Treatment Outcomes in Symptomatic Dientamoeba fragilis Infection: A Prospective Clinical and Molecular Study

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This study aimed to evaluate clinical and molecular responses to various antiparasitic regimens and to assess the association between symptom resolution and parasite eradication. Methods In this prospective, single-center observational study conducted between January 2019 and June 2023, we included 105 symptomatic patients with a positive stool PCR for DF. Patients were treated with paromomycin (PAR), nitroimidazole monotherapy (NM; metronidazole/tinidazole), or a combination of tinidazole and albendazole (T + A). Clinical and molecular outcomes were assessed one month post-treatment using a structured symptom questionnaire and repeat PCR testing. Results Of 96 patients with follow-up, 73 underwent repeat PCR testing. PAR was associated with significantly higher clinical and molecular cure rates (82.8% and 88.5%, respectively) compared to T + A (35.0% and 29.2%) and NM (4.2% and 10.0%). Combination therapy with T + A was superior to NM in clinical response but not in molecular clearance. A strong correlation was observed between clinical cure and DF eradication (p < 0.00001), reinforcing the pathogenic role of DF. Median symptom duration before referral was 9 months, indicating the chronic nature of untreated infection. Conclusions DF should be considered in patients with unexplained chronic gastrointestinal symptoms. Molecular testing is recommended for accurate diagnosis. Paromomycin appears to be the most effective treatment, with a strong association between clinical recovery and microbiological cure. Randomized controlled trials are warranted to further define optimal management strategies for DF infection. Gastrointestinal symptoms Paromomycin Metronidazole Tinidazole Albendazole Antiprotozoal therapy Molecular diagnosis PCR Clinical cure D. fragilis pathogenicity Figures Figure 1 Introduction Dientamoeba fragilis (DF) is a binucleated flagellated protozoan and one of the most commonly detected intestinal parasites worldwide. Over the past decade, the use of molecular diagnostic techniques for stool testing has increased in many countries, particularly in the evaluation of gastrointestinal (GI) symptoms. As a result, DF has gained increased recognition and is now often referred to as an emerging protozoan pathogen [ 1 ]. Despite its high global prevalence, the pathogenic potential of DF remains controversial. When Dobell and Jepps first described the organism in 1918, they concluded it was nonpathogenic—even though six of the seven patients in their report presented with diarrhea or dysentery [ 2 ]. Several subsequent observations supported this nonpathogenic view, citing DF’s lack of tissue invasion and its frequent detection in asymptomatic individuals [ 3 , 4 ]. A recent systematic review examining the causal role of DF in diarrheal illness reported conflicting results [ 5 ]. However,, DF infection may present with a broader spectrum of manifestations beyond diarrhea, including altered bowel habits, abdominal pain, bloating, and even extraintestinal features such as fatigue and eosinophilia [ 6 ]. In light of this uncertainty, treatment is still recommended for symptomatic patients in whom DF is the only identified pathogen. This recommendation is supported by the U.S. Centers for Disease Control and Prevention (CDC) and clinical platforms such as UpToDate and Medscape [ 7 ]. However, there is no consensus regarding the optimal treatment regimen. Metronidazole, historically considered first-line therapy [ 8 ], failed to demonstrate superiority over placebo in a randomized controlled trial involving children [ 9 ]. Observational studies have reported higher rates of clinical and microbiological cure with other agents, such as paromomycin, clioquinol, and ornidazole, compared to metronidazole [ 10 – 13 ]. Given these controversies, we conducted an observational study to assess the clinical and molecular response to various antiprotozoal agents commonly used in the treatment of symptomatic DF infection in routine clinical practice. Methods A single-center, prospective observational study conducted at Sheba Medical Center between January 2019 and June 2023. We included consecutive patients presenting with chronic gastrointestinal (GI) symptoms lasting longer than one month and a positive stool PCR test for Dientamoeba fragilis (DF), either as a monoinfection or co-infection