Patients with abnormalities of the biliary tract (the system that connects the liver to the small intestine and allows bile to flow) are prone to the development of strictures, dilations, or other problems that impede bile flow and promote the formation of gallstones. All of this leads to the development of infections in this anatomical region known as cholangitis. These infections typically require the patient to be admitted to the hospital, either to a general ward or even the ICU. These are, therefore, serious infections that threaten the patient's survival. Unfortunately, one-quarter of these patients experience not just one, but multiple infections of this type over an extended period of time. This condition is called acute recurrent cholangitis (ARC).

It is important to note that the antibiotics used to treat these repeated infections lead to the selection of more resistant and virulent bacteria that remain in the intestine for months or years and, in turn, promote the recurrence of these infectious episodes. ARC affects a more vulnerable population, which, combined with the proliferation of resistant bacteria, increases the risk of serious complications and a reduced response to available treatments.

Replacing these dangerous gut bacteria with bacteria from healthy donors is a strategy that has proven effective in reducing recurrent urinary tract infections. A similar approach has been successful in patients with chronic vaginal infection (vaginosis). This gut microbiota replacement is currently being investigated for a wide range of diseases affecting various sites, with promising results in some cases. There is strong evidence that this strategy may also be effective in patients with recurrent episodes of cholangitis.

The procedure is considered very safe and is subject to strict safety measures to prevent risks to the recipient of these bacteria (requirements similar to those used for blood transfusions). If this strategy proves effective, it could reduce patient suffering and even mortality, as well as save money spent on hospital stays and medications, and contribute to reducing antibiotic use and the emergence of bacterial resistance. Even if no significant clinical benefit is demonstrated in this clinical setting, the information obtained will contribute to increasing knowledge about the role of the fecal microbiota, the technical and scientific aspects of fecal microbiota transplantation, and its potential uses. It should be noted that a patient association called ALBI España (Association for the Fight Against Inflammatory Biliary Diseases) has participated in the study's design and strongly supports this research, which could lead to a reduction in mortality among its patients and a reduction in adverse effects and costs associated with hospital admissions and antibiotic use.

In summary, the use of FMT in patients with AC could have a significant impact on treatment effectiveness, reducing new episodes of cholangitis. This would improve patients' quality of life-including their physical, emotional, and social well-being-by reducing hospitalizations and associated complications. Another clear benefit would be the reduction in the proliferation of resistant bacteria and a lower use of antibiotics. The use of TMF would increase the autonomy of the National Health System (SNS) by enabling it to develop and manage non-commercial, non-profit therapies with low production costs, which would guarantee equitable access for patients to an effective and safe therapy.

Hypothesis FMT reconstitutes the physiological biodiversity of the intestinal microbiota by reducing colonization by pathogenic bacteria capable of ascending invasion of the biliary tract, thereby reducing the risk of new episodes of non-obstructive RAC compared to conventional management (TASC, intestinal decontamination, or individual episode treatment).

Objectives Primary To assess whether the time to the next episode of acute cholangitis is longer in patients with non-obstructive RAC treated with FMT compared to those receiving conventional treatment.

Secondary

To evaluate and compare the effectiveness of FMT in reducing the number of new episodes of acute cholangitis in patients with non-obstructive RAC compared to conventional treatment over 24 months.

To determine whether FMT reduces intestinal colonization by resistant bacteria present at the index episode of cholangitis.

To confirm the feasibility and safety of FMT using a model of healthy stool and blood donors (quarantined), whose release would take place after repeated negative results on microbiological testing for potentially transmissible infections.

Methods Design: Open-label, randomized phase II clinical trial. Participating institutions: Multicentre. See feasibility/work plan section.

Inclusion criteria

• Patients ≥18 years of age who have had ≥2 episodes of acute cholangitis in the past year.

Exclusion criteria

• Oral intolerance or inability to swallow capsules.
• Pregnancy or breastfeeding.
• Any clinically significant disease (other than RAC) such as disseminated malignancy or organ failure resulting in relevant cardiac, hepatic, pulmonary, or neurodegenerative disease with an expected survival of less than one year.
• Inability to understand the study, sign the informed consent, and/or collect stool samples.
• RAC due to the presence of choledocholithiasis (any number of stones in the biliary tract) or extrahepatic bile duct stricture that cannot be resolved by surgical or endoscopic treatment. Lack of resolution of extrahepatic biliary obstruction is defined as failure to remove all stones, or a residual diameter in stricture cases of less than 75% of the bile duct diameter above and below the stricture, even after balloon dilation and/or biliary stent placement

Intervention Patients will be randomized to receive FMT plus standard treatment (ST) (intervention group) or ST alone (control group).

In both groups, the time elapsed from randomization to the next episode of acute cholangitis (primary endpoint) will be assessed over a 24-month follow-up period.

