey smallpox virus by 
polymerase chain reaction (PCR) is determined in the analysis performed on the feces. 
In conclusion, the short-term efficacy and safety of FMT treatment of patients with 
recurrent CDI is well demonstrated in several clinical studies. This treatment represents an 
effective and safe way to prevent CDI recurrence and is an effective alternative treatment in 
patients who have failed antibiotic treatment (Bouza et al., 2020) or have multiple CDI 
recurrences. 
 


 
 
 
 
 
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02. STUDY OBJECTIVES 
02.01. Primary Objective 
The primary objective of the study is to evaluate the efficacy of the investigational 
medication (MBK-01) compared to the control (fidaxomicin) at eight weeks after treatment 
initiation. 
02.02. Secondary Objectives  
The secondary objectives are to assess the safety of the investigational medication and the 
quality of life of patients participating in the study. 
03. TYPE OF CLINICAL STUDY AND DESIGN 
03.01. Type of study. Global Design 
Phase III randomized, controlled, open-label clinical trial with 2 treatment arms. 
03.02. Randomization Process 
Since the study case report form is electronic (eCRF), the eCRF’s own platform has a 
randomization tool that will allow this process to be performed. 
03.03. Type of Control and Design 
I. 
Experimental group: single-dose treatment of four oral capsules of lyophilized 
fecal microbiota (MBK-01).  
II. 
Control group: treatment with fidaxomicin, 200 mg/12 hours for 10 days. 
03.04. Blinding Techniques 
There are no blinding techniques in this study; it is an open-label study. 
03.05. Study Plan 
Once the patient is considered for participation in the study due to him or her experiencing 
an episode of diarrhea (≥3 stools/24 hours), and the presence of CD is confirmed with the 
prescribed tests, the inclusion and exclusion criteria will be assessed.  
Treatment of the diarrhea episode with 3-5 days of vancomycin is permitted as long as a 
subsequent 24-hour washout is performed prior to randomization and study medication 
administration. The patient will be enrolled in the study by signing the informed consent 
(ICF) at the time the 24-hour washout period begins.  


 
 
 
 
 
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For patients who have been started on fidaxomicin but have not completed the first day of 
antibiotic treatment, treatment with fidaxomicin may be stopped and treatment of the 
diarrhea episode started with 3-5 days of vancomycin, provided that a subsequent 24-hour 
washout, prior to randomization and administration of study medication, as in the previous 
case, is performed. The patient will be included in the study upon signing the ICF, once the 
24-hour washout period begins. 
If the patient, upon enrolling in the study, is on antibiotic treatment for a medical indication 
(other than the episode of infection being studied), he/she should discontinue antibiotic 
treatment before enrolling in the study and undergo a 24-hour washout period. The patient 
will be included in the study upon signing the ICF, once the 24-hour washout period begins.  
Accordingly, in the event that patients have received previous treatment for the episode 
with vancomycin (3-5 days) and/or antibiotic treatment for any other indication, it is 
necessary that, after signing the ICF, they undergo a 24-hour washout period prior to 
randomization.  
Finally, subjects will be randomized and assigned to one of the two treatment arms. The 
patient will then be administered the study medication. Subsequently, follow-up will be 
performed for six months after treatment until the study close-out visit. 
 
03.06 Early Trial Termination 
This study may be terminated prematurely if, in the opinion of the Sponsor, there is 
sufficient cause. The investigator will receive written notification that the cessation party 
documents the reason for the suspension and provides instructions for the follow-up of 
active patients.  
In case of premature termination of the trial, the investigator will be responsible for 
ensuring the patient’s medical care, per standard of care or compassionate use. 
 
03.07. Treatment Efficacy Assessments 
 
03.07.01. Definition of Study Endpoints 
03.07.01.A. Efficacy Variables 
Primary variable: The absence of CD diarrhea without the need for re-treatment (or 
absence of recurrence as defined in Section 03.07.02), as assessed by the subject interview 
and physical examination. The primary variable is operated by the probability of 
recurrence. 
 
Other efficacy variables:  
Good/poor patient progress: The definition of good/bad evolution is detailed in Section 
03.07.02. 


 
 
 
 
 
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Time to Recurrence Based on Randomization Groups: The definition of recurrence is 
detailed in Section 03.07.02. 
Overall survival (OS): Percentage of patients still alive after a defined period of time since 
treatment was started. 
 
03.07.01.B. Safety Variables 
• 
Number of Adverse Events by randomization group. 
• 
Number of patients within each Adverse Event by randomization group. 
• 
Type of Adverse Events by randomization group. 
• 
Number of Serious Adverse Events by randomization group. 
• 
Type of Serious Adverse Events by randomization group. 
• 
Treatment-Related Adverse Events. 
• 
Severity of Adverse Events. 
• 
Adverse Events related to CDI. 
• 
Mortality associated with CDI. 
• 
Intensive Care Unit (ICU) admissions. 
• 
Adverse Events of Special Interest: 
Common Treatment-Related: 
Abdominal cramps, bloating and pain 
Flatulence 
Nausea 
Vomiting 
Transient fever (>38ºC) 
C-reactive protein increased (>20 mg/L) 
Fatigue 
Weight gain 
Headache 
Anorexia 
Constipation 
Diarrhea 
 
Uncommon (less than or equal to 1%): 
Ulcerative colitis 
Bacteremia 
Inflammatory Bowel Disease 
 
Rare or very rare or not known: 
Enteropathogenic infections 
 


 
 
 
 
 
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03.07.01.C. Quality of life variables 
Quality of life will be assessed using the indicators obtained by the SF-36 test, according to 
the design described in the literature (Ware et al., 1993; Alonso et al., 2003; Vilagut et al. 
2005; Vilagut et al, 2008). These measurements will be performed on all patients enrolled 
in the study, and data will be collected at baseline, eight weeks and six months post-
treatment. 
 
03.07.02. Definitions 
Mild to moderate CDI 
Episode of CDI with WBC count ≤15,000 cells/μL and serum creatinine less than 1.5 times 
baseline. 
Severe CDI 
CDI cases with a WBC count >15,000 cells/μL and/or a serum creatinine level equal to or 
greater than 1.5 times the baseline level. 
Complicated severe CDI 
CDI cases presenting with septic shock, ileus or megacolon, or requiring intensive care unit 
admission within seven days of episode diagnosis or death attributable to CDI. 
Episode of diarrhea 
Presence of ≥3 stools/24 hours. 
Resolution/response to treatment 
Resolution of diarrhea as <3 stools/24 hours for at least two consecutive days after the end 
of drug treatment. 
Mortality associated with CDI 
Cases in which there is clinical suspicion and, at the discretion of the treating physician, the 
death can be attributed to CDI and cannot be clearly attributed to other non-CDI causes, and 
it occurs within 10 days after the CDI episode and/or due to well-known complications of 
CDI (dehydration, renal failure, septic shock or megacolon, among others).  
Poor evolution 
The detection of poor progression 48-72 hours after starting treatment (MBK-01 or 
fidaxomicin) is given by a: 
1. Worsening of the diarrhea episode (at least one bowel movement more than at 
baseline*, with the start of study treatment (fidaxomicin or MBK-01) as baseline). 
And at least one of the following factors: 
1. C-reactive protein (CRP) elevation (>5% of baseline value*) 
2. Leukocyte absolute elevation (>5% of baseline value*) 
3. Progression to sepsis: hypotension or organ failure with no other apparent cause.  


 
 
 
 
 
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*NOTE: 
Baseline 
is 
considered 
the 
time 
of 
start 
of 
study 
treatment 
(fidaxomicin/investigational medication). 
Primary CD infection 
First episode of laboratory-confirmed CD infection.  
Recurrence 
Recurrence is the reappearance of clinical manifestations of a new episode of CDI that 
recurs within eight weeks after the onset of symptoms from a previous episode, provided 
that the symptoms of the previous episode had resolved after completion of treatment 
(Debast et al., 2014). 
 
04. STUDY POPULATION. PATIENT SELECTION 
The clinical trial will be conducted in patients who experience an episode of CD infection 
(either the first episode or subsequent recurrences). 
04.01. Inclusion Criteria 
1. Male and female patients, 18 years of age and older. 
2. Patients who experience an episode of CD infection (either a first episode or 
subsequent recurrences). 
3. Presence of an episode of diarrhea defined as ≥3 stools/24 hours, at the onset of the 
episode. 
4. Confirmation of the presence of CD toxin A and/or B in feces, by a direct toxin 
detection test or by PCR for detection of toxin-producing genes, at the beginning of 
the episode to be treated in the clinical trial (toxin test must be positive within seven 
days prior to patient’s inclusion in the trial). 
 
04.02. Exclusion Criteria 
1. Prior fecal microbiota transfer. 
2. Transplanted patients, except for those with solid organ transplant over two years, 
with good organ function.  
3. Absolute neutrophil count <500 cells/μL at the time of study inclusion. 
4. Pregnancy, breast-feeding, or intent to become pregnant during the course of the 
study.  
5. Active treatment with bile acid sequestrants (for example: cholestyramine). 
6. Human immunodeficiency virus (HIV)-positive subjects except those with CD4 T-
cell count >200 cells/μL and viral load less than 20 copies.  
7. Dysfunction when swallowing or deficit of oral-motor coordination. 
8. Patient admitted to ICU, or expected to be admitted to ICU, for severe illness. 
9. History of significant medical conditions that, in the opinion of the investigator, 
would not allow for adequate assessment or follow-up of the patient.  


 
 
 
 
 
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04.03. Expected number of patients 
Some 104 patients (52 in the control group treated with fidaxomicin and 52 patients in the 
experimental group treated with MBK-01). 
 
04.04. Withdrawal criteria and planned analysis of withdrawals 
Patients are free to withdraw from the study at any time for any reason, without any 
consequence. 
The primary reason for withdrawal from the study will be recorded on the end of study 
form. Patients who decide to withdraw will be encouraged to complete an early termination 
telephone visit to assess adverse events. Patients who withdraw will be discontinued from 
all study procedures. 
In addition, the investigator may withdraw the subject from the study for any reason, 
including the following: 
1. Lost to follow-up. 
2. Adverse event (AE) or serious adverse event (SAE). 
3. When the patient does not cooperate or comply with the requirements of the study, 
this will be considered protocol violation/non-compliance. 
4. Subject’s decision to withdraw consent. 
5. Clinical conditions of the patient that prevent continuity.  
6. Investigator decision to terminate the study. 
7. Recurrence by CD as defined in Section 03.07.02. 
 
04.05. Withdrawal criteria for lack of efficacy 
A patient’s lack of efficacy is defined as the occurrence of a new recurrence of CD infection. 
Patients who experience a new recurrence will be withdrawn from the study and treated 
with rescue medication, which will be antibiotic treatment (vancomycin or fidaxomicin).  
 
04.05.01. Discontinuation from treatment 
Patients who are considered lost before receiving the experimental treatment or antibiotic 
do not require follow-up within the clinical trial.  
Patients who are considered lost after receiving the experimental treatment or antibiotic 
will be followed until completion of the study visits (six months), or until ≥30 days after the 
start of treatment, if they discontinue their follow-up in the study. In the latter case, 
information relevant to the assessment of safety (adverse events and concomitant 
treatments) will be collected at a minimum.  
 


 
 
 
 
 
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04.06. Duration of participation 
Patients will be followed for six months from the start of study treatment.  
 
05. DESCRIPTION OF STUDY TREATMENT 
05.01. Definition of treatment 
The investigational product (MBK-01) is a capsule-containing medicinal product with a 
heterologous, healthy donor-derived, oral fecal microbiota lyophilisate. 
 
05.02. Investigational Product 
05.02.01. Description of the manufacturing technique(s) of the 
proposed therapy(ies) 
The starting samples used to perform FMT are feces from healthy donors previously tested 
by clinicians, and validated after passing microbiological screening in blood and feces, 
including tests for COVID-19, 
monkey smallpox virus, and enteropathogenic 
microorganisms and toxins (Youngster et al., 2014). The selection and exclusion criteria for 
donors are described in Table 1 and Table 2.  
Table 1. Donor Inclusion Criteria. 
Inclusion Criteria 
Age 18-50 years (50-60 if screening for negative colorectal cancer) 
Body mass index (BMI) 18-30 kg/m2 
Ability to donate 2 to 5 times a week 
Good overall health 
 
Table 2. Donor exclusion criteria. 
Exclusion Criteria 
Infectious 
History of infection or exposure to HIV, hepatitis B, or hepatitis C, syphilis, primate T-
lymphotropic viruses (PTLVs), malaria, or tuberculosis 
At-risk sexual contacts or behaviors or partner change in the past three months 
Sexual contact, in the past 12 months, with someone who has used needles for drug or 
steroid use or for anything else that has not been prescribed by a doctor 
Sexual contact, within the past 12 months, with someone with HIV, Hepatitis B, or 
Hepatitis C, with hemophilia, or receiving clotting factor concentrates. Contacting 
someone with hepatitis 


 
 
 
 
 
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Exclusion Criteria 
Infection and/or treatment of any sexually transmitted disease within the past 12 months 
Illicit drug use within the past three months 
Previous organ or tissue transplantation 
Have been in contact, within the past six months, with blood from another person, or have 
been punctured/cut with material that could be contaminated with blood or fluids from 
another person 
Tattoos, piercings, acupuncture, or accidental needle stick within the past 12 months 
Risk factors for Creutzfelt-Jakob disease 
Recent gastrointestinal infection (Rotavirus, Giardia lamblia, etc.) 
Travel in the past 12 months to tropical countries with endemic diarrheal illnesses or 
high-risk of traveler’s diarrhea 
Vaccination within the last 12 months with live attenuated vaccines or any injection  
Healthcare personnel or workers from hospitals and healthcare institutions (risk of 
transmission of multi-resistant microorganisms) or if working with animals (risk of 
transmission of zoonoses) 
Those who have been in a prison or reformatory facility within the past 12 months or 
have been under arrest for more than 72 hours 
Those who have experienced malaria, Chagas disease, or babesiosis or trypanosomasis 
Those who have suffered, or have anyone in their environment who has suffered, COVID-
19 
If you have been exposed to or are infected with monkey smallpox virus 
Fever, frequent cough, or feeling short of breath in the past two weeks 
Working with animals (to avoid the risk of transmission of zoonoses) 
Gastrointestinal comorbidities 
Inflammatory bowel disease 
Irritable bowel syndrome, chronic functional constipation, or chronic functional diarrhea 
(per ROME IV) 
Celiac disease and other chronic digestive disorders 
History or high risk for gastrointestinal cancer or polyposis 
A family member diagnosed with Crohn’s disease or ulcerative colitis (parents, siblings 
or children) 
Family history of colon cancer (parents, siblings or children) 
Diarrhea, blood in stool, abdominal pain, or other significant digestive symptomatology 
within the past three months, or routinely 
Consumption of drugs that may alter gut microbiota 
Use of antibiotics, antifungals, or antivirals within the past three months 
Use of Proton-pump inhibitors (PPIs) in the past three months 