with Blastocystis spp., Patients with elevated inflammatory markers were excluded. Molecular testing was performed using the Allplex™ GI Parasite Assay (Seegene, South Korea), a one-step real-time PCR that detects gastrointestinal parasites including: Blastocystis hominis , Cryptosporidium spp., Cyclospora cayetanensis , Dientamoeba fragilis , Entamoeba histolytica , and Giardia lamblia . At the initial visit, all patients completed a structured questionnaire that included travel history (when relevant), type and duration of symptoms, and the presence of the following gastrointestinal symptoms: loose stools, abdominal pain, bloating, flatulence and borborygmus, loss of appetite, weight loss, and fatigue (see Supplemental Appendix for questionnaire). Relevant prior investigations such as imaging studies or endoscopic procedures were also documented. Treatment regimens were determined at the discretion of the attending physician or primary care provider. Some patients were prescribed a combination of tinidazole and albendazole, based on our clinic’s policy and previous experience with other intestinal protozoal infections [ 14 ]. Patients were asked to repeat the stool PCR test approximately one month after completing treatment. Clinical cure was assessed one month post-treatment and defined as resolution or significant improvement in gastrointestinal symptoms, based on a yes/no response to the question: “In regard to all your gastrointestinal symptoms, as compared with the way you felt before you started the study medication, have you had adequate relief of your gastrointestinal symptoms?” [ 15 ]. Molecular cure was defined as a negative stool PCR for DF at least one month following completion of treatment. Patients who did not achieve clinical cure and remained PCR-positive were prescribed an alternative antiprotozoal therapy. Each treatment course, including multiple courses in the same patient, was evaluated independently. All patients with both clinical and molecular follow-up were included in the efficacy analysis. Patients without follow-up PCR were included in the symptom analysis. The first treatment course was assessed for clinical and molecular response, while all treatment courses were analyzed for correlation between clinical outcome and molecular clearance. Statistical analysis was performed using Microsoft Excel 2016 (Microsoft Corp., Redmond, WA, USA). Continuous variables are presented as mean ± standard deviation (SD) or median with interquartile range (IQR). Categorical variables are reported as frequency and percentage. Group comparisons were performed using the Chi-square test or t-test for categorical and continuous variables, respectively. For expected frequencies < 5, Fisher’s exact test was used. Binary logistic regression was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs) for clinical and molecular cure associated with each treatment. A p-value < 0.05 was considered statistically significant. The study was approved by the Institutional Review Board (IRB) of Sheba Medical Center. Results Over the four-and-a-half-year study period, 105 symptomatic patients were referred to our clinic with a positive PCR result for Dientamoeba fragilis (DF), either as monoinfection (n = 89, 84.8%) or in coinfection with Blastocystis spp. (n = 16, 15.2%). Demographic and clinical characteristics are summarized in Table 1 . Table 1 Demographic and Clinical Characteristics of the Study Population (N = 105) Characteristic Value Demographics Age, years (median, IQR) 34 (21–49) Female sex, n (%) 57 (54.3%) Travel history prior to symptom onset, n (%) 47 (44.8%) Symptoms Duration of symptoms, months (median, IQR) 9 (4–36) Abdominal pain, n (%) 81 (77.1%) Loose stools, n (%) 81 (77.1%) Bloating, n (%) 56 (53.3%) Fatigue, n (%) 58 (55.2%) Weight loss, n (%) 29 (27.6%) Values are presented as n (%) or median (interquartile range, IQR). Patient ages ranged from 2.5 to 83 years, with a median age of 34 years. Seventeen patients (16.5%) were children under 18 years of age (median age 9 years), while the median age among adults (≥ 18 years) was 39 years. Females comprised 54.3% of the cohort (n = 57). Forty-seven patients (44.8%) reported international travel prior to symptom onset. Travel destinations included the Indian subcontinent (n = 17), East and/or Southeast Asia (n = 8), Latin America (n = 7), Sub-Saharan Africa (n = 6), North Africa (n = 4), North America (n = 2), Central Europe (n = 2), and Eastern Europe (n = 1). The