Fecal Microbiota Transplantation Donor Screening: Is Dientamoeba fragilis a Valid Criterion for Donor Exclusion? A Longitudinal Study of a Swiss Cohort

Fecal Microbiota Transplantation Donor Screening: Is Dientamoeba fragilis a Valid Criterion for Donor Exclusion? 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A Longitudinal Study of a Swiss Cohort View ORCID Profile Keyvan Moser , Aurelie Ballif , View ORCID Profile Trestan Pillonel , Maura Concu , View ORCID Profile Elena Montenegro Borbolla , View ORCID Profile Beatrice Nickel , View ORCID Profile Camille Stampfli , View ORCID Profile Marie-Therese Ruf , View ORCID Profile Maxime Audry , View ORCID Profile Nathalie Kapel , Susanna Gerber , View ORCID Profile Damien Jacot , View ORCID Profile Claire Bertelli , View ORCID Profile Tatiana Galperine doi: https://doi.org/10.1101/2025.10.29.25338826 Keyvan Moser 1 Service of Infectious Diseases, Lausanne University Hospital and University of Lausanne , Lausanne, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Keyvan Moser For correspondence: keyvan.moser{at}hotmail.fr Aurelie Ballif 1 Service of Infectious Diseases, Lausanne University Hospital and University of Lausanne , Lausanne, Switzerland 3 Department of policlinics, Centre for Primary Care and Public Health (Unisanté) , Lausanne; Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site Trestan Pillonel 9 Institute of Microbiology, Lausanne University Hospital and University of Lausanne , Lausanne, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Trestan Pillonel Maura Concu 4 Swiss Tropical and Public Health Institute , Allschwil, Switzerland 5 University of Basel , Basel, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site Elena Montenegro Borbolla 1 Service of Infectious Diseases, Lausanne University Hospital and University of Lausanne , Lausanne, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Elena Montenegro Borbolla Beatrice Nickel 4 Swiss Tropical and Public Health Institute , Allschwil, Switzerland 5 University of Basel , Basel, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Beatrice Nickel Camille Stampfli 6 Service of Pharmacy, Lausanne University Hospital and University of Lausanne , Lausanne, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Camille Stampfli Marie-Therese Ruf 4 Swiss Tropical and Public Health Institute , Allschwil, Switzerland 5 University of Basel , Basel, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Marie-Therese Ruf Maxime Audry 6 Service of Pharmacy, Lausanne University Hospital and University of Lausanne , Lausanne, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Maxime Audry Nathalie Kapel 2 French Group for Fecal Microbiota Transplantation (GFTF) , Paris, France 7 Laboratoire de Coprologie fonctionnelle, APHP, GH Pitié-Salpêtrière , Paris, France 8 Université Paris Cité , INSERM S1139, Paris, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Nathalie Kapel Susanna Gerber 6 Service of Pharmacy, Lausanne University Hospital and University of Lausanne , Lausanne, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site Damien Jacot 9 Institute of Microbiology, Lausanne University Hospital and University of Lausanne , Lausanne, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Damien Jacot Claire Bertelli 9 Institute of Microbiology, Lausanne University Hospital and University of Lausanne , Lausanne, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Claire Bertelli Tatiana Galperine 1 Service of Infectious Diseases, Lausanne University Hospital and University of Lausanne , Lausanne, Switzerland 2 French Group for Fecal Microbiota Transplantation (GFTF) , Paris, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Tatiana Galperine Abstract Full Text Info/History Metrics Supplementary material Data/Code Preview PDF Abstract Introduction Dientamoeba fragilis is a protozoan of the human digestive tract, yet its transmission and pathogenic role remain poorly understood. This study aimed to evaluate its impact on the efficacy and safety of FMT in treating recurrent Clostridioides difficile infection (rCDI). Patients and methods This longitudinal cohort study analyzed stool samples from FMT donors and recipients pre-treatment and at 2 and 8 weeks post-FMT. All samples were retrospectively tested using real-time