 
 
 
 
 
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Exclusion Criteria 
Use of systemic anti-cancer agents within the past three months 
Taking medications related to gastric reflux, heartburn, within the past three months 
Use of immunosuppressive medication or chemotherapy within the past three months  
Other reasons 
Legal inability to give consent 
Active cancer process or cancer history in the past 10 years 
Consumer of >28 Alcohol Standard Drink Unit (SDU) per week 
Have had surgery for something in the past three months 
Gastroscopy or colonoscopy within the past three months 
Prior major digestive system surgery (excluding appendectomy) (parents, siblings or 
children) 
Have had a transplant within the last 12 months 
Evolved liver disease 
Chronic kidney disease 
Autoimmune and systemic diseases 
Chronic or autoimmune systemic diseases with GI involvement 
Diabetes mellitus 
Metabolic syndrome 
Neurological, neurodegenerative or psychiatric diseases 
Symptoms of eczema or psoriasis in the past eight weeks 
Food, seasonal, animal, medication, latex, dust, other allergies 
Asthma in the past 12 months 
Experienced symptoms or taken treatment in the past 12 months for attention deficit, 
hyperactivity, anxiety, and/or depression 
Pregnancy or delivery or termination of pregnancy in the last six months 
Vaccinations not correctly received per the vaccine schedule 
Significant vascular disease 
 
In addition, due to the incubation window of potential infection-causing agents, all samples 
received will pass a quarantine period and, upon donor re-screening, will be used in the 
production of gut microbiota capsules or microbiota suspension.  
Once validated by screening testing, the donor will visit our facilities to perform the stool in 
situ. The donor performs the stool in a specific stool collection container that can be 


 
 
 
 
 
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attached to the toilet. Once the sample is obtained, it will be processed in less than six hours 
after evacuation, during which time it will be stored refrigerated.  
05.02.02. Pharmacokinetics 
Depending on the extent of microbiota alteration, and subsequent bacterial exposure, the 
microbiota composition could be restored to a similar state or assume a new steady state 
consisting of different species of bacteria (Costello et al., 2012). This disruption in the 
bacterial community therefore gives rise to an opportunity for externally supplied bacteria 
to be established due to reduced competition or elimination of resident species occupying 
the same niche that need the same nutrients (Lee et al., 2013; Maldonado-Gómez et al., 
2016). 
The transplanted microbiota restores the receptor’s gut microbiota by reintroduction of 
bacterial toxins that were absent from the receptor prior to FMT, supporting expansion of 
the receptor’s own commensal microbiota and restoring, over time, an optimal microbiota 
community (Browne et al., 2017). 
To date, there is no literature describing or showing data suggesting that the transferred 
microbiota leaves the intestinal environment and colonizes other areas. 
05.02.02.A. Bioavailability of the investigational medication 
There are no studies evaluating the bioavailability of the investigational medication or other 
FMT. Most bacteria that constitute the gut microbiota reside in the large intestine, while a 
minority of bacteria are found in the small intestine and stomach (Guarner, 2011; Lawley et 
al., 2013; Clarke et al. 2019). The stomach and duodenum harbor a small number of 
microorganisms that adhere to the mucosal surface or are in transit, usually less than 103 
bacteria per gram of content. Acidic, biliary, and pancreatic secretions destroy most of the 
ingested microorganisms, and propulsive motor activity prevents stable colonization of the 
lumen of the small intestine. The number of bacteria increases progressively throughout the 
jejunum and ileum, from about 104 in the jejunum to 107 colony-forming units per gram of 
content at the ileal end. The colon is densely populated with anaerobes, and bacterial counts 
reach densities of about 1011 colony-forming units per gram of content (Guarner et al., 
2011). The hypromellose capsules of the investigational medication MBK-01 do not dissolve 
in the stomach but in the small intestine and, incrementally, into the large intestine, the area 
of the gastrointestinal tract richest in microbiota. For that reason, the microbiota (which 
constitutes the active ingredient in MBK-01) will reach this area of the body, it is estimated 
that each bacterial type could access its specific niches, to reconstitute the intestinal flora. 
05.02.03. Adverse effects 
FMT has been shown to have a high safety profile for the different administrations in all 
studies with patients with primary CD infection and with r-CDI in which it has been 
evaluated, reporting a low number of effects, mostly moderate and self-limiting and 
occurring shortly after administration (Youngster et al 2014a, Youngster et al. 2016, Staley 
et al. 2017, Camacho-Ortiz et al. 2017, Ng SCC et al. 2017, Juul et al. 2018, Cheng et al. 2019, 
Madoff et al. 2020). In addition, the type or severity of adverse effects was shown not to 


 
 
 
 
 
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differ depending on the route of administration, although patient perception is more 
positive when capsules are administered by oral administration (Kao et al., 2017, Hirsch et 
al., 2015, Staley et al., 2017, Jiang et al., 2018, Cheng et al., 2019). Even in two of these studies 
using lyophilized microbiota capsules, no adverse effects were reported (Reigadas et al., 
2019; Staley et al., 2019). The short-term safety of treatment has been demonstrated in all 
studies. Long-term safety was assessed in one of the studies, which follows patients treated 
with FMT for one year. This study reports that patients had no gastrointestinal adverse 
effects (Staley et al., 2019).  
05.02.04. Presentation and instructions for proper administration 
The investigational drug substance MBK-01 is contained in white hypromellose capsules. 
These capsules will be contained, in fours, in 35 mL white high-density polyethylene (HDPE) 
bottles with a tamper evident stopper and a built-in desiccant. The bottle will be properly 
labeled. 
The four capsules contained in each bottle correspond to the dosage established for each 
patient. 
05.02.05. Storage and receipt of study product 
Each site will be supplied with a sufficient amount of MBK-01 for the treatment of patients. 
Where applicable, the drug product will be shipped to the appropriately refrigerated 
administration site at 5 ±3°C. All finished product shipments are performed through a Good 
Distribution Practices (GDPs) certified company.  
Upon receipt of the investigational medication, it should be stored refrigerated at a 
temperature of 5 ±3°C until administration to the patient. 
05.02.06. Administration and Dosing Schedule 
The dosing regimen is four capsules in a single dose (≥2.1-2.5 x1011 microorganisms). The 
decision on timing of intake should be left to the discretion of the PI and will be swallowed 
orally, in a single intake. The investigational medication MBK-01 should be taken in a fasting 
state. In case the patient has difficulty swallowing, a gel or gel water may help to facilitate 
the swallowing process.  
05.02.07. Supportive measures during treatment 
Measures will be taken to ensure the safety of patients participating in the trial at all times.  
Use of more timely supportive measures (e.g., hydration-monitoring of patient’s 
electrolytes and antipyretics in case of fever (>38ºC)) will be permitted and as deemed by 
the treating medical team. 
05.02.08. Dose modification 
A second dose of FMT is not planned to be provided during the course of this clinical trial, 
and re-administration of the same dose will not be evaluated for a patient who has already 
received it. 


 
 
 
 
 
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05.03. Fidaxomicin 
Fidaxomicin is an antibiotic that belongs to the group of macrocyclic antibacterials, is 
bactericidal, and inhibits RNA synthesis by bacterial RNA polymerase. Interferes with RNA 
polymerase at a site other than rifampicin. Inhibition of CD RNA polymerase occurs at a 
concentration twenty-fold lower than that of the E. coli enzyme (1 μM vs. 20 μM), partially 
explaining the significant specificity of fidaxomicin activity. Fidaxomicin has been shown to 
inhibit CD sporulation in vitro. 
It is indicated for the treatment of CDI in adults and in pediatric patients weighing at least 
12.5 kg. 
The dose administered is 200 mg (one tablet), given twice daily (every 12 hours) for 10 
days.  
The most common adverse reactions are vomiting (1.2%), nausea (2.7%), and constipation 
(1.2%). 
The supply and shipment of fidaxomicin will be performed by an external company with 
Good Distribution Practices (GDPs) certificate. Fidaxomicin 200 mg tablets does not require 
any special storage conditions. 
 
05.04. Permitted and Prohibited Concomitant Treatments 
Subjects should not take any systemic antibiotics for the duration of the study.  
Antiemetics and antidiarrheals are not recommended as supportive measures. There are no 
specific dietary restrictions, except those listed in medical order. 
Apart from the listed types of medication, there is no drug contraindicated or not to be used 
concurrently with the study drug. 
 
06. STUDY ASSESSMENTS 
06.01. Medical History 
The patient’s medical history will be collected and will be part of the study documentation. 
 
06.02. Physical examination 
A comprehensive physical examination will be performed including major systems 
(cardiovascular, abdominal, neurological [sensory and motor], musculoskeletal, and limb 
systems). This examination will be performed at the baseline visit (before and after 
treatment administration) and at the in-person follow-up visits. The physical examination 


 
 
 
 
 
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that will be performed after treatment will be: measurement of your temperature and blood 
pressure.  
 
06.03. Laboratory tests 
 
06.03.01. Stool samples 
Stool samples will be collected at the Start-of-Treatment visit, and at Visit 4 (eight weeks 
after end of treatment). The Baseline and Visit 4 stool sample will be used for subsequent 
studies and will therefore be stored in a freezer (-20ºC - -80ºC). 
In case of an episode of diarrhea throughout the study, a new stool sample may be taken to 
confirm the presence of CD and rule out other pathologies. 
Tests to be performed may include: 
• 
Confirmation of CD with direct detection of GDH and/or A/B toxins. 
• 
Confirmation of CD with molecular techniques (PCR). 
 
06.03.02. Other laboratory tests 
• 
Blood count.  
• 
Thyroid profile. 
• 
Lipid profile: High-Density Lipoprotein (HDL), Low-Density Lipoprotein (LDL), total 
cholesterol, and triglycerides. 
• 
Liver profile tests: Alanine transaminase (ALT), Aspartate aminotransferase (AST), 
Gamma-glutamyltransferase 
(GGTP), 
Lactate 
dehydrogenase 
(LDH), 
alkaline 
phosphatase, bilirubin, total protein and albumin. 
• 
Renal panel tests: urea, creatinine, sodium, potassium, chloride, uric acid, total protein, 
serum albumin, blood glomerular filtrate, urine quantitative protein, urine creatinine, 
basic urine examination, and urine sediments.  
• 
C-reactive protein (CRP). 
 
06.04. Follow-up Assessments: Study Visits 
Visit 1. Baseline Visit (Randomization and Treatment) 
When a candidate patient presents with a clinical presentation consistent with an episode 
of CD infection (either first episode or subsequent recurrences), he/she will be routinely 
evaluated for CD in stool to confirm suspicion, as well as laboratory tests and a physical 
examination. In parallel, a member of the research team will review the patient’s medical 


 
 
 
 
 
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history and, if all preliminary eligibility and none of the exclusion criteria are met, the 
investigator will ask the patient and/or his/her legal representative to participate in the 
study, explaining the nature of the study, its rationale, risks and benefits, and answering any 
questions the patient asks about the study. Patients will be provided with an information 
sheet and a signed consent form. If the legal representative and/or the patient agree to 
participate in the study and sign the consent form, the patient may participate in the study. 
Once the preliminary eligibility of the patient has been determined and the above 
documents signed, study procedures will be performed.  
After the patient has given written consent to participate, he/she will undergo a 24-hour 
washout period (in case he/she has been treated in the current episode with vancomycin 3-
5 days and/or antibiotic for another indication) and will then be randomized into one of the 
two treatment groups. The subject will be assigned a four-digit screening number (first two 
digits for the site and the other two digits for the subject) in the order of their screening 
within the clinical trial. The number will be assigned by a system integrated into the eCRF. 
The site number and subject screening number will be included on all study forms and 
documentation. The subject withdrawn from the clinical trial retains his/her number. New 
subjects should always be assigned a new screening and randomization number.  
It must be confirmed that the patient meets all inclusion and none of the exclusion criteria. 
Only patients who meet all of the inclusion criteria and none of the exclusion criteria may 
be randomly assigned and receive the clinical trial treatment. 
The following procedures will be performed during this visit: 
- 
Obtain informed consent (ICF). 
- 
Evaluation of inclusion and exclusion criteria. 
- 
Pregnancy test (for women of childbearing potential). 
- 
In the event that the patient has been treated with antibiotics for the current episode 
of CD infection (either the first episode or subsequent recurrences) and/or for 
another medical indication, it should be confirmed that a 24-hour washout period is 
performed prior to randomization.  
- 
Physical examination: weight, height, BMI, blood pressure. 
- 
Assessment of patient’s quality of life using the SF-36 Test. 
- 
Assessment of concomitant medication. 
- 
Confirmation of the presence of CD in feces by direct detection test of CD toxins or 
by PCR. Tests performed within seven days prior to patient enrollment in the clinical 
trial will be valid.  
- 
Analytical Study: Blood count, lipid profile, thyroid profile, liver and kidney function. 
Tests performed within 24 hours prior to patient enrollment in the clinical trial will 
be valid.  
- 
Stool sample collection for subsequent studies. 
- 
Stool sample collection to confirm CDI (tests performed within seven days prior to 
patient enrollment in the clinical trial are valid). 
At this visit the patient will be started on treatment.  


 
 
 
 
 
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The duration of treatment will be different depending on the assignment:  
o Patients in the experimental arm will receive MBK-01: a single dose of 
four capsules in a single intake. 
o Patients in the control group will receive fidaxomicin: a dose of 200 mg 
(one tablet), administered twice a day (every 12 hours) for 10 days. 
The duration of the baseline visit has a maximum of 24 hours (from day -1 to day 0), in the 
event that patients need to undergo a 24-hour washout period prior to randomization and 
treatment. If patients do not have to undergo a washout period, the duration of this visit 
may be shorter.  
 