most frequently reported symptoms were loose stools and abdominal pain, each occurring in 76.7% of patients, with a mean symptom duration of 9 months prior to referral (ranged 1 to 240 months). Seventeen patients (16.2%) underwent gastrointestinal endoscopy before their initial clinic visit, and all examinations were reported as normal. Of the 105 patients, 96 (93.2%) returned for clinical follow-up, and 73 of these repeated also stool PCR testing. Regarding treatment, 24 patients were prescribed nitroimidazole monotherapy (NM), either metronidazole 500 mg three times daily for 7–10 days or tinidazole 1 g twice daily for 2–3 days. Forty patients received a combination of tinidazole and albendazole (T + A), consisting of tinidazole 1 g twice daily for 2 days followed by albendazole 400 mg twice daily for 5 days. Twenty-nine patients were treated with paromomycin (PAR) 500 mg twice daily for 7 days. One patient received diloxanide, and two were treated with albendazole monotherapy. Clinical and molecular cure rates following the first treatment course, stratified by treatment regimen, are presented in Table 2 . Patients treated with PAR achieved a clinical cure rate of 82.8% (24/29) and a molecular cure rate of 88.5% (23/26), both significantly higher than those treated with T + A (clinical: 35% [14/40]; molecular: 29.2% [7/24]) or NM (clinical: 4.2% [1/24]; molecular: 10% [2/20]). T + A was significantly superior to NM in terms of clinical cure (p < 0.005), but not for molecular cure (Table 2 ). Table 2 Clinical and Molecular Cure Rates According to Treatment Regimen Treatment N Clinical Cure OR⁽ᵃ⁾ (95% CI) p-value N (PCR) Molecular Cure OR⁽ᵃ⁾ (95% CI) p-value Paromomycin 29 82.8% (24/29) 110.4 (12.0–1018.5) < 0.0001 26 88.5% (23/26) 69.0 (10.4–457.9) < 0.0001 Tinidazole + Albendazole 40 35.0% (14/40) 12.4 (1.5–101.6) < 0.0001 24 29.2% (7/24) 3.7 (0.7–20.4) < 0.0001 Metronidazole/Tinidazole 24 4.2% (1/24) Reference Reference 20 10.0% (2/20) Reference Reference OR⁽ᵃ⁾ = Odds Ratio for cure compared to metronidazole/tinidazole monotherapy (reference group). Eighteen patients who failed initial treatment with NM or T + A and remained PCR-positive for DF were subsequently treated with PAR. Among them, 72.2% (13/18) achieved clinical cure, and 82.4% (14/17) achieved molecular cure. Further, we analyzed the association between clinical and molecular cure. Among 104 treatment courses (NM, T + A, or PAR) with available data on both outcomes, clinical cure was observed in 46 cases, of which 45 (97.8%) also achieved molecular cure. In contrast, among 58 treatment courses resulting in clinical failure, 49 (84.5%) remained PCR-positive for DF (p < 0.00001; Fig. 1 ). Patients with Blastocystis spp. coinfection were eligible for inclusion, as this organism is not considered pathogenic by most experts [ 21 ]. Coinfection with Blastocystis spp. was detected in 34 patients (33.0% of the cohort). Excluding these patients from analysis did not significantly alter the clinical or molecular cure rates (data not shown). Discussion Dientamoeba fragilis (DF) is frequently detected when molecular diagnostic testing is used as part of the routine workup for persistent gastrointestinal symptoms [ 16 ]. Despite ongoing debate regarding its pathogenicity, current clinical guidelines often recommend treatment in symptomatic patients with DF identified in the absence of other pathogens [ 17 ]. In our study, we observed significantly higher rates of both clinical and microbiological cure among symptomatic DF patients treated with paromomycin, compared to those treated with nitroimidazole-based therapy. The optimal treatment for DF remains controversial. Metronidazole has traditionally been considered the standard of care, largely due to its in vitro activity against the parasite [ 10 ]. However, a randomized controlled trial conducted in children more than a decade ago demonstrated no clinical benefit of metronidazole over placebo [ 9 ]. Additionally, several retrospective studies have shown superior outcomes with paromomycin compared to metronidazole, consistent with our findings [ 12 , 18 – 21 ]. Although clinical response rates with metronidazole in previous studies were somewhat higher than in our cohort, paromomycin consistently outperformed it. The relatively poor performance of metronidazole in our study may reflect increasing resistance or reduced efficacy along the years.. Interestingly, our results also suggest that combination therapy with an imidazole and