PCR. Shotgun metagenomics was also performed on selected donor-recipient pairs to explore transmission. CDI cure rates, gastrointestinal adverse events (AE), and serious adverse events (SAE) were assessed prospectively. Results A total of 53 FMT were analysed (179 samples), with 23 (43%) derived from D. fragilis -positive donor stool (4 of 10 donors, 40%). Four of 52 recipients (18.2%), initially negative and who received treatment from positive donors, tested positive post-FMT. Shotgun metagenomics could not definitely confirm transmission due to the lack of a good reference genome. No significant differences in efficacy, AE, or SAE were observed between FMT from D. fragilis -positive versus -negative donors, even in immunocompromised patients. No SAE were attributed to FMT. Conclusion D. fragilis may be transmitted via FMT without clinical impact, highlighting the need to reconsider donor exclusion and aligning with growing evidence of its questionable pathogenicity. INTRODUCTION Since 2014, fecal microbiota transplantation (FMT) has been the recommended treatment for recurrent Clostridioides difficile infections (rCDI), with success rates up to 85% and improved patient survival [ 1 – 5 ]. FMT aims to therapeutically modulate the recipient’s gut microbiome. For safety reasons, donors undergo rigorous selection procedures, including multiple clinical and biological screenings, to minimize the risk of transmitting pathogens and potential diseases associated with gut microbiota [ 6 ]. Following this selection process, only 3-10% of candidates qualify as eligible fecal microbiota donors, limiting the availability and accessibility of this proven beneficial treatment [ 7 ] [ 8 ]. Dientamoeba fragilis is an intestinal protozoan belonging to the Trichomonadidae order even though it lacks flagella [ 9 ]. D. fragilis has been detected worldwide with prevalence rates ranging from 0.2% to 82%, and often exceeding 20% in molecular epidemiological studies [ 10 ]. However, its route of transmission remains poorly understood. The pathogenicity of D. fragilis remain debated, with limited evidence linking it to gastrointestinal symptoms such as diarrhea and abdominal pain [ 11 ]. FMT serves as a valuable and reliable model for studying the impact of controversial fecal microorganisms, providing direct exposure to donor gut microbiota in recipients. For example, Blastocystis hominis , another protist with debated pathogenicity, was removed from the donor exclusion list following a study by Teerver et al., which demonstrated no adverse consequences in recipients from B. hominis -positive donors [ 12 ]. As a result of this study, the 5th edition of the Guide to the Quality and Safety of Organs for Transplantation (EDQM, 2022) no longer lists B. hominis as an exclusion criterion for donors, unlike D. fragilis . The transmission and impact of D. fragilis during FMT, however, still require further investigation. To date, real-time PCR (RT-PCR) for D. fragilis is not part of routine donor screening at the Lausanne University Hospital, the only center in Switzerland performing FMT. The primary objective of our study was to assess the prevalence of D. fragilis in stool samples from FMT donors and their paired recipients, both before and after FMT, using RT-PCR. Additionally, we aimed to investigate whether the presence of D. fragilis affects the efficacy of FMT or is associated with adverse events post-FMT. METHODS Study design and setting This observational cohort study was conducted at Lausanne University Hospital (CHUV) in Switzerland. From January 2023 to December 2024, all eligible patients receiving FMT as part of routine clinical care for rCDI and their corresponding donors involved in the production of FMT batches used for treatment were prospectively included. Molecular screening for D. fragilis was retrospectively performed using RT-PCR-based detection methods on stool samples collected from donors and from their paired recipients before and after FMT. The patient sample size was determined based on pragmatic considerations, and inclusion followed a total enumerative sampling approach, encompassing all patients who met the eligibility criteria over a two-year period. Study data were collected from patients’ electronic health records and were documented