Evaluation of fecal microbiota, live (RBL) for the prevention of recurrent Clostridioides difficile infection: Canadian subgroup analysis of an open-label phase 3 study

Abstract

Background

Fecal microbiota, live (RBL) is a single-dose, broad consortium, microbiota-based product approved in the United States and Canada for preventing recurrent Clostridioides difficile (C. diff) infection in adults following standard-of-care (SOC) treatment.

Methods

PUNCH CD3-OLS was a phase 3, open-label, multicenter study evaluating safety and effectiveness of RBL in adults, including those with comorbidities, with recurrent C. diff infection. Participants were aged ≥18 years, had recurrent C. diff infection (≥1 recurrence/≥2 severe episodes; diagnostic method was site-specific), and received SOC antibiotics before rectal administration of RBL. The primary endpoint was treatment-emergent adverse events (TEAEs) through 6 months post-administration. Secondary endpoints included treatment success at 8 weeks and sustained clinical response at 6 months. This subgroup analysis examined outcomes among Canadian participants across 5 sites.

Results

Among 117 Canadian participants who received RBL, most were female and White, with a mean age of 61.3 years; 24.8% reported a first recurrence. The enrolling C. diff infection was diagnosed via polymerase chain reaction for 70.9% of participants. TEAEs, reported in 59.8% of participants, were mostly mild-to-moderate gastrointestinal events. Serious TEAEs occurred in 5.1% of participants within 8 weeks and 6.8% between 8 weeks and 6 months. Treatment success was achieved by 75.2% of participants at 8 weeks, among whom 90.9% maintained a sustained clinical response at 6 months.

Conclusions

Safety and effectiveness outcomes in Canadian participants were consistent with those observed in the PUNCH CD3 randomized controlled trial population, supporting the use of RBL for prevention of recurrent C. diff infection in Canada.

ClinicalTrials.gov identifier

NCT03931941.

Introduction

Clostridioides difficile (C. diff) infection is the leading cause of healthcare-associated infectious diarrhea, resulting in significant morbidity and healthcare costs.¹ In Canada, healthcare-associated C. diff infection rates range between 3.57 and 3.95 infections per 10 000 patient-days, with a 30-day attributable mortality rate between 1.1% and 2.7%.² Recurrent C. diff infection remains a major challenge, with each recurrence increasing the risk of future episodes and leading to higher morbidity and treatment costs.¹ A 2021 literature review reported a median recurrence rate of 23.7% in Canada, the highest observed globally.¹ Factors that increase the risk for recurrent C. diff infection include antibiotic use, hospital admission, age ≥65 years, and underlying comorbidities.³ Antibiotic therapy with vancomycin remains one of the standard-of-care (SOC) treatments for both primary and recurrent C. diff infection,⁴ but the broad-spectrum activity of vancomycin within the intestinal lumen exacerbates gut microbiota dysbiosis and contributes to recurrence.^5–8 Fidaxomicin exhibits greater selectivity and reduces rates of recurrence by approximately 50%, but its recommended usage as the first-line treatment of C. diff infection is not widespread due to high cost and lack of insurance coverage.⁴ Prescribing this more selective antibiotic following treatment of several episodes of C. diff infection with vancomycin may reduce the risk of recurrence; however, the protective effect of this narrow-spectrum antibiotic is attenuated as the microbes to be protected are already diminished.^7,8

Microbiota-based products represent an alternative approach for preventing recurrent C. diff infection by restoring a healthy gut microbiota composition.^1,5 Fecal microbiota, live (REBYOTA, abbreviated RBL), is a single-dose, broad consortium, rectally administered product used to prevent recurrent C. diff infection following SOC antibiotics.⁹ Following administration of RBL, increases in microbial diversity and shifts in Bacteroidia and Clostridia have been observed, along with increases in secondary bile acids that inhibit C. diff spore germination and vegetative growth.¹⁰ Clonal engraftment of donor-derived strains following RBL administration has also been associated with restoration of microbiota composition from dysbiosis toward healthy patterns, supporting its role in preventing recurrent infection.¹¹ RBL was initially approved in the United States by the Food and Drug Administration (FDA) as the first microbiota-based product for recurrent C. diff infection prevention.⁹ RBL was approved by Health Canada on March 5, 2025, marking an important milestone for microbiota-based therapies in Canada.¹² This approval was based on the pivotal randomized placebo-controlled PUNCH CD3 trial, which assessed RBL in a selectively defined patient population, providing a controlled efficacy benchmark for comparison with outcomes observed in broader, real-world populations.¹³ In PUNCH CD3, higher treatment success was observed in the RBL group (70.6%) than in the placebo group (57.5%), and RBL demonstrated a favourable safety profile, with mostly mild-to-moderate adverse events reported.