albendazole leads to improved clinical outcomes compared to imidazole monotherapy. Thus, this regimen may be considered when first-line alternatives such as paromomycin or clioquinol are not available. A key finding in our study is the strong correlation between clinical cure and microbiological eradication of DF, as shown in Fig. 1 . This association lends support to the assumption that DF is a true pathogen. Although D. fragilis has been known for over a century [ 2 ] uncertainty regarding its clinical relevance persists. Much of the debate centers on observational data indicating similar detection rates of DF in symptomatic and asymptomatic individuals [ 22 ]. However, our findings, together with those from a large Finnish cohort, challenge this assumption. In the Finnish study, involving 297 episodes of DF treated with paromomycin and 84 with metronidazole, microbiological cure was significantly associated with symptom resolution (OR 5.85 [95% CI: 3.02–11.32], p < 0.001) [ 18 , 21 ]. Spontaneous clearance of DF has been described in previous studies [ 9 , 12 , 20 ], but our data highlight the chronic nature of symptomatic DF infections. The median symptom duration before presentation was nine months (IQR 4–36), suggesting that spontaneous resolution had not occurred and reflecting the hesitancy among clinicians to refer patients due to uncertainty regarding DF's pathogenic role. Our study has several limitations. It was observational, non-randomized, and conducted in a single center in a single country. However, patients were referred from across the country, and our findings are consistent with reports from other regions, particularly Europe. Moreover, although not interventional, the study reflects real-world clinical practice, which may better capture treatment effectiveness in community settings. In conclusion, D. fragilis should be considered in the differential diagnosis of patients with chronic gastrointestinal symptoms when no alternative etiology is found. Molecular testing is essential for accurate diagnosis. Our findings demonstrate a strong association between parasite eradication and symptom resolution, and paromomycin appears to be the most effective treatment currently available. However, randomized controlled trials are needed to validate these results and establish the optimal therapeutic approach for DF. Declarations Competing Interests The authors declare that they have no relevant financial or non-financial interests to disclose. Funding This study received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. Ethical Approval The study was approved by the Institutional Review Board of Sheba Medical Center . All procedures performed were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki declaration and its later amendments. Author Contributions E.S and D.Y conceptualized the study and supervised the project. Data collection was conducted by [A.B and E.S, and statistical analysis was performed by A.B. The manuscript was drafted by A.B. and critically revised by all authors. All authors read and approved the final manuscript. References Stark D, Al-Qassab SE, Barratt JLN, Stanley K, Roberts T, Marriott D, et al. Evaluation of multiplex tandem real-time PCR for detection of Cryptosporidium spp., Dientamoeba fragilis, Entamoeba histolytica, and Giardia intestinalis in clinical stool samples. J Clin Microbiol. 2011;49:257–62. Jepps MW, Dobell C. Dientamoeba fragilis n. g., n. sp., a new Intestinal Amoeba from Man. Parasitology. 1918;10:352–67. Dientamoeba fragilis. A harmless commensal or a mild pathogen? Paediatr Child Health. 1998;3:81–2. Jirků M, Kašparová A, Lhotská Z, Oborník M, Brožová K, Petrželková KJ et al. A Cross-Sectional Study on the Occurrence of the Intestinal Protist, Dientamoeba fragilis, in the Gut-Healthy Volunteers and Their Animals. Int J Mol Sci. 2022;23. Wong Z-W, Faulder K, Robinson JL. Does Dientamoeba fragilis cause diarrhea? A systematic review. Parasitol Res. 2018;117:971–80. Stark D, Barratt J, Chan D, Ellis JT. Dientamoeba fragilis, the Neglected Trichomonad of the Human Bowel. Clin Microbiol Rev. 2016;29:553–80. https:// Nagata N, Marriott D, Harkness J, Ellis JT, Stark D. In vitro susceptibility testing of Dientamoeba fragilis. Antimicrob Agents Chemother. 