If a new episode of acute cholangitis occurs in the intervention group during the follow-up period, empirical and/or targeted antibiotic treatment will be administered at the discretion of the medical team, along with a second FMT dose. In the event of a subsequent episode (2nd recurrence), no further FMT will be administered and ST will be continued.

If a new episode of cholangitis occurs in a control group patient, FMT will be offered following the same criteria as in the intervention group in the event of a second episode of cholangitis after FMT. The effect of FMT in control group patients will be studied in a separate analysis from the primary objective. Any surgical or endoscopic procedure on the biliary tract that may reduce the risk of new cholangitis episodes will be taken into account in the analysis.

Description of FMT

To be selected, donors will provide informed consent and will be evaluated through a general medical history, specific digestive and infectious background, lifestyle and risk travel history, and laboratory assessment. They must meet the following criteria:

Normal daily bowel movements. No antibiotic use in the past 6 months. No history of chronic digestive disease, including inflammatory bowel disease. No chronic diseases of any kind, including autoimmune, inflammatory, degenerative, or neoplastic conditions.

Clinically stable.

To ensure patient safety, previously established recommendations will be followed. Donor stool and blood will be tested for pathogens in accordance with local protocols and European guidelines (31).

Serological testing will include hepatitis A, B, and C; HIV-1 and HIV-2; syphilis, CMV, EBV, HTLV-1/2, and toxoplasmosis. Stool analysis will include stool culture for enteric bacterial pathogens, techniques to detect multidrug-resistant bacteria, detection of Norovirus, Adenovirus, or Rotavirus, C. difficile, parasite antigens (Giardia and Cryptosporidium), and microscopy with rapid staining for protozoa from three samples collected on alternate days, with an extended panel for immunosuppressed patients (28, 31). After an initial set of microbiological tests with negative results, the capsules will be stored. They will be released for administration after a 3-month quarantine period with newly confirmed negative microbiological results (29).

FMT capsules will be prepared starting from 100 g of stool, to which 1/5 volume of normal saline (NaCl 0.9%) will be added. After allowing the sample to hydrate for 30 minutes at room temperature, it will be homogenized in a Stomacher (230 rpm). Once sufficiently homogeneous, 30% glycerol will be added and solid residues will be removed by gentle centrifugation (400 × g for 20 minutes). A second centrifugation of the supernatant will be performed at higher speed (1000 × g for 30 minutes). The pellet from this second centrifugation will be frozen at -80°C for at least 1 hour, then lyophilized at -50°C for 18-20 hours. The resulting powder will be encapsulated in enteric-coated capsules, prepared in batches and stored in the absence of moisture at 4°C, with a shelf life of 6 months. An aliquot from all processing steps will be stored at -80°C, and sample and donor traceability will be maintained in a dedicated REDCap database.

Sample Size It is assumed that the 12-month survival function for the next episode of cholangitis in the ST arm is 0.10, and that the survival function in the FMT group is 0.50. With a 1:1 allocation, a 12-month recruitment period, 24-month follow-up, an alpha of 0.05, 80% statistical power, a one-sided hypothesis, and using the Tarone-Ware rank test, 17 patients per group (34 total) would be required. Under these assumptions, 17 events are expected in the ST group and 22 in the FMT group.

Competing events may occur during follow-up that prevent observation of the event of interest (loss to follow-up, patient death, or a surgical or endoscopic procedure modifying the risk of RAC). Although unresolved bile duct obstruction or stricture is an exclusion criterion, we define as a competing event any biliary intervention for potential complications not present at randomization but that may arise subsequently, such as biliary stent obstruction (resulting in an estimated stricture exceeding 25%) due to stone formation. Competing events will be addressed using the cause-specific hazard function (CSH). This approach is recommended for evaluating associations in etiological studies, and competing events are treated as censored observations. Assuming a 20% dropout rate, a total of 44 patients will be recruited (22 per arm).

Randomization A 1:1 randomization will be performed, stratified by center using variable block permutations of 2 and 4. Assignment will be centralized and conducted electronically via REDCap.

Clinical Follow-up Direct telephone contact will be maintained with study participants in the event of any incident related to the project. If a patient develops any symptoms of a new episode of acute cholangitis, they will present to the emergency department, where additional tests deemed necessary by the medical team will be performed, and a stool culture will be obtained to analyze possible causes of FMT failure in that participant.

The study protocol will include the follow-up visits to be conducted during the 24-month follow-up period. In both groups, the time elapsed from randomization to the next episode of acute cholangitis (primary endpoint) will be assessed.

Stool samples will be collected from all patients before randomization to detect the presence of resistant bacteria and to study bacterial quantity and diversity. Subsequent samples will be collected at one week, one month, and three months after randomization. The presence of resistant strains in the recipient will be studied at the originating hospital using stool culture and standard techniques for detection of extended-spectrum beta-lactamase-producing Enterobacteriaceae, carbapenemase-producing gram-negative bacilli, and vancomycin-resistant enterococci.