Visit 2. Clinical course assessment (f2f): 72 hours ±1 day after treatment initiation. 
The following procedures will be performed during this visit: 
- 
Physical examination: weight, height, BMI, blood pressure 
- 
Evaluation of the evolution of the patient’s clinical symptomatology 
- 
Assessment of concomitant medication 
- 
Assessment of potential adverse events 
- 
Patient’s assessment of good/bad progress  
- 
Laboratory tests for good/bad progress: CRP, and absolute leukocyte value 
- 
Assessing Patient Adherence 
 
Visit 3. Post-Treatment Assessment (F2F): three weeks ±4 days after treatment 
initiation. 
The following procedures will be performed during this visit: 
- 
Physical examination: weight, height, BMI, blood pressure 
- 
Evaluation of the evolution of the patient’s clinical symptomatology 
- 
Assessment of concomitant medication 
- 
Assessment of potential adverse events 
- 
Assessing Patient Adherence 
 
Visit 4. Post-Treatment Assessment (F2F): eight weeks ±4 days after treatment 
initiation.  
The following procedures will be performed during this visit: 
- 
Physical examination: weight, height, BMI, blood pressure 
- 
Evaluation of the evolution of the patient’s clinical symptomatology 
- 
Assessment of concomitant medication 
- 
Evaluation of potential adverse events 
- 
Quality of life assessment using SF-36 Test 


 
 
 
 
 
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- 
Analytical study: Blood count, lipid profile, thyroid profile, liver and kidney function.  
- 
Stool sample collection for subsequent studies. 
 
Visit 5. Early follow-up (telephone call): three months ±6 days after treatment start.  
The following procedures will be performed during this visit: 
- 
Evaluation of potential adverse events 
- Evaluation of the evolution of the patient’s clinical symptomatology 
- 
Assessment of concomitant medication 
 
Visit 6 (end of study). Late follow-up (phone call): six months ±6 days after treatment 
initiation 
The following procedures will be performed during this visit: 
-  
Evaluation of the evolution of the patient’s clinical symptomatology 
-  
Assessment of potential adverse events 
-  
Assessment of concomitant medication 
- 
Quality of life assessment using the SF-36 Test 
 
All patients who discontinue from the study and/or are discontinued prior to completing 
the planned follow-up should have an end of study visit ≥30 days after the start of treatment, 
where, at the very least, information relevant to the safety assessment (adverse events and 
concomitant treatments) will be collected. 
 
Unscheduled Visits  
In the event of an episode of diarrhea during the follow-up period (especially with suspected 
recurrence due to CD) and/or other clinical situation of significance at the discretion of the 
PI and the research team, unscheduled visits may be performed. All tests performed during 
these visits as well as their results should be detailed. 
 
07. MONITORING 
Monitoring shall mean the act of monitoring the progress of this clinical trial, ensuring that 
it is performed, recorded and reported in accordance with: 
• The protocol, 
• Standard Operating Procedures (SOPs) 
• Good Clinical Practice (GCP) 
• And applicable regulatory requirements. 
In this study, monitoring tasks will be performed on behalf of the Sponsor by Sermes CRO. 


 
 
 
 
 
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08. ADVERSE EVENTS 
08.01. General Considerations 
All adverse events (AEs) will be recorded in the eCRFs. All adverse events will be collected 
from the first dose of experimental or antibiotic treatment until the patient’s last follow-up 
visit, or until 30 days after the start of treatment, if they discontinue follow-up in the study.  
 
08.02. Adverse Event (AE) 
Any untoward health occurrence in a patient or clinical investigation subject treated with a 
medicinal product, even if it does not necessarily have a causal relationship with this 
treatment, is considered an AE. 
 
08.03. Adverse Drug Reaction (ADR) 
An adverse drug reaction (ADR) is any untoward and unintended reaction to an 
investigational medicinal product, regardless of the dose administered. 
 
08.04. Serious Adverse Event (SAE) 
A Serious Adverse Event (SAE) is defined as any untoward experience involving a patient, 
whether or not considered related to protocol treatment. Serious adverse events are those 
that result in: 
• 
Death 
• 
An event that threatens the patient’s life 
• 
Hospitalization or prolongation of hospitalization 
• 
Persistent or significant disability/incapacity 
• 
A congenital anomaly or birth defect 
• 
Any other condition deemed clinically relevant 
 
08.05. Suspected Unexpected Serious Adverse Reaction (SUSAR) 
A Suspected Unexpected Serious Adverse Reaction (SUSAR) is a serious adverse reaction 
whose nature, severity, or outcome is not related to the reference safety information. 
It is the Investigator’s obligation to report serious and/or unexpected adverse reactions to 
the Sponsor within 24 hours of awareness.  


 
 
 
 
 
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08.06. Causality assessment 
The causality of the events will be discussed both in relation to treatment and pathology 
under the clinical judgment of the treating physicians. 
Causality is defined by the modified Karch-Lasagna algorithm (Karch et al., 1997):  
Probability of Causality: 
- 
Definitive – The adverse reaction (AR) appears in a reasonable temporal sequence 
after administration of the drug. The effect had already been mentioned previously 
or is an expected response to the study medication, with an improvement confirmed 
after the reduction or discontinuation of the medication and a reappearance of the 
effect after re-administration of the study medication, with no other apparent 
etiological alternative. 
- 
Probable – RA appears with a reasonable temporal sequence after drug 
administration. The effect had already been referred to previously or is an expected 
response for the study medication. Improvement occurs after treatment reduction 
or discontinuation, and any other etiology is unlikely or less likely. Information on 
rechallenge is not required. 
- 
Possible – RA appears in a reasonable temporal sequence after drug administration. 
The effect had been previously referred to or is an expected response for the study 
medication. There is a possible etiological alternative that may be responsible for 
RA. No information is necessary on the suspension of the medication or it may be 
unclear. 
- 
Unlikely/doubtful – RA does not appear in a temporal sequence after drug 
administration, or if it exists, it is remote. The effect is not an expected or known 
response to the study medication. There is a possible etiological alternative that may 
be responsible for RA. 
- 
Unrelated – RA is due to an underlying or concurrent disease or the effect of another 
medication and does not meet any of the above definitions. 
 
09. ETHICAL ASPECTS 
09.01. General Considerations. Approvals 
The study will be conducted in accordance with the requirements expressed in the 
Declaration of Helsinki (review of the 64th General Assembly, Fortaleza, Brazil, October 
2013) and following the recommendations of Good Clinical Practice (CPMP/ICH/135/95) 
and the current regulation establishing the requirements for the conduct of clinical trials 
with medicinal products, Royal Decree 1090/2015 of December 4 and European Regulation 
536/2014 of April 16. 


 
 
 
 
 
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The IRB/IEC will review this protocol and all study documentation to ensure that the rights, 
safety, and well-being of the patient are protected, and only those sites approved by the 
IRB/IEC may conduct the study. 
 
09.02. Informed Consent Form (ICF) 
Subjects who wish to participate in the study will be given a written document called the 
“Patient Information Sheet”, which will include information on the most important aspects 
of the study. The Informed Consent document will detail that the subject freely and 
voluntarily grants his/her informed consent before being included in the study and that 
he/she is informed about the nature, significance, implications and risk of the study. 
The investigator will be responsible for obtaining informed consent for this study, directly 
from the subject or otherwise from a legal representative. The subject participating in this 
study or his/her representative may revoke his/her consent at any time, without a 
pronouncement of cause, and this situation will not result in any liability or harm. 
 
09.03. Confidentiality 
The processing, communication and transfer of personal data of all participating subjects 
will be in accordance with the provisions of Organic Law 3/2018, of December 5, on the 
Protection of Personal Data and guarantee of digital rights.  
The data collected for the study will be identified by a code, so that no information that can 
identify the patient is included, and only the study doctor/collaborators will be able to relate 
this data to the patient and to his/her medical history.  
All documents generated during the study, as well as the contents of the case report forms, 
will be considered strictly confidential and not be disclosed to third parties. 
 
09.04. Data Integrity and Quality Assurance 
All data collected in the study, including data obtained from the eCRFs, as well as documents 
prepared during the study, will be preserved from uses not permitted by persons outside 
the research and, therefore, will be strictly estimated confidential and not be exposed to 
third parties.  
Clinical data management defined for this study follows applicable laws and regulations. 
 
10. STATISTICAL ANALYSIS 
10.01. General Statistical Considerations 


 
 
 
 
 
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A Statistical Analysis Plan (SAP) will be developed during the execution of the study and 
prior to database lock, which will describe in detail the statistical methods to be employed, 
the strategy to be followed for missing values, and the tables and figures to be included in 
the statistical report. 
As an overview of the statistical analysis, data will be presented as absolute frequencies and 
percentages for qualitative variables. For quantitative variables, central tendency statistics, 
such as mean and median, and scatter statistics, such as standard deviation and 
interquartile range, minimum and maximum values, may be provided as indicators of the 
shape of the distribution such as asymmetry and kurtosis indices, will be used. For ordinal 
variables, depending on the number of categories, one form or another of description will 
be used. In all cases, a column should be added to the data presentation table that includes 
the number of patients with available data. The analyses will be done divided by 
randomization group. 
A descriptive analysis of all relevant variables collected at baseline will be performed, and 
this analysis will be divided by randomization groups. 
 
10.02. Determination of sample size 
For the sample size calculation, the information presented throughout the protocol was 
taken into account. The sample size calculation was performed considering: 
The confidence level (97.06%) 
Statistical power (80%) 
The effect size (𝜂∗2=0.02 for quantitative outcomes; Odds ratio=2.5 for probability analysis) 
The study design: number of visits, study groups and primary variable. 
Potential losses (50%), including patients withdrawn (5%), death (5%), patients not 
evaluable (40%). 
The estimated total of patients is 104, and considering the sample size per group, 52 
patients should be recruited in each group. 
The sample size calculation was performed with the software G*Power (version 3.1.9.7). 
 
10.03. Randomization 
Randomization will occur as follows: 
1. Arm identification (randomization groups): in this case the arms will be: 
o Experimental group: single-dose treatment of four oral capsules of 
lyophilized fecal microbiota.  
o Control group: treatment with fidaxomicin, 200 mg/12 hours for 10 days. 
2. Randomization process: each patient will be individually and independently 
randomized with equal chances of assignment between the different arms of the 


 
 
 
 
 
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study. If the study has two arms, this type of randomization would be equivalent to 
the toss of a coin to decide on the face of the resulting coin. 
 
10.04. Study Populations 
The Intent-to-Treat (ITT) population consists of all patients who have been included in 
this study, meet or fail to meet the eligibility criteria and have been randomized. In addition, 
the following ITT subpopulations will be considered for the analysis of probability of 
recurrence and time to recurrence: 
• 
ITT Subpopulation 1: It will consist of all ITT patients who have data on whether or 
not recurrence has occurred through Visit 4 or later. 
• 
ITT Subpopulation 2: It will consist of all ITT patients who have been withdrawn 
shortly before Visit 4 and are aware of whether recurrence has occurred or not. 
The Per Protocol population (PP) includes all patients who have been recruited for the 
present study, meet all inclusion and none of the exclusion criteria, and have remained in 
the study without any major protocol deviations. 
The Safety Population (SP) consists of all patients who have been randomized to either of 
the two randomization groups and who have taken at least one dose of the corresponding 
treatment. 
 
10.05. Efficacy Analyses 
The final efficacy analysis will be performed on the ITT Population as well as the PP 
Population. 
Inferential analysis of efficacy variables will be performed as follows: 
- 
Probability of recurrence based on randomization group will be studied using 
binary logistic regression analysis. The odds recurrence ratio will be reported, using 
the MBK-01 group as a reference. In addition, the confidence interval, Nagelkerke’s 
R-squared and Yule’s Q-coefficient will be included, as well as the critical level. The 
number of diarrhea episodes by visit and group will be analyzed, if data are 
available, using mixed models of analysis of variance or relevant robust/non-
parametric models. 
- 
Time to recurrence (weeks) by group will be analyzed using Cox regression. An 
event is defined as a recurrence, censoring cases without recurrence.  
- 
Good/bad patient evolution will be analyzed using logistic regression procedures 
and incorporating the same indicators as described for the analysis of the 
probability of recurrence. 
- 
Overall survival in weeks will be analyzed by Cox regression including 
randomization group as an independent variable.  
 


 
 
 
 
 
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Due to the trial characteristics, the hypotheses may be hypothesized as either non-
inferiority or superiority. Since the trial is Phase III, both hypotheses can be tested in the 
same clinical trial as long as the α risk is controlled; or, alternatively, it is confirmed that 
there are significant differences when, in fact, at the population level, there are no such 
differences. It is important to remember that, in order to avoid false positives, it is required 
that the type I or α error be distributed across the different hypotheses, resulting in a lower 
value of the critical level or p to find statistically significant results (Ferreira-Gonzalez, 
2014). 
Although the overall significance level will be alpha=0.05, because more than one data 
analysis will be performed, it will be adjusted to 0.0294 to avoid inflation of the type I error 
probability (Geller and Pocock, 1987). If multiple pairwise comparison analyses are used, 
the statistical report will describe the procedure used. Outcome effect size measures will be 
incorporated. Detailed adjustments and procedures for each statistical analysis as well as 
treatment of missing values and sensitivity analysis will be described in detail in the SAP. 
 
10.06. Safety Analyses 
Safety variables are operated with the number of adverse events documented and classified 
by frequency, severity, seriousness and causality as assessed by the patient interview and 
physical examination.  
For the analysis of Adverse Events, a descriptive analysis (frequency and percentages) of 
them will be performed throughout the study, and a list of them will be presented in which 
they will be grouped according to gravity and severity. These events will be listed by patient 
and randomization group. Recording of adverse reactions will be done using Verbatim 
described in the eCRF.  
Adverse Events and concomitant medication will be coded using a standardization system. 
For Adverse Events, the most current version of the MedDRA system will be used, and the 
ATC code will be used for concomitant medication. 
The analysis of physical examination variables will be done by frequencies and percentages 
and divided by randomization group. 
The safety analysis, both interim and final, will be conducted on the Safety Population (SP).  
 
10.07. Variables and Subgroups 
The table presented below (Table 3) lists the different variables and subgroups to be 
considered for statistical analysis. 


 
 
 
 
 
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Table 3. Study variables and subgroups to be considered in the statistical analysis. 
 