2012;56:487–94. Röser D, Simonsen J, Stensvold CR, Olsen KEP, Bytzer P, Nielsen HV, 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;58:1692–9. Schure JMA, de Ter M, Weel JFL, van Roon EN, Faber TE. Symptoms and treatment of Dientamoeba fragilis infection in children, a retrospective study. Pediatr Infect Dis J. 2013;32:e148–50. Hazenberg HMJL, Mank TG, Band C, Euser SM, van Soest EJ. A prospective analysis of clinical and parasitological outcomes after treatment or a wait-and-see approach of Dientamoeba fragilis infection in an adult general practice population. Eur J Clin Microbiol Infect Dis. 2025;44:143–50. Burgaña A, Abellana R, Yordanov SZ, Kazan R, Pérez Ortiz AM, Ramos CC, et al. Paromomycin is superior to metronidazole in Dientamoeba fragilis treatment. Int J Parasitol Drugs Drug Resist. 2019;11:95–100. Kurt O, Girginkardeşler N, Balcioğlu IC, Ozbilgin A, Ok UZ. A comparison of metronidazole and single-dose ornidazole for the treatment of dientamoebiasis. Clin Microbiol Infect. 2008;14:601–4. Meltzer E, Lachish T, Schwartz E. Treatment of giardiasis after nonresponse to nitroimidazole. Emerg Infect Dis. 2014;20:1742–4. Rifaximin for Irritable Bowel Syndrome without Constipation. N Engl J Med. 2011;364:1467–8. Peretz A, Azrad M, Ken- Dror S, Strauss M, Sagas D, Parizada M et al. The epidemiology of intestinal protozoa in the Israeli population based on molecular stool test: a nationwide study. Microbiol Spectr. 2024;12. van Gestel RS, Kusters JG, Monkelbaan JF. A clinical guideline on Dientamoeba fragilis infections. Parasitology. 2019;146:1131–9. Pietilä J-P, Häkkinen TA, Pakarinen L, Ollgren J, Kantele A. Treatment of Dientamoeba fragilis: A retrospective Finnish analysis of faecal clearance and clinical cure comparing four antiprotozoal drugs. New Microbes New Infect. 2023;54:101179. Clemente L, Pasut M, Carlet R, Ruscio M, Fontana F. Dientamoeba fragilis in the North-East of Italy: Prevalence study and treatment. Parasitol Int. 2021;80:102227. van Hellemond JJ, Molhoek N, Koelewijn R, Wismans PJ, van Genderen PJJ. Is paromomycin the drug of choice for eradication of Dientamoeba fragilis in adults? Int J Parasitol Drugs Drug Resist. 2012;2:162–5. Menéndez C, Fernández-Suarez J, Boga Ribeiro JA, Rodríguez-Pérez M, Vázquez F, Gonzalez-Sotorrios N, et al. Epidemiological and clinical characteristics of Dientamoeba fragilis infection. Enferm Infecc Microbiol Clin. 2019;37:290–5. Holtman GA, Kranenberg JJ, Blanker MH, Ott A, Lisman-van Leeuwen Y, Berger MY. Dientamoeba fragilis colonization is not associated with gastrointestinal symptoms in children at primary care level. Fam Pract. 2017;34:25–9. Supplementary Material The Supplementary Appendix file is not available with this version. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 23 Feb, 2026 Read the published version in Infection → Version 1 posted Editorial decision: Revision requested 15 Jan, 2026 Reviewers agreed at journal 12 Nov, 2025 Reviews received at journal 11 Nov, 2025 Reviewers agreed at journal 10 Oct, 2025 Reviews received at journal 19 Jul, 2025 Reviewers agreed at journal 16 Jul, 2025 Reviewers invited by journal 16 Jul, 2025 Editor assigned by journal 04 Jun, 2025 Submission checks completed at journal 04 Jun, 2025 First submitted to journal 03 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6814590","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":486361668,"identity":"57ffd73f-c70d-44c2-abdc-ae4e4a834f38","order_by":0,"name":"Asaf Biber","email":"","orcid":"","institution":"Sheba Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Asaf","middleName":"","lastName":"Biber","suffix":""},{"id":486361669,"identity":"ec902b8a-048e-4ef6-b53d-1174f6a30770","order_by":1,"name":"Dafna Yahav","email":"","orcid":"","institution":"Sheba Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Dafna","middleName":"","lastName":"Yahav","suffix":""},{"id":486361670,"identity":"983596d5-334a-4782-b724-b47d1206d2c8","order_by":2,"name":"Eli Schwartz","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAElEQVRIiWNgGAWjYDACdsYGBiBi4EcTl5DBqYUZqkWyDaoUpoUHtxYgBmkxOIaqhQGnFn5m5uYPP3cclje+3/xM4mMbQx3/jATGDz8YLHBqkWxmbJPsPXPYcNsxNjPJGWcYJCRuJDBL9uBxmMFhxjZmxrbbjNuOMZhJ81QAHXYjgUEan1/sDzM2fwZqsd/cxv5N+o8Bg4Q80Jbf+LQYAENMGqglcQMbj5k0A9AWgxsJbHhtkTgM8kvb/+QZx3KKLXvOSEhuPPOwzbLHALcW/vb2xx9+tqXZ9jcf33jjZ5sNv9zx5MM3flTUyeHSgmErAzSaiNUwCkbBKBgFowAbAAAB+ExAFdBfZgAAAABJRU5ErkJggg==","orcid":"","institution":"Sheba Medical Center","correspondingAuthor":true,"prefix":"","firstName":"Eli","middleName":"","lastName":"Schwartz","suffix":""}],"badges":[],"createdAt":"2025-06-03 21:38:01","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6814590/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6814590/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s15010-026-02746-6","type":"published","date":"2026-02-23T15:57:55+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":87361141,"identity":"9e7d1cc7-791f-4df8-ae3b-e7d33c8bda67","added_by":"auto","created_at":"2025-07-23 05:51:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":154912,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMolecular PCR result by clinical outcome.