and managed using REDcap electronic data capture tools hosted at CHUV. This manuscript was prepared in accordance with the STROBE statement for cohort studies. [ 13 ]. Study participants Patients – rCDI Eligibility criteria required patients to be at least 18 years old and to have undergone FMT, with the indication aligned with the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) guidelines for Clostridioides difficile treatment [ 1 ]. Patients received at least 10 days of pretreatment with fidaxomicin or vancomycin. FMT was administered to inpatients either via endoscopy (colonoscopy or jejunostomy) following bowel lavage with 2 liters of macrogol solution the day prior or through oral ingestion of 20 capsules per day over two consecutive days (40 in total). In severe cases, the dosage was increased to 30 capsules per day (60 in total). Patient variables were standardized, including age (continuous variable), sex (female or male), and immunosuppression status (binarized). Severe immunosuppression status was defined as current or foreseeable neutropenia (<⍰1500 neutrophils/µl) within the next 14 days; scheduled or recent (<⍰110 days) allogeneic stem cell transplantation; active graft-versus-host disease (GvHD) requiring immunosuppressive treatment; or ongoing chemotherapy. To assess the clinical success of FMT, cure was defined as the absence of CDI recurrence at 8 weeks post-FMT. Criteria for severe, complicated and recurrent CDI followed the ESCMID guidelines [ 1 ]. Gastrointestinal adverse events (AE) following FMT, including bloating, nausea, abdominal pain, and changes in bowel movements, were prospectively monitored and documented at weeks 2 and 8. Follow-up assessments were conducted via phone at 6 months, 12 months, and annually for up to 5 years. Serious adverse events (SAE) were evaluated in accordance with the ICH E2A guidelines [ 14 ]. The causal relationship between FMT and SAE was evaluated using categories defined in the Fecal Microbiota Transplant National Registry of the American Gastroenterological Association (AGA) [ 15 ]. Donors All microbiota donors were healthy, unpaid, volunteers aged 18 to 50 years and were submitted to a detailed biological screening (Supplementary Table 1). Stool donations were collected at the CHUV FMT center and processed individually, without pooling between donations or donors (see Supplementary material for detailed FMT production). The donors included in this study were those who provided stool used in the production of the treatment for the enrolled patients. In all samples, direct microscopic examination for D. fragilis yielded negative results. Native stool collection, D. fragilis detection Donor native stool used for treatment was collected prior to treatment production. Stool samples from FMT recipients were obtained 24-48 hours before FMT, as well as at 2 and 8 weeks post-FMT, as part of routine care for traceability purposes. All samples were aliquoted and biobanked at -80°C (BIOSTOOL, BB_041). The diagnosis of D. fragilis was conducted using two in-house RT-PCRs at the Diagnostic Center of Swiss Tropical and Public Health Institute (Swiss TPH) in Allschwil (see Supplementary Material for detailed detection methods). A cut-off cycle threshold (Ct) of <40 was used for both PCRs. The analysis of shotgun metagenomics sequences of positive paired donor and recipient stools was performed at the Genomics and Metagenomics laboratory of the CHUV (see Supplementary Material for detailed methods). Outcomes The primary outcome was the assessment of the prevalence of D. fragilis in stool samples using RT-PCR from both donors and corresponding recipients before FMT, and at weeks 2 and 8 post-FMT. Secondary outcomes included comparing the efficacy, gastrointestinal AE and SAE associated with FMT, depending on the D. fragilis status of donor batches. Statistics For the primary objective, the prevalence of D. fragilis –positive RT-PCR in donors and recipients were expressed as rates. With regard to the secondary objectives, the analysis focused on identifying differences in CDI recurrence rates and gastrointestinal AE and SAE between recipients of D. fragilis -positive and D. fragilis -negative treatments, using Chi-square or Fisher’s exact test. A p-value below 0.05 was considered indicative