Efficacy Variables 
Safety Variables 
Quality of life variables 
Subgroups* 
Primary variable: Probability of recurrence. 
Number of Adverse Events by Randomization Group 
SF-36 Dimension Score 
Primary episode of CDI. 
Number of patients within each Adverse Event by randomization 
group. 
Good/Poor patient progress.  
Type of Adverse Events by Randomization Group 
 
Recurrent episodes of CDI. 
Time to recurrence 
Number of Serious Adverse Events by Randomization Group 
Patients without prior vancomycin. 
 
Type of Serious Adverse Events by Randomization Group 
 
 
Overall survival 
Relationship to treatment 
 
Severity of the Adverse Event 
Relationship to CDI 
Mortality associated with CDI 
Adverse Events of Special Interest (AESI) – See Section 
03.07.01.B. 
 
 
Suspected Unexpected Serious Adverse Reaction (SUSARs) 
 
 
*These subgroups will be included for the analysis of the probability of recurrence, time to recurrence, and the analysis of the patient’s good/bad outcome. 


 
 
 
 
 
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10.08. Interim Statistical Analysis 
Two interim analyses are planned in this study.  
1. The first interim analysis will be performed when approximately 50% of patients 
have completed treatment. 
2. The second interim analysis will be performed when approximately 80% of 
patients have completed V4.  
All interim analyses will incorporate a descriptive analysis of patient baseline 
characteristics, an efficacy analysis, and a safety analysis as described above. 
Analysis of baseline and efficacy and safety variables will be performed by comparing the 
randomization groups.  
The interim statistical analysis will be performed on the ITT population for efficacy and on 
the PS in the safety analyses section. 
The results of the interim statistical analysis could lead to early discontinuation of the study 
if large differences between the two randomization groups are revealed after the study, or 
obvious futility is shown. In addition to saving time and resources, this could reduce the 
exposure of study participants to a treatment that is inferior or shows no difference from 
the comparator.  
It should be noted that, in the event that large and clear differences between the 
randomization groups in the interim statistical analysis or a clear futility are observed, early 
study discontinuation should be assessed. The study of the differences between the 
randomization groups should be accompanied by measures of effect size, confidence 
intervals, and not just statistical significance.  
 
 


 
 
 
 
 
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11. REFERENCES 
Abreu, Y. Abreu A. T., J. A. Velarde-Ruiz Velasco, M. R. Zavala-Solares, J. M. Remes-Troche, R. 
I. Carmona-Sanchez, J. M. Aldana-Ledesma, A. Camacho-Ortiz, R. Contreras-Omana, R. Diaz-
Seoane, C. T. Elizondo-Vazquez, E. Garza-Gonzalez, G. Grajales-Figueroa, O. Gomez-
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Risk Factors for Unfavorable Clinical Outcomes: Results of a Hospital-Based Study in 
Barcelona, Spain.” J Clin Microbiol 51, no. 5 (May 2013): 1465-73.  
 
Seekatz, A. M., J. Aas, C. E. Gessert, T. A. Rubin, D. M. Saman, J. S. Bakken, and V. B. Young. 
“Recovery of the Gut Microbiome Following Fecal Microbiota Transplantation.” mBio 5, no. 
3 (Jun 17 2014): e00893-14.  
 
Seekatz, A. M., C. M. Theriot, C. T. Molloy, K. L. Wozniak, I. L. Bergin, and V. B. Young. “Fecal 
Microbiota Transplantation Eliminates Clostridium Difficile in a Murine Model of Relapsing 
Disease.” Infect Immun 83, no. 10 (Oct 2015): 3838-46.  
 
Song, Y., S. Garg, M. Girotra, C. Maddox, E. C. von Rosenvinge, A. Dutta, S. Dutta, and W. F. 
Fricke. “Microbiota Dynamics in Patients Treated with Fecal Microbiota Transplantation for 
Recurrent Clostridium Difficile Infection.” PLoS One 8, no. 11 (2013): e81330.  
 
Staley, C., M. J. Hamilton, B. P. Vaughn, C. T. Graiziger, K. M. Newman, A. J. Kabage, M. J. 
Sadowsky, and A. Khoruts. “Successful Resolution of Recurrent Clostridium Difficile 
Infection Using Freeze-Dried, Encapsulated Fecal Microbiota; Pragmatic Cohort Study.” Am 
J Gastroenterol 112, no. 6 (Jun 2017): 940-47.  
 
Staley, C., T. Kaiser, B. P. Vaughn, C. Graiziger, M. J. Hamilton, A. J. Kabage, A. Khoruts, and M. 
J. Sadowsky. “Durable Long-Term Bacterial Engraftment Following Encapsulated Fecal 
Microbiota Transplantation to Treat Clostridium Difficile Infection.” mBio 10, no. 4 (Jul 23 
2019).  
 
Staley, C., T. Kaiser, B. P. Vaughn, C. T. Graiziger, M. J. Hamilton, T. U. Rehman, K. Song, A. 
Khoruts, and M. J. Sadowsky. “Predicting Recurrence of Clostridium Difficile Infection 
Following Encapsulated Fecal Microbiota Transplantation.” Microbiome 6, no. 1 (Sep 18 
2018): 166.  
 
Tan, X. and S. Johnson. “Fecal Microbiota Transplantation (Fmt) for C. Difficile Infection, Just 
Say ‘No’.” Anaerobe 60 (Dec 2019): 102092.  
 
Tenover, F. C., E. J. Baron, L. R. Peterson, and D. H. Persing. “Laboratory Diagnosis of 
Clostridium Difficile Infection Can Molecular Amplification Methods Move Us out of 
Uncertainty?” J Mol Diagn 13, no. 6 (Nov 2011): 573-82.  


 
 
 
 
 
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van Nood, E., A. Vrieze, M. Nieuwdorp, S. Fuentes, E. G. Zoetendal, W. M. de Vos, C. E. Visser, 
E. J. Kuijper, J. F. Bartelsman, J. G. Tijssen, P. Speelman, M. G. Dijkgraaf, and J. J. Keller. 
“Duodenal Infusion of Donor Feces for Recurrent Clostridium Difficile.” N Engl J Med 368, 
no. 5 (Jan 31 2013): 407-15.  
 
Vilagut, G., M. Ferrer, L. Rajmil, P. Rebollo, G. Permanyer-Miralda, J. M. Quintana, R. Santed, 
J. M. Valderas, A. Ribera, A. Domingo-Salvany, and J. Alonso. “[the Spanish Version of the 
Short Form 36 Health Survey: A Decade of Experience and New Developments].” Gac Sanit 
19, no. 2 (Mar-Apr 2005): 135-50.  
 
Vilagut, G., J. M. Valderas, M. Ferrer, O. Garin, E. Lopez-Garcia, and J. Alonso. “[Interpretation 
of Sf-36 and Sf-12 Questionnaires in Spain: Physical and Mental Components].” Med Clin 
(Barc) 130, no. 19 (May 24 2008): 726-35.  
 
Voth, D. E. and J. D. Ballard. “Clostridium Difficile Toxins: Mechanism of Action and Role in 
Disease.” Clin Microbiol Rev 18, no. 2 (Apr 2005): 247-63.  
 
Wang, S., M. Xu, W. Wang, X. Cao, M. Piao, S. Khan, F. Yan, H. Cao, and B. Wang. “Systematic 
Review: Adverse Events of Fecal Microbiota Transplantation.” PLoS One 11, no. 8 (2016): 
e0161174.  
Ware, J.E., K.K. Snow, M. Kosinski, and B. Gandek. “SF-36 Health Survey Manual & 
Interpretation Guide” MA: The Health Institute, New England Medical Center, 1993 (pp.6:1-
6:22). 
 
Wilcox, M. H., D. N. Gerding, I. R. Poxton, C. Kelly, R. Nathan, T. Birch, O. A. Cornely, G. Rahav, 
E. Bouza, C. Lee, G. Jenkin, W. Jensen, Y. S. Kim, J. Yoshida, L. Gabryelski, A. Pedley, K. Eves, R. 
Tipping, D. Guris, N. Kartsonis, M. B. Dorr, I. Modify, and Modify Ii Investigators. 
“Bezlotoxumab for Prevention of Recurrent Clostridium Difficile Infection.” N Engl J Med 
376, no. 4 (Jan 26 2017): 305-17.  
 
Youngster, I., J. Mahabamunuge, H. K. Systrom, J. Sauk, H. Khalili, J. Levin, J. L. Kaplan, and E. 
L. Hohmann. “Oral, Frozen Fecal Microbiota Transplant (Fmt) Capsules for Recurrent 
Clostridium Difficile Infection.” BMC Med 14, no. 1 (Sep 9 2016): 134.  
 
Youngster, I., G. H. Russell, C. Pindar, T. Ziv-Baran, J. Sauk, and E. L. Hohmann. “Oral, 
Capsulized, Frozen Fecal Microbiota Transplantation for Relapsing Clostridium Difficile 
Infection.” JAMA 312, no. 17 (Nov 5 2014): 1772-8.  
 
Youngster, I., J. Sauk, C. Pindar, R. G. Wilson, J. L. Kaplan, M. B. Smith, E. J. Alm, D. Gevers, G. 
H. Russell, and E. L. Hohmann. “Fecal Microbiota Transplant for Relapsing Clostridium 
Difficile Infection Using a Frozen Inoculum from Unrelated Donors: A Randomized, Open-
Label, Controlled Pilot Study.” Clin Infect Dis 58, no. 11 (Jun 2014): 1515-22.  
 
 


 
 
 
 
 
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ANNEX 1: SUMMARY OF ICD-01 PROTOCOL AMENDMENTS 
Summary and justification of Substantial Modification No.01  
(07/30/2021) 
➢ Substantial modification of the clinical trial protocol: 
Changes have been performed to the Exclusion Criteria of the Clinical Trial (see the 
section “Study population. Patient selection. 2. Exclusion criteria” of the Protocol) that have 
a direct impact on the recruitment capacity of the study. The modification of the exclusion 
criteria was motivated by the following causes:  
1. The modification or the elimination of some of the initially proposed exclusion 
criteria significantly promotes the recruitment capacity of the centers that are 
involved in the study.  
2. The proposed modifications in the exclusion criteria do not affect the fulfillment of 
the objectives (primary and secondary) of the study. The increase in the recruitment 
capacity of the study promotes the fulfillment of the study objectives within the 
established period.  
3. The sample of treated patients in the clinical trial is more heterogeneous, and 
therefore more representative of the population that is intended to be treated with 
MBK-01. 
 
In conclusion, the changes contained in this substantial modification would help to achieve 
the objectives of the clinical trial within the established period. 
 
Summary and justification of Substantial Modification No.02 
(11/18/2021) 
Substantial Modification No. 02 of November 18, 2021, consists of the addition of the 
following participating centers: 
- 
Hospital General Universitario de Alicante – Dra. Esperanza Merino de Lucas 
- 
Hospital Universitario La Paz – Dra. Alicia Rico Nieto 
- 
Hospital General Universitario Gregorio Marañón – Dra. Patricia Muñoz García 
- 
Hospital Universitari de Girona Dr. Josep Trueta – Dr. Antoni Castro Guardiola 
The number of centers has been increased to expand the recruitment options so that the 
number of patients under study can be reached in the estimated time.  


 
 
 
 
 
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Summary and justification of Substantial Modification No.03 
(12/20/2021) 
➢ Substantial modification of the clinical trial protocol: 
Modifications have been performed to the Inclusion and Exclusion Criteria of the clinical 
trial (see the section “Study population. Patient selection”). These changes have a direct 
impact on some aspects of the study design: by allowing the use prior antibiotic treatment 
for the patients included in the study, it is necessary to consider a washout period prior to 
the treatment.  
The modification of the inclusion and exclusion criteria was motivated by the following 
causes: 
1. Due to the low recruitment rate, and because the modification of the inclusion and 
exclusion criteria directly impacts the recruitment capacity, it is intended to 
significantly promote the recruitment capacity of the involved centers. 
2. The proposed modifications do not affect the fulfillment of the objectives of the 
study. The higher recruiting capacity will favor the fulfillment of objectives within 
the established period. 
3. The sample to be treated in the study is more representative of the population that 
is intended to be treated with MBK-01. 
In addition, the timing of the interim analysis of the study has been modified, so that it will 
be performed when half of the patients have completed treatment. Thus, it is intended to 
know beforehand the partial efficacy results of the study.  
In conclusion, for everything stated above, the changes contained in this substantial 
modification would help to achieve the objectives of the clinical trial within the established 
period. 
➢ Patient Information Sheet – Informed consent form 
The changes produced in the new V4.0 protocol version results in the update of the Patient 
Information Sheet-Informed Consent form, then generating the new 3.0 version of 
December 17, 2021. 
➢ Study schedule 
Due to the low recruitment rate achieved so far, and as stated in the new version of the 
protocol, the inclusion period has been changed from August-December 2021 to August 
2021 – June 2022. 
 


 
 
 
 
 
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Summary and justification of Substantial Modification No.04 
(04/18/2022) 
The substantial modification No.04 of April 18, 2022 consists of the addition of the 
following participating centers: 
- 
Hospital Universitario Quirónsalud Madrid – Dr. Daniel Carnevali 
- 
Hospital Quirónsalud Barcelona – Dr. Fernando Cereto Castro 
The number of centers has been increased to expand the recruitment options so that the 
number of patients under study can be reached in the estimated time.  
 
Summary and justification of Substantial Modification No.05 
(08/05/2022) 
➢ Substantial modification of the clinical trial protocol: 
The clinical trial population has been expanded, and the study inclusion criteria have been 
modified (see the section “Study population. Patient selection”). This modification is 
motivated by the following causes: 
1. Due to the low recruitment rate, and because the modification of the inclusion 
criteria directly impacts the recruitment capacity, it is intended to significantly 
promote the recruitment capacity of the centers involved.  
2. The proposed modifications do not affect the fulfillment of the objectives of the 
study. The increase in the recruiting capacity will promote the fulfillment of 
objectives within the established period. 
3. The changes allow an additional group of patients with Clostridioides difficile (CD) 
infection to have access to a potentially effective treatment, while still using the same 
patient population as before, without compromising the data that has already been 
collected in the study. 
4. The extension of the period of detection of the Clostridioides difficile toxin within 7 
days prior to the inclusion of the patient in the trial has been carried out to take into 
consideration the duration of treatment with vancomycin and the washout period 
that some participants may require.  
The statistical analysis has been adapted to include the evaluation of an additional 
secondary safety variable, which will provide more information on the safety of the 
investigational medicinal product.  
The sample size has been adapted, without this change affecting the fulfillment of the study 
objectives. 