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eProportion of molecular PCR results by clinical outcome. Patients with clinical cure showed a markedly higher proportion of PCR-negative results (98%) compared to those with clinical failure (15.5%).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6814590/v1/e50f6cd6673680f172d0b4ea.png"},{"id":103765662,"identity":"3d5e2415-44ec-46bf-8f57-e22463d30260","added_by":"auto","created_at":"2026-03-02 16:06:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":583711,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6814590/v1/e3eef7bd-6fc4-4cd2-83e8-8c836a9b0120.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Treatment Outcomes in Symptomatic Dientamoeba fragilis Infection: A Prospective Clinical and Molecular Study","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cem\u003eDientamoeba fragilis\u003c/em\u003e (DF) is a binucleated flagellated protozoan and one of the most commonly detected intestinal parasites worldwide. Over the past decade, the use of molecular diagnostic techniques for stool testing has increased in many countries, particularly in the evaluation of gastrointestinal (GI) symptoms. As a result, DF has gained increased recognition and is now often referred to as an emerging protozoan pathogen [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eDespite its high global prevalence, the pathogenic potential of DF remains controversial. When Dobell and Jepps first described the organism in 1918, they concluded it was nonpathogenic\u0026mdash;even though six of the seven patients in their report presented with diarrhea or dysentery [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Several subsequent observations supported this nonpathogenic view, citing DF\u0026rsquo;s lack of tissue invasion and its frequent detection in asymptomatic individuals [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eA recent systematic review examining the causal role of DF in diarrheal illness reported conflicting results [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However,, DF infection may present with a broader spectrum of manifestations beyond diarrhea, including altered bowel habits, abdominal pain, bloating, and even extraintestinal features such as fatigue and eosinophilia [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn light of this uncertainty, treatment is still recommended for symptomatic patients in whom DF is the only identified pathogen. This recommendation is supported by the U.S. Centers for Disease Control and Prevention (CDC) and clinical platforms such as UpToDate and Medscape [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. However, there is no consensus regarding the optimal treatment regimen. Metronidazole, historically considered first-line therapy [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], failed to demonstrate superiority over placebo in a randomized controlled trial involving children [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Observational studies have reported higher rates of clinical and microbiological cure with other agents, such as paromomycin, clioquinol, and ornidazole, compared to metronidazole [\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eGiven these controversies, we conducted an observational study to assess the clinical and molecular response to various antiprotozoal agents commonly used in the treatment of symptomatic DF infection in routine clinical practice.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eA single-center, prospective observational study conducted at Sheba Medical Center between January 2019 and June 2023. We included consecutive patients presenting with chronic gastrointestinal (GI) symptoms lasting longer than one month and a positive stool PCR test for \u003cem\u003eDientamoeba fragilis\u003c/em\u003e (DF), either as a monoinfection or co-infection with \u003cem\u003eBlastocystis\u003c/em\u003e spp., Patients with elevated inflammatory markers were excluded.