Fecal Microbiota Transplant vs Standard Treatment for Recurrent Non-Obstructive Cholangitis

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

• Number of recurrent acute cholangitis (RAC) episodes [Срок оценки: 24 months]
• Intestinal colonization by resistant bacteria [Срок оценки: Three months]