 
 
 
 
 
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Some aspects of the study design have been modified, allowing the inclusion in the trial of 
patients who were previously treated with fidaxomicin, if the duration of treatment is not 
therapeutic (see the “Study plan” section).  
In addition, it has been included that the interim analysis of the study could lead to the early 
interruption of the study with the aim of maintaining the safety of the participants. 
In conclusion, for everything stated above, the changes contained in this substantial 
modification would help to achieve the objectives of the clinical trial within the established 
period. 
➢ Patient Information Sheet – Informed consent form 
The changes produced in the new V5.0 protocol version result in the update of the Patient 
Information Sheet-Informed Consent form, generating the new 5.0 version on July 29, 2022. 
➢ Investigator´s brochure (IB) 
The changes produced in the new V5.0 protocol version result in the update of the 
Investigator´s brochure, generating the new 2.0 version on July 29, 2022. 
➢ Study schedule 
Due to the low recruitment rate achieved so far, and as stated in the new version of the 
protocol, the inclusion period has varied from August 2021 - June 2022 to August 2021 - 
December 2022, so that the total duration of the study will last approximately 22 months.  
 
Summary and justification of Substantial Modification No.06 
(06/14/2023) 
➢ Substantial modification of the clinical trial protocol: 
The procedures adopted in October 2022 have been included in the protocol, following the 
recommendations of the guidance document “Safety Alert Regarding Use of Fecal Microbiota 
for Transplantation and Additional Safety Protections Pertaining to Monkeypox Virus”, 
issued by the FDA in 2022, and which were notified to the AEMPS through an Urgent 
Security Notification on 10/21/2022. 
The donor inclusion criteria have been modified in accordance with the procedures above 
mentioned (see the section “Product under investigation. Description of the manufacturing 
technique(s) of the proposed therapy(s)”).  
The sample size has been adapted from 66 to 82 patients to adjust the expected number of 
patients recruited to the actual observed dropout rate from the study, without this change 
affecting the fulfillment of the study objectives. 


 
 
 
 
 
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A new planned study schedule has been established, taking into consideration the changes 
performed to the number of patients to be recruited. 
Some non-relevant explanatory editions have been made with the aim of facilitating the 
understanding and application of the protocol. 
➢ Patient Information Sheet – Informed consent form 
The changes produced in the new V6.0 protocol version result in the update of the Patient 
Information Sheet-Informed Consent form, then generating the new 6.0 version of May 18, 
2023. 
➢ Non-substantial modification of the IB and Investigational Medicinal Product 
Dossier (IMPD): 
Both documents have been modified to include the above explained safety procedures for 
the prevention of monkeypox transmission at the IB and IMPD. 
The IMPD annexes have been modified to adapt the structure to the new regulation, and 
incorporating the recommendations made by the AEMPS in tests with the same product in 
clinical research phase for other indications, regarding the quality section. 
 
Summary and justification of Substantial Modification No.07 
(08/07/2023) 
➢ 
Substantial modification of the clinical trial protocol:  
Reformulation of main and secondary variables, and adaptation of the statistical plan 
Revision to the definition of the study variables to add the use of odds ratios and logistic 
regression, in addition to the mixed ANOVA, in the analysis of the main efficacy variable, 
thus allowing to perform the study of the probability of recurrence, in addition to the 
number of diarrhea episodes. Given that if the patient has a recurrence he or she is removed 
from the study, and it is not possible to model recurrences using ANOVA, but it is possible 
to model recurrences using binary logistic regression. 
Furthermore, the variable “Duration of hospitalization” is eliminated given that many 
patients receive outpatient treatment or the reason for admission was not CD infection. The 
variable “Duration of treatment” is also removed given that the duration of treatment with 
MBK-01 is equal to one day for all patients, and in the case of Fidaxomicin the duration of 
the treatment is 10 days, except in cases of withdrawal of the study prior to the completion.  
Complementary ITT subpopulations are added for the analysis of the probability of 
recurrence and time to recurrence (ITT-1: allows patients withdrawn before V4 to be 
considered; ITT-2: allows increasing the precision of recurrence monitoring).  


 
 
 
 
 
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The covariate analysis is removed since no information has been collected on CD infection 
severity, and information on the number of previous recurrences in some patients is not 
available or is 0 in primary episodes. Furthermore, it is of interest to the promoter to study 
the added subgroups.  
Additionally, a clarifying and non-substantive revision of the definition of CD infection 
recurrence was included.  
Review of sample size calculation  
The reformulation of the main variable results in the need to review the statistical 
calculation of the sample size, which will be based on a confidence level of 97.06%, Odds 
Ratio of 2.5, statistical power of 80% and loss rate of 50%, then generating a sample size of 
104 subjects (52 in each group). The dropout rate remains at a value close to that observed 
to date (50%).  
Premature trial termination  
Criteria that allow premature termination of the clinical trial are defined, including 
unforeseen risks, changes in product development plans, difficulties in recruitment, or 
significant and evident interim results of superiority/futility of the investigational drug 
compared to the control.  
The possibility of a premature termination could provide advantages such as minimizing 
risks to subjects, a higher efficiency in the management of research resources, and/or, 
where appropriate, accelerating the development of the product and its access to the 
market. 
Interim analyses 
The possibility of a premature termination of the trial makes it necessary to plan a second 
interim statistical analysis to evaluate trends in efficacy and safety results. In addition, the 
variables that will enter in these interim analyses are reviewed and completed.  
Safety monitoring (withdrawn patients) 
The high rate of follow-up loss, as well as the possibility of a premature trial termination, 
make it necessary to define a strategy for the follow-up of patients withdrawn before 
completing the scheduled follow-up. Thus, it is defined that all patients withdrawn before 
completing the planned follow-up must undergo an end-of-study visit ≥30 days after 
starting treatment, in which, at least, relevant safety information (events) will be collected 
(adverse events and concomitant treatments).  
The results of the first interim analysis of the trial show a low-risk safety profile of MBK-01, 
with a frequency of treatment-related adverse events (AEs) of 0% (0/19 in MBK-01 and 
0/14 in the Fidaxomicin). The most frequent AEs were diarrhea and elevated C-reactive 
protein. On July 17, after a review of the safety information, it was observed that there are 
no serious adverse events (SAEs) related to the treatments (0% for both the MBK-01 group 
and the group receiving Fidaxomicin). In both the MBK-01 group and the Fidaxomicin 


 
 
 
 
 
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group, the most frequent adverse event was diarrhea, with this happening in all cases before 
8 weeks after treatment. 
In addition, small explanatory and non-relevant format editions have been made with the 
aim of facilitating the understanding and application of the protocol.  
➢ Patient Information Sheet – Informed consent form 
The changes produced in the new V7.0 protocol version result in the update of the Patient 
Information Sheet-Informed Consent form, then generating the new 7.0 version on August 
4, 2023.  
➢ The IB and IMPD of the study have not been modified 

SAP_001.pdf: 
ICD-01. Randomized, controlled, open-label 
phase III clinical trial in patients with primary or 
recurrent Clostridioides difficile (CD) infection to 
evaluate the efficacy and safety of freeze-dried fecal 
microbiota capsules vs Fidaxomicin.
Statistical Analysis Plan. 
Version 3.0. (06/10/2023)
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ICD-01. Statistical Analysis Plan v3.0 (06/10/2023)
Annex I SOP 6002 - Approved on 30-03-2022
ICD-01. Randomized, controlled, open-label phase III clinical trial 
in patients with primary or recurrent Clostridioides difficile (CD) 
infection to evaluate the efficacy and safety of freeze-dried fecal 
microbiota capsules vs Fidaxomicin. Statistical Analysis Plan. 
Version 3.0. 03/10/2023.
CONTENTS
1.
Introduction...........................................................................................................................4
1.1.
Objectives of the study..................................................................................................5
1.1.1.
Main objective. ......................................................................................................5
1.1.2.
Secondary objectives. ............................................................................................5
1.2.
Study variables...............................................................................................................5
1.2.1.
Efficiency variables.................................................................................................5
1.2.2.
Safety variables......................................................................................................6
1.2.3.
Quality of life variables. .........................................................................................7
1.2.4.
Subgroup analysis. ...............................................................................................16
1.3.
Analysis populations. ...................................................................................................17
1.3.1
Intention-to-treat (ITT) population......................................................................17
1.3.2
Population per Protocol (PP). ..............................................................................17
1.3.3
Security Population (PS).......................................................................................17
1.4.
Study outline................................................................................................................18
2
Description of statistical analysis.........................................................................................19
2.1
Disposition and flow of patients..................................................................................19
2.2
Statistical analysis according to protocol.....................................................................20
2.3
Descriptive statistics. ...................................................................................................20
2.3.1
Baseline descriptive statistics. .............................................................................20
2.3.2
Efficiency analysis. ...............................................................................................22
2.3.3
Security analysis...................................................................................................22
2.4
Inferential analysis.......................................................................................................24
2.4.1
Efficiency analysis. ...............................................................................................24
2.4.2
Security analysis...................................................................................................25
2.5
Statistical analysis steps...............................................................................................25


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ICD-01. Statistical Analysis Plan v3.0 (06/10/2023)
Annex I SOP 6002 - Approved on 30-03-2022
2.6
Procedure for accounting for missing data, treatment of outliers and case allocation.
 28
2.7
Procedures for multiplicity analysis.............................................................................29
2.8
Intermediate statistical analysis ..................................................................................30
2.9
Subgroup analysis........................................................................................................32
2.10
Deviations from the SAP..............................................................................................32
3.
Summary of statistical analysis............................................................................................33
4.
References...........................................................................................................................35


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ICD-01. Statistical Analysis Plan v3.0 (06/10/2023)
Annex I SOP 6002 - Approved on 30-03-2022
1. Introduction.
This document describes the statistical analysis plan (SAP) foreseen for the ICD-01 study. The 
proposed analyses use as a reference what is stated in the study protocol (version 7, dated 
04/08/2023), regarding the objectives. The objectives of the sponsor and the PI will also be 
taken into account in order to arrive at a final version of this SAP.
Once approved by the promoter, the statistical analysis will adhere to the contents of this 
document. Any additional request not included in this document may entail an additional 
budget from Sermes CRO.
Optionally, and in order to ensure the repeatability of the results obtained, the compendium of 
the statistical syntax used will be attached to the final statistical report if the software used is 
governed by a programming language.
The recommended statistical software for statistical analysis is R (R Core Team, 2023) with the 
R Studio interface (version 2023.06.2). Apart from the installed functions such as Rbase, and 
among other available data processing and analysis packages, it is suggested to use the ones 
described in the table below:
Recommended R packages for data processing and analysis.
Package
Version
Target
References
readxl
1.4.3
Reading Excel files
Wichham and Bryan (2019)
psych
2.3.6
Descriptive analysis
Revelle (2023)
ggplot2
3.4.3
Graphic design
Wickham (2016)
coin
1.4-2
Non-parametric tests
Hothorn et al. (2006, 2008)
gmodels
2.18.1
Contingency table analysis
Warnes et al. (2018)
survival
3.5-7
Survival analysis
Therneau (2021)
survminer
0.4.9
Survival charts
Kassambara et al. (2021)
sqldf
0.4-11
SQL Queries
Grothendieck, G. (2017).
For any questions regarding the Statistical Analysis Plan, the SERMES CRO Biometrics 
Department is at your disposal.


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ICD-01. Statistical Analysis Plan v3.0 (06/10/2023)
Annex I SOP 6002 - Approved on 30-03-2022
1.1.
Objectives of the study.
Note: The objectives are specified in the protocol (page 14).
1.1.1.
Main objective.
The main objective is to evaluate the efficacy of fecal microbiota transfer compared to the 
control (Fidaxomicin) at 8 weeks after initiation of treatment.
1.1.2.
Secondary objectives.
Secondary objectives involve assessing the safety of the investigational drug and the quality of 
life of the patients participating in the study.
1.2.
Study variables.
Note: The study variables are described in the protocol (pages 14-15).
1.2.1.
Efficiency variables.
The primary variable is the absence of diarrhea absence of CD diarrhea without retreatment 
(or absence of recurrence as defined in Section 03.07.02 of the protocol), as assessed by 
subject interview and physical examination. The primary variable is operationalized by the 
probability of recurrence.
Recurrence is defined as the reappearance of clinical manifestations of a new episode of CDI 
that recurs within 8 weeks after the onset of symptoms of a previous episode, provided that 
the symptoms of the previous episode had resolved after completing treatment (Debast et al., 
2013).


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Important note. A patient who recurs will be withdrawn from the study. Failure of a patient to 
be effective is defined as the occurrence of a new recurrence of CD infection. Patients with a 
new recurrence will be withdrawn from the study and treated with rescue medication, which 
will be antibiotic treatment (vancomycin or fidaxomicin).
Other efficacy variables:
o
Good/bad evolution of the patient: Bad evolution is understood as the detection 
48-72 hours after the start of treatment (MBK-01 or Fidaxomicin) of a worsening of 
the diarrhea episode (at least one stool more than at baseline*, with baseline 
being understood as the time of the start of study treatment (MBK-01 or 
Fidaxomicin). In addition, at least one of the following factors must be detected:
1.
Elevation of C-reactive protein (CRP) value (>5% of baseline*).
2.
Elevation of the absolute value of leukocytes ( >5% of the value a t  
baseline*).
3.
Progression to sepsis: hypotension or organ failure with no other 
apparent cause.
*Note: baseline is considered to be the time of initiation of study treatment 
(Fidaxomicin /MBK-01).
o
Time to recurrence according to randomization groups: Recurrence is defined as 
the reappearance of clinical manifestations of a new CDI episode in a patient with 
a treated and cured CDI episode within 8 weeks after the end of treatment. If the 
reappearance of a new CDI episode is later than 8 weeks, it would be a possible 
case of reinfection.
o
Overall survival (OS): percentage of patients who are still alive after a defined 
period of time since starting treatment. This will take into account the start date of 
treatment and the end date of the study if the patient has not died and the date of 
exitus if the patient has died. If the patient leaves the study prematurely, the last 
available visit date will be considered.
1.2.2.
Safety variables.
•
Number of Adverse Events per randomization group.
•
Number of patients within each Adverse Event per randomization group.
•
Type of Adverse Events by randomization group.
•
Number of Adverse Events of Special Interest by randomization group.
•
Number of patients within each Adverse Event of Special Concern per randomization 
group.