PUNCH CD3-OLS is the largest study to date that evaluated the safety and effectiveness of RBL in preventing recurrent C. diff infection across adults with recurrent C. diff infection in the United States and Canada.⁵ Previous prospective, double-blinded, randomized studies of RBL excluded participants with comorbidities such as inflammatory bowel disease (IBD; Crohn’s disease and ulcerative colitis) and immunocompromising conditions.^13–16 PUNCH CD3-OLS included these participants, making the study population more representative of a real-world clinical setting.⁵ In PUNCH CD3-OLS, 47.3% of participants reported RBL- or administration-related treatment-emergent adverse events (TEAEs) within 8 weeks of RBL administration. Most TEAEs were mild or moderate in severity. The treatment success rate at 8 weeks was 73.8%; of participants with treatment success, 91.0% had a sustained clinical response through 6 months after RBL administration. With the approval of RBL in Canada, this subgroup analysis focuses on the Canadian participants in PUNCH CD3-OLS, providing insights into regional safety and effectiveness outcomes.

Methods

Study design

PUNCH CD3-OLS was a phase 3, open-label, single-arm, prospective study designed to evaluate the safety and effectiveness of RBL for preventing recurrent C. diff infection in adults with common comorbidities following SOC treatment. Each site obtained approval of the protocol from its institutional review board/research ethics board. Detailed information on the institutional review board/research ethics board has been published previously.⁵ The study was conducted in accordance with the ethical standards of the 1964 Declaration of Helsinki and received a Health Canada No Objection Letter. Prior to initiation of any study procedures, all participants provided informed consent to participate. Additional study design details and statistical analysis methods have been previously published.⁵

Study population

PUNCH CD3-OLS enrolled adults aged 18 years or older in the United States (46 study sites) and Canada (5 study sites) with a history of at least one recurrence of C. diff infection confirmed by the treating physician or ≥2 episodes of severe C. diff infection resulting in hospitalization.⁵ The method for recurrent C. diff infection diagnosis was not prespecified and the treating physicians could use methods customary to the study sites. These methods included polymerase chain reactions (PCR) and enzyme immunoassays for C. diff glutamate dehydrogenase antigen and toxins A and B. Exclusion criteria included those with continued C. diff infection diarrhea despite antibiotic treatment, prior participation in a clinical study evaluating RBL, and the use of antibiotics for conditions other than C. diff infection through the 8-week follow-up assessment. Full inclusion/exclusion criteria have been published previously.⁵

Participants received SOC antibiotics for recurrent C. diff infection before undergoing rectal administration of a single dose of RBL within 24-72 hours of antibiotic completion. Participants were eligible to receive a second course of RBL within 21 days if they met the criteria for treatment failure. Treatment failure was defined as the return of diarrhea due to C. diff infection within 8 weeks of RBL administration. Antibiotic treatment before a second course of RBL was at the discretion of the treating physician; however, if antibiotics were given to control symptoms, a 24- to 72-hour washout period was required prior to administration of RBL.

Study outcomes

The primary endpoint was the incidence of TEAEs, including severity and relatedness to RBL, through 6 months after administration. The secondary endpoints were treatment success at 8 weeks, defined as the absence of C. diff infection (defined as ≥3 unformed stools in ≤24 hours for at least 2 consecutive days, and a positive stool test result for C. diff as determined by the central laboratory), and sustained clinical response at 6 months among those who achieved treatment success. The safety and effectiveness outcomes of Canadian participants were compared with those of the US study population.

Statistical analysis

Descriptive statistics are reported for all outcomes because PUNCH CD3-OLS was not designed to statistically test hypotheses in this exploratory subgroup analysis. Safety analyses were performed on the safety population, which included all participants who received RBL. Effectiveness analyses were based on the modified intent-to-treat population, defined as participants who received RBL but excluded those who did not complete the study or discontinued before treatment success or failure could be evaluated. Two-sided 95% confidence intervals (CI) were calculated for effectiveness outcomes using the Clopper-Pearson method.