of statistical significance in all comparisons. All analyses were performed using RStudio, version 4.2.1. Ethical statement Ethical approval for this study was obtained from the Canton of Vaud Ethics Committee (ID: CER-VD 2024-00418). Written informed consent was obtained for anonymized patients and donors for participation in the research and reuse of biological samples. RESULTS RT-PCR detection of D. fragilis in stools from donors and FMT recipients A total of 179 stool samples were analyzed by RT-PCR for D. fragilis including 37 samples from 10 donors and 142 samples from 47 recipients. All donors and recipients were of European ethnic origin. The characteristics of the study population are summarized in Table 2 . Details of recipients’ characteristics, clinical follow-up, RT-PCR results, and FMT data are provided in Supplementary Table 2. View this table: View inline View popup Download powerpoint Table 1. Fecal microbiota transplantation with D. fragilis -positive donor stool and recipient D. fragilis status View this table: View inline View popup Download powerpoint Table 2. Demographic characteristics of fecal microbiota transplantation recipients and comparison between transfer of D. fragilis -positive and -negative donor stool D. fragilis was detected in the stool of 4 out of 10 donors (40%), with Ct values for RT-PCR ranging from 19.3 to 36.1 ( Table 1 ). Fecal material from the 10 donors was used to produce a total of 233 oral FMT preparations (9,320 capsules) and 54 suspensions. Batches derived from D. fragilis -positive donor stool accounted for 46 of the 233 oral preparations (1,840 capsules, 19.7%) and 37 of the 54 suspensions (68.5%). In total, 53 FMT treatments were administered, of which 23 (43%) originated from D. fragilis -positive donor stool. Prior to FMT, D. fragilis was negative in 52 of 53 (98%) recipient stool samples. One recipient (1/47, 2%) was tested positive before FMT and received a treatment coming from D. fragilis -positive donor stool. There is a significant difference in the prevalence of D. fragilis between donors and recipients in our sample (p<0.05). Among the 22 patients who tested negative before FMT and received stool from D. fragilis -positive donors, only four (18.2%) tested positive by RT-PCR at day 15 post-FMT, all of whom remained positive at week 8. All four patients received FMT via colonoscopy, with transplants thawed using a standardized warm water bath method (34.5°C). Despite varying immunosuppression statuses, these patients tolerated the procedure well and were considered cured at the 8-week follow-up. The D. fragilis PCR Ct values in stool samples from positive donors used for their treatments ranged from 19.3 to 31.6. In positive recipients’ stool, Ct ranged from 21 to 34.5. Notably, four other patients who received FMT from the same donor batches under identical conditions did not test positive for D. fragilis at any point during follow-up. Shotgun metagenomic sequencing was performed on one stool sample from donor DL01 and two samples from the FMT recipient RL42, as this pair presented the highest D. fragilis concentration (lowest Ct). From the 36 to 51 million reads generated per sample, only 19 (DL01), 380 (RL42 D15), and 31 (RL42 W8) reads mapped to the available D. fragilis transcriptome. Due to the extremely low number of mapped reads, we were unable to confirm with confidence the transmission of D. fragilis from donor to recipient. Efficacy at 8 weeks according to D. fragilis positivity of the donors At week 8, CDI resolution was achieved in 44/47 (93.6%) patients after a single FMT, increasing to 46/47 (97.9%) after two FMTs and 47/47 (100%) after three. Notably, all post-FMT CDI recurrences occurred in recipients of stool from D. fragilis -negative donors. The presence of D. fragilis in donor stools had no impact on FMT efficacy (p=0.12) ( Table 2 ). The median follow-up at the time of analysis was 13.3 months (range 3-25). Even though the cohort included individuals with significant morbidity and severe immunosuppression (see Supplementary Table 2), no new episodes of CDI were reported during the studied period after the 8 weeks follow-up. AE and SAE at 15 days and 8 weeks post FMT Patients could experience multiple gastrointestinal AE associated with a single FMT. A