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ICD-01. Statistical Analysis Plan v3.0 (06/10/2023)
Annex I SOP 6002 - Approved on 30-03-2022
•
Type of Adverse Events of Special Interest by randomization group.
•
Number of Serious Adverse Events by randomization group.
•
Type of Serious Adverse Events by randomization group.
•
Adverse events related to treatment.
•
Severity of Adverse Events.
•
Adverse Events in relation to ICD.
•
Mortality associated with CDI.
•
Admissions to the Intensive Care Unit (ICU).
•
Adverse Events of Special Interest (AESI) by frequency of occurrence.
1.2.3.
Quality of life variables.
Quality of life is measured by the SF-36 questionnaire which consists of 36 questions (items) 
that assess both positive and negative states of quality of life.
These 36 items cover the following areas:
•
Physical function: Degree to which health limits physical activities such as self-care, 
walking, climbing stairs, bending, picking up or carrying weights, and moderate to 
strenuous exertion.
•
Physical Role: Degree to which physical health interferes with work and other daily 
activities, including less than desired performance, limitation in the type of activities 
performed, or difficulty in performing activities.
•
Bodily pain: The intensity of the pain and its effect on the usual work, both away from 
home and at home.
•
General health: Personal assessment of health that includes current health, future health 
prospects and resistance to illness.
•
Vitality: Feeling of energy and vitality, as opposed to feeling tired and exhausted.
•
Social function: Degree to which physical or emotional health problems interfere with 
usual social life.
•
Emotional role: Degree to which emotional problems interfere with work or other daily 
activities, including reduced time spent on those activities, lower than desired 
performance, and decreased care at work.
•
Mental health: General mental health, including depression, anxiety, behavioral and 
emotional control and overall positive affect.
In addition, the SF-36 includes a transition item that asks about the change in general health 
status with respect to the previous year. This item is not used for the calculation of any of the 
scales, but provides important information about the perceived change in health status during 
the year prior to the application of the SF-36 instrument.


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ICD-01. Statistical Analysis Plan v3.0 (06/10/2023)
Annex I SOP 6002 - Approved on 30-03-2022
The number of items per dimension and the interpretation of the scores are presented in the 
table below:
Dimension
Number of items
Worst score (0)
Best score (100)
Physical function
10
Very limited to carry out all 
physical activities including 
bathing or showering, due to 
health
Performs all types of 
physical activities including 
the most strenuous 
without any limitation due 
to
health
Physical role
4
Problems with work or other daily 
activities due to physical health
No problems with work or 
other daily activities due 
to health
physics
Body pain
2
Very intense pain and
extremely limiting
No pain or limitations
due to him
General health
5
Evaluates its own health as bad and
believes it is likely to get worse
Evaluates one's own health 
as
excellent
Vitality
4
Feeling tired and exhausted all the 
time
It feels very dynamic and full 
of energy all the time.
time
Social function
2
Extreme and very frequent 
interference with normal social 
activities, due to physical or 
emotional problems
Carries out normal social 
activities without any 
interference due to 
physical problems
or emotional
Emotional role
3
Problems with work and other 
daily activities due to emotional 
problems
No problem with work and 
other daily activities due to 
problems
emotional
Mental health
5
Feelings of anguish and 
depression all the time.
Feeling of happiness,
tranquility and 
calmness all the time
Health transition item
1
He believes his health is much 
worse now than it was a year 
ago.
He believes that his overall 
health is much better now 
than it is now.
one year ago
Note: Source: Vilagut et al.,2005.


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The table below shows schematically the physical and mental health phenomena that are 
evaluated by the SF-36:
Physical scale
Mental Scale
Scale
Functionality
Welfare
Disability
Personal 
evaluatio
n
Functionality
Welfare
Disability
Personal 
evaluatio
n
Physical 
function
X
Physical role
X
Body pain
X
X
General health
X
X
Vitality
X
X
Social function
X
X
Emotional role
X
Mental health
X
X
Note: Translated from the SF-36 interpretation manual (Ware, 1993).
The items and dimensions of the SF-36 are scored so that the higher the score the better the 
health status. The table presented below indicates how the scores for each area should be 
obtained.


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Dimension
Item number
Text
Response option
Pre-coded 
value
Final value
Score
3a
Intense exertion, such as running, lifting heavy 
objects, or participating in sports
exhausting
3b
Moderate exertion, such as moving a table, 
vacuuming, bowling, or walking
more than one hour
3c
Picking up or carrying the shopping bag
3d
Climbing several flights of stairs
3e
Climbing a single flight of stairs
3f
Crouching or kneeling
3g
Walking one kilometer or more
3h
Walk several blocks (several hundred blocks) to
meters)
3i
Walk a single block (about 100 meters)
Physical 
function
3j
Bathing or dressing oneself
•
Yes,
me
very 
limited
•
Yes, it limits me a 
little
•
No, it does not 
limit me
• 1
• 2
• 3
• 1
• 2
• 3
Calculate the 
algebraic sum of 
the final value of 
items
4a
Did you have to reduce the amount of time spent 
at work, or
to your daily activities?
4b
Did you do less than you would have liked to do?
4c
Did you have to stop doing some tasks at your
work or in your daily activities?
Physical role
4d
Did you have difficulty doing your work or daily 
activities (e.g., did you find it difficult to do your 
job)?
more than normal)?
•
Always
•
Almost always
•
Many times
•
Sometimes
•
Only sometime
•
Never
• 1
• 2
• 3
• 4
• 5
• 6
• 1
• 2
• 3
• 4
• 5
• 6
Calculate the 
algebraic sum of 
the final value of 
the items


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Dimension
Item number
Text
Response option
Pre-coded 
value
Final value
Score
7
Did you have pain in any part of your body during 
the last 4 weeks?
•
No, none
•
Yes, very little
•
Yes, a little
•
Yes, moderate
•
Yes, very much
•
Yes, very much
•
1
•
2
•
3
•
4
•
5
•
6
•
6
•
5
•
4
•
3
•
2
•
1
Body pain
8
During the last 4 weeks, to what extent has the 
pain made your usual work (including work 
outside the home and housework) difficult?
•
Nothing
•
A little
•
Regular
•
Quite
•
Much
•
1
•
2
•
3
•
4
•
5
•
5
•
4
•
3
•
2
•
1
Calculate the 
algebraic sum of 
the final value of 
items
1
In general, you would say that your health is
•
Excellent
•
Very good
•
Good
•
Regular
•
Mala
•
1
•
2
•
3
•
4
•
5
•
5
•
4
•
3
•
2
•
1
11a
I think I get sick more easily than other people.
•
Absolutely true
•
Quite true
•
I do not know
•
Quite false
•
Totally false
•
1
•
2
•
3
•
4
•
5
•
1
•
2
•
3
•
4
•
5
General health
11b
I am as healthy as anyone else
•
Absolutely true
•
Quite true
•
I do not know
•
Quite false
•
Totally false
•
1
•
2
•
3
•
4
•
5
•
5
•
4
•
3
•
2
•
1
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algebraic sum of 
the final value of 
items


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Dimension
Item number
Text
Response option
Pre-coded 
value
Final value
Score
11c
I think my health is going to get worse
•
Absolutely true
•
Quite true
•
I do not know
•
Quite false
•
Totally false
•
1
•
2
•
3
•
4
•
5
•
1
•
2
•
3
•
4
•
5
11d
My health is excellent
•
Absolutely true
•
Quite true
•
I do not know
•
Quite false
•
Totally false
•
1
•
2
•
3
•
4
•
5
•
5
•
4
•
3
•
2
•
1
9a
Did you feel full of vitality?
•
Always
•
Almost always
•
Many times
•
Sometimes
•
Only sometime
•
Never
•
1
•
2
•
3
•
4
•
5
•
6
•
6
•
5
•
4
•
3
•
2
•
1
9e
Did you have a lot of energy?
•
Always
•
Almost always
•
Many times
•
Sometimes
•
Only sometime
•
Never
•
1
•
2
•
3
•
4
•
5
•
6
•
6
•
5
•
4
•
3
•
2
•
1
Vitality
9g
Did you feel exhausted?
•
Always
•
Almost always
•
Many times
•
Sometimes
•
Only sometime
•
Never
•
1
•
2
•
3
•
4
•
5
•
6
•
1
•
2
•
3
•
4
•
5
•
6
Calculate the 
algebraic sum of 
the final value of 
items


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Dimension
Item number
Text
Response option
Pre-coded 
value
Final value
Score
9i
Did you feel tired?
•
Always
•
Almost always
•
Many times
•
Sometimes
•
Only sometime
•
Never
•
1
•
2
•
3
•
4
•
5
•
6
•
1
•
2
•
3
•
4
•
5
•
6
6
During the past 4 weeks, to what extent have 
your physical health or emotional problems 
made your usual social activities with family, 
friends, neighbors or others difficult?
•
Nothing
•
A little
•
Regular
•
Quite
•
Much
•
1
•
2
•
3
•
4
•
5
•
5
•
4
•
3
•
2
•
1
Social function
10
During the past 4 weeks, how often has physical 
health or emotional problems made it difficult 
for you to engage in social activities (such as 
visiting friends or family)?
•
Nothing
•
A little
•
Regular
•
Quite
•
Much
•
1
•
2
•
3
•
4
•
5
•
1
•
2
•
3
•
4
•
5
Calculate the 
algebraic sum of 
the final value of 
items
5a
Did you have to reduce the time you spent at 
work or in your daily activities because of a 
problem?
emotional?
5b
Did you do less than you would have liked to do, by
any emotional problems?
Emotional role
5c
Did you not go about your work or your daily 
activities as carefully as usual, by
any emotional problems?
•
Always
•
Almost always
•
Many times
•
Sometimes
•
Only sometime
•
Never
•
1
•
2
•
3
•
4
•
5
•
6
•
1
•
2
•
3
•
4
•
5
•
6
Calculate the 
algebraic sum of 
the final value of 
items


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Dimension
Item number
Text
Response option
Pre-coded 
value
Final value
Score
9b
were you very nervous?
•
Always
•
Almost always
•
Many times
•
Sometimes
•
Only sometime
•
Never
•
1
•
2
•
3
•
4
•
5
•
6
•
1
•
2
•
3
•
4
•
5
•
6
9c
Did he feel so low in morale that nothing could 
cheer him up?
•
Always
•
Almost always
•
Many times
•
Sometimes
•
Only sometime
•
Never
•
1
•
2
•
3
•
4
•
5
•
6
•
1
•
2
•
3
•
4
•
5
•
6
9d
Did you feel calm and collected?
•
Always
•
Almost always
•
Many times
•
Sometimes
•
Only sometime
•
Never
•
1
•
2
•
3
•
4
•
5
•
6
•
6
•
5
•
4
•
3
•
2
•
1
Mental health
9f
Did you feel discouraged and sad?
•
Always
•
Almost always
•
Many times
•
Sometimes
•
Only sometime
•
Never
•
1
•
2
•
3
•
4
•
5
•
6
•
1
•
2
•
3
•
4
•
5
•
6
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algebraic sum of 
the final value of 
items


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Dimension
Item number
Text
Response option
Pre-coded 
value
Final value
Score
9h
Did you feel happy?
•
Always
•
Almost always
•
Many times
•
Sometimes
•
Only sometime
•
Never
•
1
•
2
•
3
•
4
•
5
•
6
•
6
•
5
•
4
•
3
•
2
•
1
Declared 
health 
evolution
2
How would you say your health is today 
compared to a year ago?
•
Much better now 
than a year ago
•
Slightly 
better 
now than a year 
ago
•
About the same as 
a year ago
•
Slightly 
worse 
now than a year 
ago
•
Much worse now 
than a year ago
•
1
•
2
•
3
•
4
•
5
•
5
•
4
•
3
•
2
•
1
The score for this 
item is inverted as 
a final value.


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The calculation of the total scores for each SF-36 scale is shown in the table below:
Scale
Final sum after recoding
Minimum and 
maximum
(direct scoring)
Possible range in
direct scores
Physical function
3a+3b+3c+3d+3e+3f+3g+3h+3i+3j
10, 30
20
Physical role
4a+4b+4c+4d
4, 20
16
Body pain
7+8
2, 12
10
General health
1+11a+11b+11c+11d
5, 25
20
Vitality
9a+9e+9g+9i
4, 24
20
Social function
6+10
2, 10
8
Emotional role
5a+5b+5c
3, 15
12
Mental health
9b+9c+9d+9f+9h
5, 30
25
After obtaining the direct scores, they should be transformed to a scale from 0 to 100 using the 
following formula (Alonso et al., 2003):
𝑃𝑢𝑛𝑡𝑢𝑎𝑐𝑖ó𝑛 𝑡𝑟𝑎𝑛𝑠𝑓𝑜𝑟𝑚𝑎𝑑𝑎 =
𝑃𝑢𝑛𝑡𝑢𝑎𝑐𝑖ó𝑛 𝑑𝑖𝑟𝑒𝑐𝑡𝑎 - 𝑃𝑢𝑛𝑡𝑢𝑎𝑐𝑖ó𝑛 𝑚í𝑛𝑖𝑚𝑎
𝑅𝑎𝑛𝑔𝑜 𝑑𝑒 𝑙𝑎 𝑝𝑢𝑛𝑡𝑢𝑎𝑐𝑖ó𝑛 𝑑𝑖𝑟𝑒𝑐𝑡𝑎
· 100
For example, if a patient scores 21 points on the Physical Function scale, his or her score out of 
100 would be as follows:
𝑃𝑢𝑛𝑡𝑢𝑎𝑐𝑖ó𝑛 𝑡𝑟𝑎𝑛𝑠𝑓𝑜𝑟𝑚𝑎𝑑𝑎 =
21 - 10
20
- 100 = 55
Transformed scale scores are not calculated for the Declared Evolution of Health item. It is 
recommended to treat responses to this item as ordinal level data and to analyze the 
percentage of respondents selecting each response option.
1.2.4.
Subgroup analysis.
All efficacy variables will be studied in the following subgroups:
-
Patients with primary episode of CDI.
-
Patients with recurrent episodes of CDI.
-
Patients not receiving vancomycin prior to receiving study treatment.