Results

Participant demographics and baseline characteristics

Of the 697 participants in PUNCH CD3-OLS, 117 Canadian participants and 580 US participants received RBL. Baseline characteristics and demographics were similar between Canadian and US participants. The mean (SD) age of Canadian participants was 61.3 (17.4) years and of US participants was 61.1 (17.3) years (Table S1). Approximately half of the Canadian (49.6% [58/117]) and US (48.3% [280/580]) participants were aged 65 years or older. The majority of Canadian and US participants were female, accounting for 70.1% (82/117) and 69.8% (405/580) of the subgroups, respectively. Most participants in the Canadian and US cohorts identified as White (91.5% [107/117] and 94.3% [547/580], respectively). Only 24.8% (29/117) of Canadian participants and 27.1% (157/580) of US participants received RBL for their first recurrence (ie, second C. diff infection episode), while 75.2% (88/117) and 72.2% (419/580), respectively, had multiple recurrences (ie, ≥3 C. diff infection episodes). The enrolling C. diff infection episode was diagnosed via PCR for 70.9% (83/117) of Canadian participants and 60.2% (349/580) of US participants. Vancomycin was the most commonly used antibiotic for the enrolling C. diff infection in both Canadian (86.3% [101/117]) and US (84.7% [491/580]) participants. Finally, 17.1% (20/117) of Canadian participants and 10.3% (60/580) of US participants were hospitalized for their qualifying C. diff infection episode.

Safety

Treatment-emergent adverse events were reported in 59.8% (70/117) of Canadian participants and 44.8% (260/580) of US participants within 8 weeks of RBL administration (Table 1). The majority of TEAEs occurred within the first 8 weeks following RBL administration, with a lower incidence reported between 8 weeks and 6 months (Canadian participants: 29.9% [35/117]; US participants: 21.6% [125/580]) (Table 1). Most TEAEs were mild to moderate in severity (Table 1), with 48.7% (57/117) in Canadian participants and 25.5% (148/580) in US participants being gastrointestinal in nature (Table S2). The most frequently reported gastrointestinal TEAEs included diarrhea, abdominal pain, nausea, and abdominal distension. TEAEs related to infections were assessed during the safety review process and determined to be unrelated to RBL. Serious TEAEs occurred in 5.1% (6/117) of Canadian participants and 3.6% (21/580) of US participants within the first 8 weeks following administration (Table 1). Between 8 weeks and 6 months, serious TEAEs were reported in 6.8% (8/117) of Canadian participants and 2.9% (17/580) of US participants. The majority of serious TEAEs were related to preexisting conditions.

Table 1.

Summary of TEAEs within 8 weeks and between 8 weeks and 6 months follow-up of RBL administration (safety population).

Participants, n (%)	Within 8 weeks (N = 697)		8 weeks-6 months (N = 697)
	Canada	United States	Canada	United States
	n = 117	n = 580	n = 117	n = 580
Any TEAE	70 (59.8)	260 (44.8)	35 (29.9)	125 (21.6)
TEAE by severity
Mild	21 (17.9)	121 (20.9)	5 (4.3)	46 (7.9)
Moderate	34 (29.1)	104 (17.9)	23 (19.7)	56 (9.7)
Severe	14 (12.0)	32 (5.5)	5 (4.3)	21 (3.6)
Potentially life-threatening	1 (0.9)	3 (0.5)	2 (1.7)	2 (0.3)
Any serious TEAE	6 (5.1)	21 (3.6)	8 (6.8)	17 (2.9)
Serious TEAE by relatednessa
Related to RBL	1 (0.9)b	0	0	0
Related to administration procedure	0	0	0	0
Related to C. diff infection	0	1 (0.2)	0	1 (0.2)
Related to a preexisting condition	5 (4.3)	14 (2.4)	7 (6.0)	11 (1.9)
TEAEs leading to withdrawal from study	0	4 (0.7)	1 (0.9)	0
TEAEs leading to death	0	3 (0.5)	1 (0.9)	0

Abbreviations: C. diff infection, Clostridioides difficile infection; RBL, fecal microbiota, live; TEAE, treatment-emergent adverse event.

^aRelatedness categories are not mutually exclusive and may have included more than one relatedness category (eg, possibly related to C. diff infection, definitely related to a preexisting condition).