total of 53 gastrointestinal AE and 12 SAE were recorded and are detailed in Supplementary Table 2. None of the SAE were related to FMT. Four SAE (33%) occurred in recipients with transplants from D. fragilis -positive donors. There were no significant differences in the incidence of gastrointestinal adverse events or severe adverse events between patients treated with FMT from D. fragilis -positive or -negative donors ( Table 3 ). View this table: View inline View popup Download powerpoint Table 3. Adverse events occurring at least once during follow-up and comparison between D. fragilis -positive and -negative donor stool recipients DISCUSSION The framework of FMT represents an excellent opportunity to study the debated pathogenicity of the enteric protozoan D. fragilis . To assess the implication of D. fragilis colonization on gastrointestinal health, we assessed the presence of D. fragilis in stools from donors as well as their paired FMT recipients alongside their mid-term clinical symptoms. To our knowledge, this is the first report of D. fragilis detection by RT-PCR in stool samples of previously negative recipients, following FMT. Although many donors tested positive for D. fragilis (40%), the new detection of this protozoan in recipients post-FMT via RT-PCR was rare, occurring in only 4 out of 22 cases. Neither the safety as measured by the occurrence of AE, nor the efficacy of FMT was affected by the detection of D. fragilis donors within this cohort. The life cycle of D. fragilis remains incompletely understood. While rare putative cyst and precyst forms have been observed in clinical specimens, the precise modes of human transmission remain unclear [ 9 , 16 ]. While direct fecal-oral transmission has yet to be confirmed [ 9 ], our results are suggestive of some level of transmission by this route and may support the hypothesis of the existence of a cyst stage in D. fragilis [ 17 ]. This finding, however, is contrary to previous studies who found no evidence of D. fragilis transmission from donor to recipient via FMT in rCDI patients [ 18 ]. Moreover, the lack of a reproducible animal model for D. fragilis infection has significantly hindered research into its biology and pathogenicity. Given the fragility of this protozoan, its survival during FMT production is questionable. As demonstrated by Hurych et al. , a dramatic decrease in the viability of this protist after deep freezing is measured, with no viable organisms detectable in culture media after a single freeze-thaw cycle [ 19 ]. It is important to note that while PCR is a highly sensitive screening tool, it does not provide information on the viability or infectivity of D. fragilis but only detects the presence of nucleic acids. However, the persistent finding of DNA for several weeks after FMT would suggest a successful colonization. Our findings, showing that 81.8% of recipients remained RT-PCR negative after receiving a D. fragilis -positive FMT, along with existing evidence, suggest that D. fragilis only occasionally remains viable in the context of FMT. While we attempted a shotgun sequencing approach to confirm the donor to recipient transmission of D. fragilis via FMT, the low concentration of the protozoan in stools as well as the lack of a good reference genome for D. fragilis complicated the analysis. The number of D. fragilis sequences recovered was too low to assert the genetic similarity of donor and FMT recipient strains. While unlikely, the four recipients who tested positive may also have been colonized by another natural source within 15 days post-FMT. However, the critical question is not merely whether transmission occurs, but what the clinical consequences for recipients may be. Indeed, the pathogenicity of D. fragilis remains controversial. While it has been associated with gastrointestinal symptoms such as diarrhea and abdominal pain, the evidence supporting this link is inconsistent. A randomized controlled trial in Denmark by Röser et al. evaluated metronidazole for D. fragilis infections in 96 children, finding no significant symptom improvement over placebo, though parasitological eradication was more frequent in the metronidazole group [ 20 ]. More recently, a case-control study found no significant association between the detection of