Outcome Variables

Primary efficacy endpoint:

Time to the next episode of cholangitis from patient randomization. Patients who do not experience cholangitis or other competing events will be censored at 24 months from study initiation. Competing events will be treated as censored observations in order to estimate the cause-specific hazard function using Cox regression.

Secondary efficacy endpoints:

Number of RAC episodes based on medical diagnosis over the following 24 months. Recurrences will be diagnosed based on the patient's symptoms and the results of relevant cultures. This analysis will include patients in the ST arm who received FMT treatment following a cholangitis episode.

Proportion of recurrence-free patients at 6 months and annual recurrence rate (number of recurrences per patient per year).

Secondary safety endpoints:

Incidence of AEs, serious AEs, AEs leading to treatment discontinuation or inability to receive a second FMT dose due to a contraindication at that time, AEs leading to study discontinuation, and AEs causing participant death.

Assessment through stool cultures of the emergence of multidrug-resistant bacteria.

Ethical and Regulatory Aspects The study will be submitted for evaluation and approval by the Research Ethics Committee of the coordinating center (HUPH), both regarding donors and their informed consent, as well as the study itself. A certificate of facility suitability and management approval will be obtained for each participating center, and investigators will notify their center's committees and other structures according to local procedures. Since the approval of the European SoHo Regulation, FMT research is no longer considered investigational medicinal product research but rather research involving substances of human origin; therefore, evaluation and approval will be sought from the ONT (Committee for the Evaluation of Innovation with Human Cells, Tissues, and Derived Products), with notification to the AEMPS for information purposes. In accordance with current requirements for this technology, compliance will be maintained with cell and tissue regulations regarding donors, traceability, and biovigilance, and with applicable ICH guidelines for medicinal products regarding trial conduct (GCP standards) and monitoring of adverse events in participating patients. Data used in the development of this project will be processed in accordance with GDPR 2016/679 and Organic Law 3/2018 on the Protection of Personal Data. Research ethical principles will be followed, including the updated Declaration of Helsinki (2024).

Statistical Analysis Demographic variables will be described using absolute and relative frequencies for categorical variables, and mean and standard deviation or median and 25th-75th percentiles for numerical variables, depending on whether the normality assumption is met. Two analysis populations will be defined: the ITT population (intention-to-treat analysis) and the AT population (as-treated). The ITT population will consider the randomly assigned treatment arm and will record the time to the next RAC episode. In the AT population, the treatment actually received by the patient will be taken into account, and patients in the ST arm who received FMT following a cholangitis episode will be considered as FMT-treated for the secondary analyses. The primary analysis will be conducted in the ITT population, with a secondary analysis in the AT population. The primary analysis will estimate the cause-specific hazard function using Cox regression in the ITT population. Effect size will be assessed using the Hazard Ratio (HR) with its corresponding 95% CI, as the exponentiated coefficients measure the association between the randomized arm and the event rate. The cumulative incidence function (CIF) in each arm will also be estimated with corresponding 95% confidence intervals. The recurrence rate ratio (RRR) for each group will be analyzed using Poisson regression. P-values below 0.05 will be considered statistically significant.

As a complement to the protocol, a Statistical Analysis Plan (SAP) will be developed detailing the statistical techniques and methods to be used. The SAP will be developed and finalized before database lock. Given the nature of the study, no interim analysis for efficacy, safety, or futility is considered justified. However, the SAP will include an interim analysis to assess the number of events before recruitment is completed, in order to evaluate a possible sample size modification.

The SAP and statistical analysis will be conducted within the SCReN Platform using SCReN-established procedures and software: SAS version 9 for Windows or Stata v.18, if performed by the Biostatistics Unit of IIS Puerta de Hierro.

Limitations and Potential Difficulties Although the COLANBIOTA study includes a control group, it does not employ a double-blind design, which may represent a methodological limitation and could introduce bias in the evaluation of treatment efficacy and safety. The decision not to use blinding is based on the nature of FMT itself, which makes it difficult to create an indistinguishable placebo. To mitigate this potential bias, objective variables will be used (occurrence or non-occurrence of a new episode of acute cholangitis) with pre-established criteria. Furthermore, the decision to hospitalize patients for acute cholangitis will be made by personnel independent of the research team. Finally, the protocol ensures that the treatment and follow-up of both arms are pre-specified and identical.

Although the calculated sample size is sufficient to address the hypothesis, it is a small sample that may limit population representativeness, given the heterogeneity and complexity of affected patients. Another theoretical limitation for the generalizability of results relates to the impact of different donors at each center, as well as the manufacturing process used. Standardization of donor selection and manufacturing according to EDQM-established criteria will mitigate this issue.