^bA flare of preexisting ulcerative colitis (assessed as definitely related to a preexisting condition and possibly related to RBL).²⁶

The safety profile observed in Canadian participants was similar to that of the US population. Although the proportion of Canadian participants experiencing TEAEs was slightly higher than in the US cohort, the types and severity of adverse events remained consistent across both groups. No safety concerns were identified by the medical monitor, data safety monitoring board, the FDA, or Health Canada.

Effectiveness

Treatment success at 8 weeks was achieved by 75.2% (88/117; 95% CI, 66.4%-82.7%) of Canadian participants and 73.5% (411/559; 95% CI, 69.7%-77.1%) of US participants (Figure 1). Among participants who achieved treatment success, 90.9% (80/88; 95% CI, 82.9%-96.0%) of Canadian participants and 91.0% (374/411; 95% CI, 87.8%-93.6%) of US participants maintained a sustained clinical response at 6 months.

Figure 1.

Eight-week treatment success and 6-month sustained clinical response (mITT population). CI, confidence interval; mITT, modified intent-to-treat. ^aTreatment success was defined as the absence of C. diff infection diarrhea through 8 weeks after RBL administration. ^bEffectiveness outcomes were indeterminate if the C. diff infection test was inconclusive or missed at time of visit. ^cSustained clinical response was defined as the treatment success of the presenting C. diff infection and no new C. diff infection through 6 months.

Discussion

PUNCH CD3-OLS, the largest open-label study to date of a broad-consortium microbiota-based product, evaluated the safety and effectiveness of RBL in a real-world population that included individuals with comorbidities that are typically excluded from clinical trials.⁵ This subgroup analysis of PUNCH CD3-OLS aimed to assess the safety and effectiveness outcomes of RBL in Canadian participants to provide insights into the integration of RBL into Canadian clinical practice.

The results of this Canadian subgroup analysis of PUNCH CD3-OLS are consistent with outcomes of other studies in the RBL clinical development program. No new safety signals were identified, and serious TEAEs were infrequent in Canadian participants in PUNCH CD3-OLS. While the proportion of Canadian participants in PUNCH CD3-OLS experiencing gastrointestinal TEAEs was higher than in the US cohort, this difference may reflect the smaller Canadian subgroup size, the limited number of Canadian study sites, and possible site-level differences in reporting practices. Additionally, a higher proportion of Canadian participants were hospitalized for their qualifying C. diff infection episode, which may indicate differences in baseline disease severity and contribute to the TEAEs reported following administration of RBL. Importantly, the type and severity of adverse events remained consistent across both groups, with most TEAEs being mild to moderate in severity. These results are consistent with those reported in the more selectively defined PUNCH CD3 randomized trial, where most TEAEs were mild or moderate in severity, and serious TEAEs occurred at rates similar to those observed in the Canadian subgroup of PUNCH CD3-OLS.¹³ Safety findings from an integrated analysis of the entire RBL clinical trial program comprising 1061 participants across 3 phase 2 (PUNCH CD, PUNCH CD2, and PUNCH Open-Label) and 2 phase 3 (PUNCH CD3 and PUNCH CD3-OLS) prospective trials similarly showed that most TEAEs were mild to moderate in severity, with no evidence of transmission of infections related to RBL.^17,18

The effectiveness of RBL in this Canadian subgroup analysis of PUNCH CD3-OLS was demonstrated by the treatment success rate at 8 weeks and sustained clinical response through 6 months. Similar rates of treatment success and sustained clinical response were reported for the Canadian and the overall study population in PUNCH CD3-OLS.⁵ These treatment success rates were numerically higher than the 70.6% treatment success rate reported in the pivotal phase 3 randomized controlled trial PUNCH CD3, which evaluated RBL with stringent criteria that excluded participants with comorbidities such as IBD and immunosuppression.¹³ The results of this subgroup analysis therefore suggest RBL may be used in preventing recurrent C. diff infection in a broad Canadian patient population.

Several other oral microbiota-based products for preventing recurrent C. diff infection, including donor-derived fecal microbiota spores, live-brpk (SER-109), full spectrum donor stool-based CP101, and the live biotherapeutic product VE303, have also demonstrated effectiveness in preventing recurrent C. diff infection.¹⁹ While several of these studies enrolled Canadian participants, subgroup outcomes specific to these participants were not available.^20,21 The effectiveness outcome observed with RBL in Canadian participants was comparable to that reported for CP101 in the overall study population.²² Although higher treatment success rates have been reported for SER-109 and VE303, sustained clinical responses through 6 months were similar between RBL and these therapies.^21,23 While multiple microbiota-based products may be effective in preventing recurrent C. diff infection, RBL is currently the only Health Canada-approved product. RBL offers a distinct approach through its standardized, broad-consortium formulation delivered directly to the colon via rectal administration. Another advantage is that this route of delivery can be offered to those with dysphagia. As RBL is administered rectally, in-clinic administration by trained healthcare personnel is required to ensure appropriate delivery compared with oral microbiota-based products.