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ICD-01. Statistical Analysis Plan v3.0 (06/10/2023)
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1.3.
Analysis populations.
The clinical trial will be conducted in patients presenting with an episode of CD infection (either 
the first episode or subsequent recurrences).
In the present study 104 patients will be enrolled (52 in the control group treated with 
Fidaxomicin and 52 patients in the experimental group treated with the investigational 
product).
Note: The analysis populations have been defined in the protocol (page 92).
1.3.1
Intention-to-treat (ITT) population.
The Intention-to-treat population consists of all patients who have been included in the study, 
whether or not they meet the selection criteria and have been randomized.
The efficacy analysis will be carried out with the Intention to Treat (ITT) population.
1.3.2
Population per Protocol (PP).
The Protocol Population includes all those patients who have been recruited for the study, met 
all the inclusion criteria, none of the exclusion criteria and have continued in the study without 
any major deviation from the protocol.
1.3.3
Security Population (SP).
The Safety Population consists of all those patients who have been randomized into either of 
the two randomization groups and who have taken at least one dose of the corresponding 
treatment.
The security analysis will be carried out with the Security Population (SP).


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ICD-01. Statistical Analysis Plan v3.0 (06/10/2023)
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1.4.
Study outline.
Visit
Baseline 
(randomization 
and treatment)
Visit 2
Visit 3
Visit 4
Visit 5
Visit 6 (end 
of study)
Weather
(-1 a 0)
72h after the start 
of treatment
3
weeks
8
weeks
3 months
6 months
Type of visit
On-site
On-site
On-site
On-site
Telefónica
Telefónica
Window period
N/A
±1 day
±4 days
±4 days
±6 days
±6 days
Procedures
Informed consent
X
Washing period1
X
Inclusion/exclusion criteria
X
Randomization
X
Treatment
X
Medical history and history of 
ICD
X
Physical examination
X
X
X
X
Pregnancy test
X
Laboratory tests
X
X
Stool sample
X2
X1
Concomitant treatment of 
patients
X
X
X
X
X
X
Evaluation of therapeutic 
compliance
X
X
Assessment of diarrhea (main 
variable)
X
X
X
X
X
X
Evaluation of length of 
hospitalization
X
X
X
X
Evaluation of good / bad 
evolution of the patient
X
X
Evaluation of adverse effects
X
X
X
X
X
X
SF36
X
X
X
1 in case the patients have been treated with vancomycin with a 3 to 5 day regimen for the recurrence to be treated in the 
study and/or antibiotic for other medical indication, it is necessary that they undergo a 24h washout period.
2A first sample is collected (up to 72 hours before inclusion of the patient in the study) to confirm the diagnosis of CD and a 
second sample is collected once the patient has been included in the study and before receiving the medication to be stored 
for subsequent studies;3 : A stool sample will be collected for subsequent studies.


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ICD-01. Statistical Analysis Plan v3.0 (06/10/2023)
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2
Description of the statistical analysis.
2.1
Disposition and flow of patients.
Regarding patient disposition, descriptive data on the following items will be included:
•
Number of patients selected.
•
Number of selection failures.
•
Number and percentage of patients in each of the analysis populations.
•
Number of patients excluded from each analysis population along with the reason for 
exclusion.
•
Completion of the study:
o
Number and percentage of patients who completed treatment.
o
Number and percentage of patients who did not complete treatment.
o
Number of patients who completed the study.
o
Number of patients who did not complete the study.
o
Listing of all withdrawals along with the reason for termination.
A patient flow diagram following the structure of the CONSORT report including the following 
information will be made and presented in the statistical report:
•
Selected patients.
•
Selection errors. Reasons.
•
Patients randomized to the different treatment groups.
•
Patients withdrawn. Reasons.
•
Patients analyzed.


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ICD-01. Statistical Analysis Plan v3.0 (06/10/2023)
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An example of a patient flow format used by the CONSORT system is given below:
2.2
Statistical analysis according to protocol.
The protocol (pages 89-97) provides a general description of the planned analyses. In the 
present document, these analyses are detailed in their corresponding sections.
2.3
Descriptive statistics.
2.3.1
Baseline descriptive statistics.
The protocol (page 91) specifies: "A descriptive analysis of all relevant variables collected at 
baseline will be performed, and this analysis will be divided by randomization groups.
The treatment of both types of variables is illustrated as an example:


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ICD-01. Statistical Analysis Plan v3.0 (06/10/2023)
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•
Categorical variables (nominal, dichotomous or polytomous): Distribution of absolute 
and relative frequencies (transformed into percentages). Cumulative relative 
frequency distribution in the case of variables with ordered categories.
Frequency distribution of the sex variable (simulated data).
Man
Woman
Group
N
%
N
%
p-value
Treaties
2
50.0
2
50.0
Controls
5
57.1
4
42.9
Total
7
53.8
6
46.2
1
•
Quantitative variables: mean, standard deviation, minimum and maximum. If 
appropriate, other descriptive statistics such as skewness and kurtosis indices should 
be added, 95% confidence intervals should be added, and an analysis of the normality 
of the distribution should be included. In those variables that do not meet the 
assumption of normality, it will be convenient to use the median instead of the mean, 
and the interquartile range (or the AVERAGE) instead of the standard deviation. On 
occasions where the distribution of scores is peculiar, a histogram should be added for 
better understanding. When it is reasonable to compare groups of subjects (e.g., 
controls and treated), the graphical representation will be made by box-and-whisker 
plots. Where possible, a summary table in which each variable is on one line should be 
presented to facilitate tracking of that variable throughout the trial.
Age (in years) of participants (simulated data).
Group
Media
Median
DT
MAD
As
K
Min.
Max.
Treaties
39.10
21.00
12.97
11.12
0.24
-0.78
18.00
64.00
Control
31.80
38.00
16.93
7.41
0.77
-1.35
19.00
60.00
Note. SD: Standard Deviation. MAD: Mean Absolute Deviation. As: Asymmetry. K: Kurtosis. 
Min:
Min. Max: Maximum.


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2.3.2
Efficiency analysis.
The efficacy analysis involves the application of a descriptive analysis at the beginning, which 
will be complemented with the inferential analysis (if applicable). In addition, the possible 
missing values that exist in each variable being analyzed for the determination of efficacy will 
be accounted for.
For descriptive analysis, data will be presented as absolute frequencies and percentages in the 
case of qualitative variables. For quantitative variables, central tendency statistics, such as 
mean and median, and dispersion statistics, such as standard deviation and interquartile 
range, minimum and maximum values, will be used, and indicators of the shape of the 
distribution such as skewness and kurtosis indexes can be provided. In the case of ordinal 
variables, depending on the number of categories, one or another form of description will be 
used. In all cases, a column should be added to the data presentation table including the 
number of patients with available data. The analyses will be divided by randomization group.
2.3.3
Security analysis.
The protocol (pages 94-95) states that "Safety variables are operationalized with the number of 
documented adverse events ranked by frequency, severity, seriousness and causality as 
assessed by subject interview and physical examination.
For the analysis of the Adverse Events, a descriptive analysis (frequencies and percentages) of 
these events throughout the study will be performed, presenting a list of them, grouped 
according to severity and intensity. These events will be listed by patient and randomization 
group. Adverse reactions will be recorded using the Verbatim described in the eCRD.
Adverse Events and concomitant medication will be coded using a standardization system. The 
most current version of the MedDRA system will be used for Adverse Events and the ATC code 
will be used for concomitant medication.
The analysis of the physical examination variables will be done by frequencies and percentages 
and divided by randomization group.
The safety analysis will be conducted on the Safety Population (SP)."
Safety analyses will be carried out incorporating these lists:
•
Adverse events (AEs).
•
Serious adverse events (SAEs).
•
Adverse Events of Special Interest (AESI).


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•
Events related to the investigational drug or comparator: Those whose causal 
relationship is "probable" or "related" will be considered.
•
Events leading to discontinuation of study therapy.
•
Events with grades of "serious", "life-threatening" or "death".
For all such events, this information will be provided:
•
Number of events: this information will also be displayed by
o
SOC and PT codes.
o
Grade.
o
Progression.
o
Action taken.
o
Relationship with MBK-01 or with Fidaxomicin.
•
Type of events: this information will be displayed by SOC and PT codes.
•
Number of patients within each event category: this information will also be displayed 
by:
o
Organ classification system (OCS) and preferred term (PT).
o
Grade.
o
Progression.
o
Action taken.
o
Relationship with MBK-01 or with 
Fidaxomicin. The variables related to patient exitus are:
o
Patients who died.
o
Cause of exitus.
o
Days since last dose of MBK-01 or Fidaxomicin. Variables 
related to concomitant medication:
o
Number of concomitant medications.
o
Number of patients who required concomitant medication.
o
Number of concomitant medications per patient.
o
Number of concomitant medications related to Adverse Events for patients overall 
and per patient.
In addition, details on concomitant medications per patient will be displayed:
o
ATC code and name.
o
Dosage.
o
Route of administration.
o
Duration of treatment: difference in days between the end date and the start date.
o
Reason for treatment.
o
Relationship with adverse event.


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Annex I SOP 6002 - Approved on 30-03-2022
2.4
Inferential analysis.
2.4.1
Efficiency analysis.
The protocol (page 92) specifies that the efficacy analysis will be conducted in the Intention-to-
Treat Population.
Page 72 of the protocol describes that The probability of recurrence as a function of 
randomization group will be studied by binary logistic regression analysis. The odds ratio of 
recurrence will be reported, using the MBK-01 group as a reference. In addition, the 
confidence interval, Nagelkerke's R-squared and Yule's Q coefficient will be included, as well as 
the critical level. The reference group to be used will be MBK-01 (expecting that in Fidaxomicin 
the probability will be equal or higher compared to MBK-01).
The number of diarrhea episodes per visit and group will be analyzed, if assessable data are 
available, using analysis of variance mixed models or relevant robust/nonparametric models.
The good/bad evolution of patients will be analyzed using logistic regression procedures 
incorporating the same indicators as described for the analysis of the probability of recurrence.
Overall survival in weeks will be analyzed by Cox regression including the randomization group 
as an independent variable. Overall survival will be measured in weeks.
Due to the characteristics of the trial, it is possible to state the hypotheses as either non-
inferiority or superiority. Given that the trial is a Phase III trial, both hypotheses can be tested 
in the same clinical trial as long as the α risk is controlled, or, in other words, to state that 
there are significant differences when in reality, at the population level, there are no such 
differences. It is important to remember that, in order to avoid false positives, the type I or α 
error must be distributed among the different hypotheses, which means that, in order to find 
statistically significant results, the critical level or p value must be lower (Ferreira-Gonzalez, 
2014).
Although the overall significance level will be alpha=0.05, because more than one analysis of 
the data will be performed, it will be adjusted to 0.0294 to avoid inflation of the probability of 
type I error (Geller and Pocock, 1987). If multiple pairwise comparisons analyses are employed, 
the statistical report will describe the procedure used. Measures of the effect size of the 
results will be incorporated.


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2.4.2
Security analysis.
The statistical significance between the randomization group and the intensity and treatment-
relatedness of both Adverse Events and Serious Adverse Events will be studied, whenever 
feasible, using the χ2 test for association between categorical variables. If possible, the odds 
ratio of occurrence of Serious Adverse Events will be calculated and the 95% confidence 
interval of this odds ratio will be provided. The Fidaxomicin group will be used as the reference 
group.
2.5
Statistical analysis steps.
The following tables describe the techniques to be applied according to the objectives and 
variables affected.
In the case of qualitative variables, a series of analyses based on the research objectives is 
established for the statistical analysis.
Objectives
Variables affected
Applied technique
Expected results
Determination of adverse 
events and serious adverse 
events.
▪Grouping variables:
- Randomization group.
▪Variables to be analyzed:
- Presence of AAG and AA.
- Gravity.
- Relationship to treatment.
Descriptive analysis: 
frequencies and 
percentages.
Inferential analysis: Chi-
square test. Odds ratio 
analysis.
The incidence will be minimal.
Determination of Adverse 
Events of Special Interest (AESI)
▪Grouping variables:
- Randomization group.
▪Variables to be analyzed:
- Presence of AESI.
- Gravity.
- Relationship to treatment.
Descriptive analysis: 
frequencies and 
percentages.
Inferential analysis: Chi-
square test. Odds ratio 
analysis.
The incidence will be minimal.
Determination of adverse 
events in relation to the ICD
▪Grouping variables:
- Randomization group.
▪Variables to be analyzed:
- Presence of adverse events 
related to ICD.
- Gravity.
- Relationship to treatment.
Descriptive analysis: 
frequencies and 
percentages.
Inferential analysis: Chi-
square test. Odds ratio 
analysis.
The incidence will be minimal.
Determination of mortality 
associated with CDI.
▪Grouping variables:
- Randomization group.
▪Variables to be analyzed:
Descriptive analysis: 
frequencies and 
percentages.
Inferential analysis: Chi-
Mortality will be minimal.


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square test.