Distribution of FMT Capsules The adequate storage of lyophilized enteric bacteria capsules at room temperature facilitates their distribution. Delivery to any participating hospital in the study will be carried out within 24-48 hours.

Definitions

Acute cholangitis: acute inflammation of the biliary tract. The patient must meet the Tokyo criteria (27):

A. Systemic inflammation A-1. Fever and/or chills A-2. Laboratory findings: evidence of inflammatory response B. Cholestasis B-1. Jaundice B-2. Laboratory findings: abnormal liver function tests C. Imaging findings C-1. Biliary dilatation C-2. Evidence of etiology on imaging (stricture, stone, stent, etc.) Definite acute cholangitis: one criterion from A + one from B + one from C Suspected acute cholangitis: one criterion from A + one from B or C A-2: Abnormal white blood cell count, elevated serum C-reactive protein levels, and other changes indicating inflammation.

B-2: Elevated serum levels of any of the following: alkaline phosphatase, GGT, ALP, AST, or ALT.

Fever: Axillary or oral temperature ≥ 38°C. Evidence of inflammatory response: leukocyte count <4,000/µL or >10,000/µL, or C-reactive protein >1 mg/dL (or equivalent concentration).

Jaundice: Total bilirubin ≥ 2 mg/dL.

Elevated liver function tests:

Alkaline phosphatase (IU) >1.5 times the upper limit of normal Gamma-glutamyl transpeptidase (IU) >1.5 times the upper limit of normal Aspartate aminotransferase (IU) >1.5 times the upper limit of normal Alanine aminotransferase (IU) >1.5 times the upper limit of normal

Acute cholangitis ruled out: when the clinical findings and ancillary test results can be explained by another cause, such as acute hepatitis, infection at another site, or a condition clearly distinct from acute cholangitis.

Recurrent acute cholangitis: recurrent episodes of acute cholangitis. For the purposes of this study, patients with two or more episodes of acute cholangitis within the past year are considered to have recurrent acute cholangitis.

Non-obstructive recurrent acute cholangitis: includes patients with biliodigestive anastomoses such as hepaticojejunostomy. Also includes cases in which, having had stones in the extrahepatic bile duct or strictures, patients have undergone surgical or endoscopic treatment (including biliary stent placement), with documented removal of all stones and a residual diameter - in cases of stricture - greater than 75% of the bile duct diameter above and below the stricture, following balloon dilation with or without stent implantation.

Obstructive recurrent acute cholangitis: patients with biliary obstruction due to stones or benign or malignant stricture in whom the transverse diameter of the stricture does not exceed 75% of the caliber of the unaffected adjacent bile duct.

Antibiotic-resistant bacteria: resistant bacterial species including extended-spectrum beta-lactamase-producing Enterobacteriaceae, carbapenemase-producing gram-negative bacilli, and vancomycin-resistant enterococci.

Chronic suppressive antibiotic therapy: administration of one or more oral antibiotics on a continuous, alternating, intermittent, or cyclic basis to reduce episodes of RAC.

Inclusion criteria - Patients ≥18 years of age who have had ≥2 episodes of acute cholangitis in the past year.

Exclusion criteria

• Oral intolerance or inability to swallow capsules.
• Pregnancy or breastfeeding.
• Any clinically significant disease (other than RAC) such as disseminated malignancy or organ failure resulting in relevant cardiac, hepatic, pulmonary, or neurodegenerative disease with an expected survival of less than one year.

Inability to understand the study, sign the informed consent, and/or collect stool samples.

- RAC due to the presence of choledocholithiasis (any number of stones in the biliary tract) or extrahepatic bile duct stricture that cannot be resolved by surgical or endoscopic treatment. Lack of resolution of extrahepatic biliary obstruction is defined as failure to remove all stones, or a residual diameter in stricture cases of less than 75% of the bile duct diameter above and below the stricture, even after balloon dilation and/or biliary stent placement

Individual participant data (IPD) underlying the results reported in this study will be shared after appropriate de-identification. The data will be available starting 6 months after publication of the main results and for a period of up to 5 years.

Access to the data will be granted to qualified researchers who provide a methodologically sound research proposal. Requests will be reviewed by the study team and will be subject to the signing of a data use agreement.

Supporting documents, such as the study protocol and statistical analysis plan, will also be made availabl

Individual participant data (IPD) and supporting documents will be available starting 6 months after publication of the primary results and will remain available for up to 5 years.

Access to IPD and supporting information will be granted to qualified researchers who provide a methodologically sound research proposal. Requests will be reviewed by the study investigators. Approved researchers will be required to sign a data use agreement. Data will be shared in a de-identified format to protect participant confidentiality and will be provided via secure transfer methods.