Health Canada’s approval of RBL is a critical step toward making microbiota-based therapies accessible to Canadian patients.¹² Access to fecal microbiota transplantation in Canada remains challenging in most provinces, as it has been made available via a small group of committed academic clinicians at several tertiary care hospitals, primarily on a research basis, without formal provincial or territorial government support.^24,25 Over 30% of Canadian and US participants in PUNCH CD3-OLS had 4 or more episodes of C. diff infection before the administration of RBL. Therefore, this approval fills an important gap in recurrent C. diff infection prevention, offering an alternative to repeated use of antibiotics, which do not address the underlying dysbiosis. With RBL now available in Canada,¹² future real-world studies and practice assessments can capture clinical safety, effectiveness, and integration into Canadian healthcare settings. The findings from PUNCH CD3-OLS support the use of RBL in a broad range of patients, including those with comorbidities such as IBD and immunocompromising conditions.^5,26,27 The results also provide reassurance to Canadian clinicians, reinforcing the safety and effectiveness of RBL in a patient population reflective of real-world clinical practice.

This subgroup analysis has several limitations. The predominantly White population (>90%) may limit the generalizability of findings within this relatively small Canadian subgroup. Furthermore, the number of participants with comorbid conditions in the Canadian population was limited; therefore, generalizability of these findings to patients with underlying comorbidities should be interpreted with caution. TEAEs may have been related to recurrent C. diff infection and/or preexisting conditions rather than RBL, making it difficult to determine causality for these events. Despite these limitations, the study has notable strengths. The inclusion of a broader patient population with comorbidities in PUNCH CD3-OLS better reflects real-world practice. PUNCH CD3-OLS remains the largest study of a microbiota-based therapy conducted to date, further supporting the safety and effectiveness data of RBL in Canadian patients.

Conclusion

RBL was well tolerated and effective in preventing recurrent C. diff infection among Canadian participants in PUNCH CD3-OLS. The safety and effectiveness outcomes in the Canadian subgroup were comparable to those observed in the pivotal PUNCH CD3 study population.^5,13 The findings from this subgroup analysis may support the integration of RBL into Canadian clinical practice as a safe and effective prevention strategy for patients with recurrent C. diff infection. Further research, such as real-world effectiveness studies, may help to elucidate long-term outcomes and inform optimal implementation within Canadian healthcare settings.

Author contributions

All authors contributed to the conceptualization, methodology, and investigation of the study. S.S. conducted the formal analysis. All authors were involved in drafting, reviewing, and editing the manuscript, and approved the final version for submission.

Supplementary material

Supplementary material is available at Journal of the Canadian Association of Gastroenterology online.

Funding

This study was sponsored by Ferring Pharmaceuticals.

Conflicts of interest

Conflict of interest disclosure forms (ICMJE) have been collected for all co-authors and can be accessed as supplementary material.

Data availability

The datasets generated and/or analyzed during the current study are not publicly available but may be available from the corresponding author on reasonable request.

Ethics approval and consent to participate

PUNCH CD3-OLS was conducted across the United States and Canada under a US FDA Investigational New Drug Application and a Health Canada Clinical Trial Application. Each site obtained approval of the protocol from its institutional review board/research ethics board. Detailed information on the institutional review board/research ethics board is available in the previously published PUNCH CD3-OLS publication. The study was conducted in accordance with the ethical standards of the 1964 Declaration of Helsinki and received a Health Canada No Objection Letter. Prior to initiation of any study procedures, all participants provided informed consent to participate.

Consent for publication

Not applicable.

Medical writing, editorial, and other assistance

Medical writing support, under the guidance of the authors, was provided by Mariska Hattingh, PhD, Paul Sobol, PhD, and Michelle Boland, PhD (ApotheCom, Yardley, PA, USA), and was funded by Ferring Pharmaceuticals, Parsippany, NJ, USA.