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Objectives
Variables affected
Applied technique
Expected results
- Determination of mortality associated 
with CDI.
Odds ratio analysis.
ICU admissions
▪Grouping variables:
- Randomization group.
▪Variables to be analyzed:
- Determination of ICU admissions
Descriptive analysis: 
frequencies and 
percentages.
Inferential analysis: Chi-
square test. Odds ratio 
analysis.
Exploratory objective
Absence of CD diarrhea with 
no need for retreatment
▪Grouping variables:
- Randomization group.
- Visit number.
▪Variables to be analyzed:
- Resolution of the diarrhea episode.
Descriptive analysis: 
frequencies and 
percentages.
Inferential analysis: Chi-
square test.
Odds ratio analysis.
The incidence of diarrhea will 
be minimal.
Determination of good/bad 
patient outcome
▪Grouping variables:
- Randomization group.
- Visit number.
▪Variables to be analyzed:
- Good/bad patient evolution
Descriptive analysis: 
frequencies and 
percentages.
Inferential analysis: Chi-
square test. Odds ratio 
analysis.
The evolution of the patients 
will be good.
For inferential analysis involving quantitative variables, a series of analyses are established 
based on the research objectives.
Objectives
Variables affected
Applied technique
Expected results
Duration of hospitalization
▪
Grouping variables:
-Randomization group
▪
Variable under analysis:
-Duration of 
hospitalization (time 
between the beginning of 
hospitalization and the date of 
discharge)
t-test for independent 
samples
In case of non-compliance 
with assumptions: Mann-
Whitney U test
The duration will be shorter 
for patients in the 
experimental group.
Duration of treatment
▪
Grouping variables:
-Randomization group
▪
Variable under analysis:
- 
Duration 
of 
treatment 
(time 
between the start of treatment 
and the end date of treatment)
t-test for independent 
samples
In case of non-compliance 
with assumptions: Mann-
Whitney U test
The duration will be shorter 
for patients in the 
experimental group.
Time to recurrence
▪
Grouping variables:
-Randomization group.
▪
Variable under analysis:
-
Start date of treatment.
-
Recurrence.
-
Recurrence date.
▪
End date of study or date of 
patient withdrawal in case the 
patient has not had
recurrence.
Survival analysis.
Exploratory objective


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Overall survival (OS)
▪
Grouping variables:
-Randomization group.
▪
Variable under analysis:
Survival analysis.
Exploratory objective


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Objectives
Variables affected
Applied technique
Expected results
-
Start date of treatment.
-
Exitus.
-
Date of exitus.
-
End date of study or date of 
patient withdrawal in case the 
patient is not an exitus.
Changes in quality of life 
variables
▪
Grouping variables:
-
Randomization group
-
Visit number
▪
Variable under analysis:
▪
SF-36 score and its 
dimensions
Mixed ANOVA.
In case of non-compliance 
with assumptions, Brunner 
and Langer's test
Exploratory objective
Number of adverse events
▪
Grouping variables:
-Randomization group
▪
Variable under analysis:
-Number of adverse events
t-test for independent 
samples
In case of non-compliance 
with assumptions: Mann-
Whitney U test
The number of adverse 
events will be lower in 
the experimental group.
Number of serious adverse 
events
▪
Grouping variables:
-Randomization group
▪
Variable under analysis:
-Number of serious 
adverse events
t-test for independent 
samples
In case of non-compliance 
with assumptions: U-test of
Mann-Whitney
The number of serious 
adverse events will be 
lower in the experimental 
group.
Where possible, illustrative figures, such as those given below as examples, should be 
incorporated:
-
Figure representing the evolution of each individual patient (simulated data) as a 
function of visits:
-
Figure showing the overall results in terms of visits (simulated data):


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-
Figure showing the growth trend by group (simulated data):
-
Figure showing the passage of time for each randomization group (simulated data):
2.6
Procedure for accounting for missing data, treatment of 
outliers and case imputation.
The number of missing data that cannot be retrieved or obtained (missing values) will be 
reported in the presentation of results. From the moment patients have completed their 
participation in the study, no additional data will be collected (except as required by the Query 
plan and for the resolution of possible new queries arising from the initial analysis of the 
database and study variables). The data collected will be used for efficacy and safety analysis.


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Note: The protocol does not establish any procedure for case allocation.
The statistical report will incorporate information on the count of missing values for each 
variable studied and according to the study group. Optionally, the possible origin of the 
missing values will be studied to determine if they are completely random losses (MCAR), 
random losses (MAR) or non-random losses (MNAR).
In case of detecting the need to apply imputation methods in this study, both the method and 
its reference shall be specified and a study shall be made of the number of missing values and 
assess whether it is feasible to perform the imputation or not, and the decision taken shall be 
justified at all times. As far as possible, an indicator of the possible presence of bias in the 
imputation shall be provided by means of a sensitivity analysis. Whichever imputation method 
is selected, it shall be correctly identified and referenced.
On the other hand, statistical outliers will be detected from a graphical perspective, by means 
of box and whisker plots, and more complex mathematical procedures may be performed if 
necessary. The presence of these values may require the application of robust inferential 
and/or non-parametric tests that should be correctly specified and referenced in the final 
statistical report.
2.7
Procedures for multiplicity analysis.
The protocol does not specify a procedure for correcting the significance level associated with 
multiple comparison analyses. The more extrapolations that are made in simultaneous 
inferences, the more likely it is that erroneous inferences will occur. Several statistical 
techniques have been developed to prevent this from happening, allowing direct comparison 
of significance levels for single and multiple comparisons. These techniques generally require a 
tighter significance threshold for single comparisons to compensate for the number of 
inferences that are made. Methods that could be considered for multiple comparisons are, for 
example, Bonferroni correction or Holm's adjustment. For a detailed description of multiple 
comparison methods, see Chen, Feng, and Yi (2017).
Due to the characteristics of the trial, it is possible to state the hypotheses as either non-
inferiority or superiority. Given that the trial is a Phase III trial, both hypotheses can be tested 
in the same clinical trial as long as the α risk is controlled, or, in other words, to state that 
there are significant differences when in reality, at the population level, there are no such 
differences. It is important to remember that, to avoid false positives, it is required to 
"distribute" the type I or α error between the different hypotheses, which means that, to find 
statistically significant results, the value of the critical level or p must be lower (Ferreira-
Gonzalez, 2014).


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In the case of using any procedure for multiple comparisons, it must be correctly specified and 
referenced in the final statistical report.
2.8
Intermediate statistical analysis.
Pages 77 and 78 of the protocol specify that two intermediate analyses are planned for this 
study.
1.
The first interim analysis will be performed when approximately 50% of the patients 
have completed treatment.
2.
The second interim analysis will be performed when approximately 80% of the patients 
have performed V4.
All interim analyses will incorporate a descriptive analysis of baseline patient characteristics, an 
efficacy analysis and a safety analysis. The analysis of baseline variables and the efficacy and 
safety analyses will be performed by comparing the randomization groups.
Both intermediate analyses will be done in the ITT population in the efficacy section and in the 
PS in the safety analysis section.
The results could lead to early discontinuation of the study if large differences between the 
two randomization groups are revealed after the study, or obvious futility is shown, so that 
there is no possibility of showing that continuing the study until 100% of enrolled patients are 
reached would result in a clinically meaningful effect. In addition to saving time and resources, 
this could reduce the exposure of study participants to a treatment that is inferior or shows no 
difference from the comparator.
If large and clear differences between the randomization groups or clear futility are observed, 
early discontinuation of the study should be considered. The study of differences between 
randomization groups should be accompanied by measures of effect size, confidence intervals 
and not only statistical significance.
Obviously, given the variety of statistical analyses to be carried out, the final decision to 
establish futility or obvious efficacy must be agreed upon after reviewing the results obtained 
from the intermediate analyses.
2.8.1
First interim analysis.
This analysis will include these variables:
•
Efficiency analysis:
o
Recurrence probability.
o
Time to recurrence.


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o
Good/bad evolution of the patient.
o
Analysis by subgroups.
•
Security analysis:
o
Number of Adverse Events per randomization group.
o
Number of patients within each Adverse Event per randomization group.
o
Type of Adverse Events by randomization group.
o
Number of Adverse Events of Special Interest by randomization group.
o
Number of patients within each Adverse Event of Special Concern per 
randomization group.
o
Type of Adverse Events of Special Interest by randomization group.
o
Number of Serious Adverse Events by randomization group.
o
Type of Serious Adverse Events by randomization group.
o
Adverse events related to treatment.
o
Severity of Adverse Events.
o
Adverse Events in relation to ICD.
o
Mortality associated with CDI.
o
Admissions to the Intensive Care Unit (ICU).
This analysis will be versioned as version 1.0. If justified modifications are required, it will be 
versioned as 1.1, 1.2 and so on.
2.8.2
Second interim analysis.
This analysis will include these variables:
•
Efficiency analysis:
o
Recurrence probability.
o
Time to recurrence.
o
Good/bad evolution of the patient.
o
Overall survival.
o
Analysis by subgroups.
•
Security analysis:
o
Number of Adverse Events per randomization group.
o
Number of patients within each Adverse Event per randomization group.
o
Type of Adverse Events by randomization group.
o
Number of Adverse Events of Special Interest by randomization group.
o
Number of patients within each Adverse Event of Special Concern per 
randomization group.


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o
Type of Adverse Events of Special Interest by randomization group.
o
Number of Serious Adverse Events by randomization group.
o
Type of Serious Adverse Events by randomization group.
o
Adverse events related to treatment.
o
Severity of Adverse Events.
o
Adverse Events in relation to ICD.
o
Mortality associated with CDI.
o
Admissions to the Intensive Care Unit (ICU).
o
Analysis of concomitant medication:
▪
Number of drugs per randomization group.
▪
Number of medications per patient.
▪
Type of drugs per randomization group and per patient.
•
Analysis of quality of life: Analysis of the scores of the areas evaluated by the SF-36 
questionnaire.
This analysis will be versioned as version 2.0. If justified modifications are required, it will be 
versioned as 2.1, 2.2 and so on.
2.9
Subgroup analysis.
The analysis of the efficacy variables will be studied in the following subgroups:
-
Patients with primary episode of CDI.
-
Patients with recurrent episodes of CDI.
-
Patients not receiving prior vancomycin.
The analysis of the probability of recurrence and good/bad evolution of the patients will be 
performed by binary logistic regression analysis. The odds ratio of recurrence will be reported, 
using the MBK-01 group as reference. In addition, the confidence interval, Nagelkerke's R-
squared and Yule's Q coefficient will be included, as well as the critical level.
Time to recurrence will be studied using Cox regression procedures.
2.10 Deviations from the SAP.
Any deviation from the original SAP must be duly described and justified in the statistical 
report.


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3. Summary of statistical analysis.
The main objective of the study is to evaluate the efficacy of the investigational product (MBK-
01) compared to the control (Fidaxomicin) at 8 weeks after initiation of treatment.
Secondary objectives involve assessing the safety of the investigational product and the quality 
of life of the patients participating in the study.
Summary of statistical analysis.
Overall 
objectiv
es
Specific 
objectives
Independent 
variable
Operationalization 
and level of 
measurement of 
the independent 
variable in
the database
Dependent 
variable
Operationalization 
and level of 
measurement of 
the dependent 
variable in the
database
Determination of 
adverse events, 
their degree, and 
relationship with 
the product 
under 
investigation.
Randomization 
group
RANDGRP. Qualitative
AA.
*An active search 
for potential AESEV 
AAs will be 
conducted.
AEREL
Determination of 
serious adverse 
events, their 
severity, and 
relationship to 
the product in
research.
Randomization 
group
RANDGRP. Qualitative
Presence of AAG.
AESER. 
AESEV 
AEREL
Determination of 
adverse events in 
relation to the 
ICD.
Randomization 
group
RANDGRP. Qualitative
Presence of AA in 
relation to ICD.
*An active search 
will be made for 
possible AAs 
related to the ICD.
Determination of 
adverse events of 
special interest, 
their degree, and 
relationship with 
the product in
research.
Randomization 
group
RANDGRP. Qualitative
Presence of AESI.
*An active search 
for potential AESIs 
will be conducted.
Security
ICU admissions
Randomization 
group
RANDGRP. Qualitative
ICU admissions
*An active search will 
be made for those 
AEs/SAEs involving 
admission to the ICU.


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Summary of statistical analysis.
Overall 
objectiv
es
Specific 
objectives
Independen
t variable
Operationalization 
and level of 
measurement of 
the independent 
variable in
the database
Dependent 
variable
Operationalization and level 
of measurement of the 
dependent variable in the 
database
Resolution of the
diarrhea episode
Group of
randomization
RANDGRP. Qualitative
Resolution of the
diarrhea episode
RESOL_FAYN
Good/Bad 
evolution of
patient
Randomization 
group
RANDGRP. Qualitative
Good/Bad 
evolution of
patient
EVAL_RS
Time to 
recurrence
Randomization 
group
RANDGRP. Qualitative
Presence of 
recurrence 
Date of 
recurrence
recurrence
RECU_RSYN 
RECU_RSDAT
Overall survival
Randomization 
group
RANDGRP. Qualitative
Exitus
End date of study 
Exit date
*For calculating the time 
variable
TRTDEC
TRTDECDAT
Efficiency
Quality of life 
variables
Randomization 
group
RANDGRP. Qualitative
Dimensions of quality 
of life
*For calculating each 
dimension based on the 
answers.
to the variables


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ICD-01. Statistical Analysis Plan v3.0 (06/10/2023)
Annex I SOP 6002 - Approved on 30-03-2022
4. References.
Brunner E, Domhof S, Langer F (2002). Nonparametric Analysis of longitudinal Data in Factorial 
Experiments. John Wiley & Sons, New York.
Chen, S.-Y., Feng, Z., & Yi, X. (2017). A general introduction to adjustment for multiple 
comparisons. 
Journal 
of 
Thoracic 
Disease, 
9(6), 
1725-1729. 
doi:10.21037/jtd.2017.05.34.
Del Re, A. C. (2013). compute.es: Compute Effect Sizes. R package version 0.2-2. URL 
http://cran.r-project.org/web/packages/compute.es.
Grothendieck, G. (2017). sqldf: Manipulate R Data Frames Using SQL. R package version 0.4-11. 
https://CRAN.R-project.org/package=sqldf.
Kassambara, K. (2020). ggpubr: 'ggplot2' Based Publication Ready Plots. R package version 
0.4.0. https://CRAN.R-project.org/package=ggpubr.
Kassambara, A., Kosinski, M. & Biecek, P. (2021). survminer: Drawing Survival Curves using 
'ggplot2'. R package version 0.4.9. URL https://CRAN.R-project.org/package=survminer.
Navarro, D. J. (2015) Learning statistics with R: A tutorial for psychology students and other 
beginners. (Version 0.5) University of Adelaide. Adelaide, Australia.
Noguchi, K., Gel Y. R., Brunner, E., and Konietschke, F. (2012). nparLD: An R Software Package 
for the Nonparametric Analysis of Longitudinal Data in Factorial Experiments. Journal 
of Statistical Software, 50(12), 1-23. URL http://www.jstatsoft.org/v50/i12/.
R Core Team (2023). R: A language and environment for statistical computing. R Foundation for 
Statistical Computing, Vienna, Austria. URL https://www.R-project.org/.
Therneau T (2021). A Package for Survival Analysis in R. R package version 3.2-13, URL 
https://CRAN.R-project.org/package=survival.
Terry M. Therneau, Patricia M. Grambsch (2000). Modeling Survival Data: Extending the Cox 
Model. Springer, New York. ISBN 0-387-98784-3.
Warnes, G. R., Bolker, B., Lumley, T., & Johnson, R. C. (2018). gmodels: Various R Programming 
Tools 
for 
Model 
Fitting. 
R 
package 
version 
2.18.1. 
https://CRAN.R- 
project.org/package=gmodels.
Vilagut, G., M. Ferrer, L. Rajmil, P. Rebollo, G. Permanyer-Miralda, J. M. Quintana, R. Santed, J.
M. Valderas, A. Ribera, A. Domingo-Salvany, and J. Alonso. "[the Spanish Version of the


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