D. fragilis and the development of gastrointestinal symptoms [ 21 ]. Similarly, several case-control studies have failed to establish a link between D. fragilis and conditions such as irritable bowel syndrome (IBS) or celiac disease, aligning with findings for B. hominis [ 22 - 24 ]. Advances in molecular detection have demonstrated that D. fragilis is commonly found in asymptomatic individuals, consistent with the results observed in our donor cohort. A Danish study found D. fragilis prevalence at 43% in adults and 63% in children, indicating its high occurrence in the general population [ 25 ]. Likewise, in our cohort, stool donors—selected based on stringent safety criteria—frequently tested positive for D. fragilis . These findings support the hypothesis that D. fragilis may be a commensal organism within the normal gut microbiota, rather than a pathogenic entity. In our cohort, the low prevalence of these microorganisms in patients with dysbiosis before FMT further supports this model (2% in recipients versus 40% in healthy donors, p<0.05), and even suggest that D. fragilis colonization may rather reflect gastrointestinal health. Recruiting stool donors for FMT is inherently challenging due to stringent screening criteria. As noted, D. fragilis is commonly found in asymptomatic individuals and is often a reason for donor exclusion [ 8 ]. In our study, had we applied D. fragilis positivity as an exclusion criterion, 40% of potential donors would have been excluded, resulting in the discarding of 287 FMT treatments derived from these donors. Given the complexity of donor selection, this would have significantly compromised the feasibility of producing this treatment and would have deprived patients of valuable treatments, as all recipients of D. fragilis -positive FMT of this study were cured. Our data suggest that D. fragilis colonization in donors, comparably to what has been demonstrated for B. hominis by Terveer et al., did not adversely affect recipient outcomes, including in high-risk patients [ 12 ]. This further supports the reconsideration of D. fragilis as an exclusion criterion for FMT donors. CONCLUSIONS Although D. fragilis has traditionally been considered pathogenic, its role in human health remained debated. FMT offered a valuable model for evaluating the pathogenicity of such protozoans with unclear or controversial clinical significance. Our findings suggest that FMT derived from D. fragilis -positive donor stools does not lead to gastrointestinal adverse events or severe outcomes in recipients, including those who are severely immunocompromised. These findings support reconsidering the exclusion of donors based on D. fragilis positivity, in line with the precedent set for B. hominis in the EDQM guidelines. Rationalizing donor screening processes could improve the availability of FMT without compromising safety, provided that appropriate clinical follow-up of recipients is ensured. Data Availability The data that support the findings of this study are openly available in Mendeley data at https://data.mendeley.com/datasets/3f37czt42n/1 , DOI: 10.17632/3f37czt42n.1. Data supporting the findings is also available in the article's supplementary materials. https://data.mendeley.com/datasets/3f37czt42n/1 Author contributions T.G., N.K and C.B. conceived and designed the study. K.M., E.M.B, T.G., A.B. and M. A. collected the data and samples. M.C., B.N. and M.-T.R. performed molecular analyses such as RT-PCR. T.P. and C.B. performed the shotgun sequencing and analysis of stools. C.B., S.G., D.J., N.K., T.G., K.M. and A.B. contributed to analysis and interpretation of results. T.G. and K.M. contributed equally to manuscript writing. All coauthors reviewed the results and approved the final version of the manuscript. Personal acknowledgments The authors thank Alexandra Mitouassiwou-Samba, Valérie Sormani, Fabienne Aparicio and Benoit Guery as well as the sequencing platform of the DMLP for their excellent technical support. Generative AI During the preparation of this work the authors used ChatGPT (OpenAI, 2025) in order to rephrase certain sentences. After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication. Download figure Open in new tab Acknowledgment Footnotes ( aurelie.ballif{at}unisante.ch ) ( trestan.pillonel{at}chuv.ch ) ( maura.concu{at}swisstph.ch ) ( elena.montenegro{at}chuv.ch ) ( beatrice.nickel{at}swisstph.ch ) ( camille.stampfli{at}chuv.ch ) ( therese.ruf{at}swisstph.ch ) ( maxime.audry{at}chuv.ch ) ( nathalie.kapel{at}aphp.fr ) ( susanna.gerber{at}chuv.ch ) ( damien.jacot{at}chuv.ch ) ( claire.bertelli{at}chuv.ch ) ( Katerina-tatiana.galperine{at}chuv.ch ) Funding: This work was supported by internal funding at the Lausanne University Hospital (CHUV). The salary of K.M. and E.M.B. was supported as a part of NCCR Microbiomes, a National Centre of Competence in Research, funded by the Swiss National Science Foundation (grant number 51NF40 180575). Potential conflicts of interest: The authors declare that they have no known competing financial interests or personal relationships that have appeared to influence the work reported in this paper. Data: The data that support the findings of this study are openly available in Mendeley data at https://data.mendeley.com/datasets/3f37czt42n/1 , DOI: 10.17632/3f37czt42n.1. Data supporting the findings is also available in the article’s supplementary materials. The reads of the shotgun metagenomic sequencing have been made available at the European Nucleotide Archive (ENA) under project number PRJEB89891. Ethical statement: Ethical approval for this study was obtained from the Canton of Vaud Ethics Committee (ID: CER-VD 2024-00418). Written informed consent was obtained for anonymized patients and donors for participation in the research and reuse of biological samples. Abbreviations used in this paper AE Adverse Events AGA American Gastroenterological Association B. hominis Blastocystis hominis CDI Clostridioides difficile Infection CHUV Centre Hospitalier Universitaire Vaudois Ct Cycle threshold D. fragilis Dientamoeba fragilis EDQM European Directorate for the Quality of Medicines & HealthCare ESCMID European Society of Clinical Microbiology and Infectious Diseases FMT Fecal Microbiota Transplantation GvHD Graft-versus-Host Disease IBS Irritable Bowel Syndrome ICH E2A International Conference on Harmonisation, article E2A (Clinical Safety) ID Identifier rCDI Recurrent CDI REDCap REDCap, Research Electronic Data Capture software RT-PCR Real-Time Polymerase Chain Reaction SAE Serious Adverse Events REFERENCES 1. ↵ Prehn J van , Reigadas E , Vogelzang EH , et al. European Society of Clinical Microbiology and Infectious Diseases: 2021 update on the treatment guidance document for Clostridioides difficile infection in adults . Clin Microbiol Infect 2021 ; 27 : S1 – S21 . OpenUrl 2. Peery AF , Kelly CR , Kao D , et al. AGA Clinical Practice Guideline on Fecal Microbiota–Based Therapies for Select Gastrointestinal Diseases . Gastroenterology 2024 ; 166 : 409 – 434 . OpenUrl CrossRef PubMed 3. McDonald LC , Gerding DN , Johnson S , et al. Clinical Practice Guidelines for Clostridium difficile Infection in Adults and Children: 2017 Update by the Infectious Diseases Society of America (IDSA) and Society for Healthcare Epidemiology of America (SHEA) . Clin Infect Dis 2018 ; 66 : e1 – e48 . OpenUrl CrossRef PubMed 4. Ianiro G , Maida M , Burisch J , et al. Efficacy of different faecal microbiota transplantation protocols for Clostridium difficile infection: A systematic review and meta-analysis . United Eur Gastroenterol J 2018 ; 6 : 1232 – 1244 . OpenUrl CrossRef PubMed 5. ↵ Quraishi MN , Widlak M , Bhala N , et al. Systematic review with meta-analysis: the efficacy of faecal microbiota transplantation for the treatment of recurrent and refractory Clostridium difficile infection . Aliment Pharmacol Ther 2017 ; 46 : 479 – 493 . OpenUrl CrossRef PubMed 6. ↵ Cammarota G , Ianiro G , Kelly CR , et al. International consensus conference on stool banking for faecal microbiota transplantation in clinical practice . Gut 2019 ; 68 : 2111 . OpenUrl Abstract / FREE Full Text 7. ↵ Terveer EM , Vendrik KE , Ooijevaar RE , et al. Faecal microbiota transplantation for Clostridioides difficile infection: Four years’ experience of the Netherlands Donor Feces Bank . UEG J 2020 ; 8 : 1236 – 1247 . OpenUrl 8. ↵ Bénard MV , Bruijn CMA de , Fenneman AC , et al. Challenges and costs of donor screening for fecal microbiota transplantations . PLoS ONE 2022 ; 17 : e0276323 . OpenUrl PubMed 9. ↵ Hall LM , Munasinghe VS , Vella NGF , Ellis JT , Stark D. Observations on the transmission of Dientamoeba fragilis and the cyst life cycle stage . Parasitology 2024 ; 151 : 337 – 345 . OpenUrl PubMed 10. ↵ Cacciò SM. Molecular epidemiology of Dientamoeba fragilis . Acta Trop 2018 ; 184 : 73 – 77 . OpenUrl PubMed 11. ↵ Shasha D , Grupel D , Treigerman O , et al. The clinical significance of Dientamoeba fragilis and Blastocystis in human stool—retrospective cohort study . Clin Microbiol Infect 2024 ; 30 : 130 – 136 . OpenUrl PubMed 12. ↵ Terveer EM , Gool T van , Ooijevaar RE , et al. Human Transmission of Blastocystis by Fecal Microbiota Transplantation Without Development of Gastrointestinal Symptoms in Recipients . Clin Infect Dis 2019 ; 71 : 2630 – 2636 . OpenUrl 13. ↵ von Elm E , Altman DG , Egger M , Pocock SJ , Gotzsche PC , Vandenbroucke JP . The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) Statement: guidelines for reporting observational studies . 14. ↵ ICH E2A Clinical safety data management: definitions and standards for expedited reporting - Scientific guideline | European Medicines Agency [Internet] . 1995 [cited 2025 Mar 26 ]. Available from: https://www.ema.europa.eu/en/ich-e2a-clinical-safety-data-management-definitions-standards-expedited-reporting-scientific-guideline 15. ↵ American Gastroenterological Association . Fecal Microbiota Transplant National Registry [Internet] . 2017 [cited 2025 Mar 26 ]. Available from: https://cdn.clinicaltrials.gov/large-docs/55/NCT03325855/Prot_000.pdf 16. ↵ Stark D , Garcia LS , Barratt JLN , et al. Description of Dientamoeba fragilis Cyst and Precystic Forms from Human Samples . J Clin Microbiol 2014 ; 52 : 2680 – 2683 . OpenUrl Abstract / FREE Full Text 17. ↵ Stensvold CR , Tomiak J , Seyoum Y , Nielsen HV , Mark. Letter to the Editor: Comment to “Assessment of Dientamoeba fragilis interhuman transmission by fecal microbiota transplantation” by Moreno-Sabater et al. (2025) . Int J Antimicrob Agents 2025 ; 107541 – 1 . 18. ↵ Moreno-Sabater A , Sintes R , Truong S , Lemoine K , Camou O , Kapel N , et al. Assessment of Dientamoeba fragilis interhuman transmission by fecal microbiota transplantation . Int J Antimicrob Agents 2025 ; 66 ( 2 ): 107504 OpenUrl PubMed 19. ↵ Hurych J , Vodolanova L , Vejmelka J , et al. Freezing of faeces dramatically decreases the viability of Blastocystis sp . and Dientamoeba fragilis. Eur J Gastroenterol Hepatol 2022 ; 34 : 242 – 243 . OpenUrl PubMed 20. ↵ Röser D , Simonsen J , Stensvold CR , et al. Metronidazole Therapy for Treating Dientamoebiasis in Children Is Not Associated With Better Clinical Outcomes: A Randomized, Double-Blinded and Placebo-Controlled Clinical Trial . Clin Infect Dis 2014 ; 58 : 1692 – 1699 . OpenUrl CrossRef PubMed 21. ↵ Bamini GT , Charpentier E , Guemas E , et al. No evidence of pathogenicity of Dientamoeba fragilis following detection in stools: A case-control study . Parasite 2024 ; 31 : 40 . OpenUrl PubMed 22. ↵ Boer MD-de , Schuurs TA , Vermeer M , et al. Distribution and relevance of Dientamoeba fragilis and Blastocystis species in gastroenteritis: results from a case-control study . Eur J Clin Microbiol Infect Dis 2020 ; 39 : 197 – 203 . OpenUrl PubMed 23. Kalleveen MW van , Budding AE , Benninga MA , et al. Intestinal Microbiota in Children With Symptomatic Dientamoeba fragilis Infection: A Case-control Study . Pediatr Infect Dis J 2020 ; 40 : 279 – 283 . OpenUrl 24. ↵ MIzrak M , Sarzhanov F , Demirel F , Dinç B , Filik L , Dogruman-Al F. Detection of Blastocystis sp. and Dientamoeba fragilis using conventional and molecular methods in patients with celiac disease . Parasitol Int 2024 ; 101 : 102888 . OpenUrl PubMed 25. ↵ Röser D , Simonsen J , Nielsen HV , Stensvold CR , Mølbak K. Dientamoeba fragilis in Denmark: epidemiological experience derived from four years of routine real-time PCR . Eur J Clin Microbiol Infect Dis 2013 ; 32 : 1303 – 1310 . OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted November 04, 2025. Download PDF Supplementary Material Data/Code Email Thank you for your interest in spreading the word about medRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Fecal Microbiota Transplantation Donor Screening: Is Dientamoeba fragilis a Valid Criterion for Donor Exclusion? 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