\u003c/p\u003e\u003cp\u003eMolecular testing was performed using the Allplex\u0026trade; GI Parasite Assay (Seegene, South Korea), a one-step real-time PCR that detects gastrointestinal parasites including: \u003cem\u003eBlastocystis hominis\u003c/em\u003e, \u003cem\u003eCryptosporidium\u003c/em\u003e spp., \u003cem\u003eCyclospora cayetanensis\u003c/em\u003e, \u003cem\u003eDientamoeba fragilis\u003c/em\u003e, \u003cem\u003eEntamoeba histolytica\u003c/em\u003e, and \u003cem\u003eGiardia lamblia\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eAt the initial visit, all patients completed a structured questionnaire that included travel history (when relevant), type and duration of symptoms, and the presence of the following gastrointestinal symptoms: loose stools, abdominal pain, bloating, flatulence and borborygmus, loss of appetite, weight loss, and fatigue (see Supplemental Appendix for questionnaire). Relevant prior investigations such as imaging studies or endoscopic procedures were also documented.\u003c/p\u003e\u003cp\u003eTreatment regimens were determined at the discretion of the attending physician or primary care provider. Some patients were prescribed a combination of tinidazole and albendazole, based on our clinic\u0026rsquo;s policy and previous experience with other intestinal protozoal infections [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Patients were asked to repeat the stool PCR test approximately one month after completing treatment.\u003c/p\u003e\u003cp\u003eClinical cure was assessed one month post-treatment and defined as resolution or significant improvement in gastrointestinal symptoms, based on a yes/no response to the question:\u003c/p\u003e\u003cp\u003e\u0026ldquo;In regard to all your gastrointestinal symptoms, as compared with the way you felt before you started the study medication, have you had adequate relief of your gastrointestinal symptoms?\u0026rdquo; [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eMolecular cure was defined as a negative stool PCR for DF at least one month following completion of treatment.\u003c/p\u003e\u003cp\u003ePatients who did not achieve clinical cure and remained PCR-positive were prescribed an alternative antiprotozoal therapy. Each treatment course, including multiple courses in the same patient, was evaluated independently. All patients with both clinical and molecular follow-up were included in the efficacy analysis. Patients without follow-up PCR were included in the symptom analysis. The first treatment course was assessed for clinical and molecular response, while all treatment courses were analyzed for correlation between clinical outcome and molecular clearance.\u003c/p\u003e\u003cp\u003eStatistical analysis was performed using Microsoft Excel 2016 (Microsoft Corp., Redmond, WA, USA). Continuous variables are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) or median with interquartile range (IQR). Categorical variables are reported as frequency and percentage. Group comparisons were performed using the Chi-square test or t-test for categorical and continuous variables, respectively. For expected frequencies\u0026thinsp;\u0026lt;\u0026thinsp;5, Fisher\u0026rsquo;s exact test was used. Binary logistic regression was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs) for clinical and molecular cure associated with each treatment. A p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\u003cp\u003e The study was approved by the Institutional Review Board (IRB) of Sheba Medical Center.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eOver the four-and-a-half-year study period, 105 symptomatic patients were referred to our clinic with a positive PCR result for \u003cem\u003eDientamoeba fragilis\u003c/em\u003e (DF), either as monoinfection (n\u0026thinsp;=\u0026thinsp;89, 84.8%) or in coinfection with \u003cem\u003eBlastocystis\u003c/em\u003e spp. (n\u0026thinsp;=\u0026thinsp;16, 15.2%). Demographic and clinical characteristics are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDemographic and Clinical Characteristics of the Study Population (N\u0026thinsp;=\u0026thinsp;105)\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCharacteristic\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eValue\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eDemographics\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAge, years (median, IQR)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e34 (21\u0026ndash;49)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFemale sex, n (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e57 (54.3%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTravel history prior to symptom onset, n (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e47 (44.8%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eSymptoms\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDuration of symptoms, months (median, IQR)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e9 (4\u0026ndash;36)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAbdominal pain, n (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e81 (77.1%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLoose stools, n (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e81 (77.1%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBloating, n (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e56 (53.3%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFatigue, n (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e58 (55.2%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWeight loss, n (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e29 (27.6%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"2\"\u003e\u003cem\u003eValues are presented as n (%) or median (interquartile range, IQR).\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003ePatient ages ranged from 2.5 to 83 years, with a median age of 34 years. Seventeen patients (16.5%) were children under 18 years of age (median age 9 years), while the median age among adults (\u0026ge;\u0026thinsp;18 years) was 39 years. Females comprised 54.3% of the cohort (n\u0026thinsp;=\u0026thinsp;57).\u003c/p\u003e\u003cp\u003eForty-seven patients (44.8%) reported international travel prior to symptom onset. Travel destinations included the Indian subcontinent (n\u0026thinsp;=\u0026thinsp;17), East and/or Southeast Asia (n\u0026thinsp;=\u0026thinsp;8), Latin America (n\u0026thinsp;=\u0026thinsp;7), Sub-Saharan Africa (n\u0026thinsp;=\u0026thinsp;6), North Africa (n\u0026thinsp;=\u0026thinsp;4), North America (n\u0026thinsp;=\u0026thinsp;2), Central Europe (n\u0026thinsp;=\u0026thinsp;2), and Eastern Europe (n\u0026thinsp;=\u0026thinsp;1).\u003c/p\u003e\u003cp\u003eThe most frequently reported symptoms were loose stools and abdominal pain, each occurring in 76.7% of patients, with a mean symptom duration of 9 months prior to referral (ranged 1 to 240 months). Seventeen patients (16.2%) underwent gastrointestinal endoscopy before their initial clinic visit, and all examinations were reported as normal.\u003c/p\u003e\u003cp\u003eOf the 105 patients, 96 (93.2%) returned for clinical follow-up, and 73 of these repeated also stool PCR testing.\u003c/p\u003e\u003cp\u003eRegarding treatment, 24 patients were prescribed nitroimidazole monotherapy (NM), either metronidazole 500 mg three times daily for 7\u0026ndash;10 days or tinidazole 1 g twice daily for 2\u0026ndash;3 days. Forty patients received a combination of tinidazole and albendazole (T\u0026thinsp;+\u0026thinsp;A), consisting of tinidazole 1 g twice daily for 2 days followed by albendazole 400 mg twice daily for 5 days. Twenty-nine patients were treated with paromomycin (PAR) 500 mg twice daily for 7 days. One patient received diloxanide, and two were treated with albendazole monotherapy.\u003c/p\u003e\u003cp\u003eClinical and molecular cure rates following the first treatment course, stratified by treatment regimen, are presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Patients treated with PAR achieved a clinical cure rate of 82.8% (24/29) and a molecular cure rate of 88.5% (23/26), both significantly higher than those treated with T\u0026thinsp;+\u0026thinsp;A (clinical: 35% [14/40]; molecular: 29.2% [7/24]) or NM (clinical: 4.2% [1/24]; molecular: 10% [2/20]). T\u0026thinsp;+\u0026thinsp;A was significantly superior to NM in terms of clinical cure (p\u0026thinsp;\u0026lt;\u0026thinsp;0.005), but not for molecular cure (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eClinical and Molecular Cure Rates According to Treatment Regimen\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTreatment\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eN\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eClinical Cure\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eOR⁽ᵃ⁾ (95% CI)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003ep-value\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eN (PCR)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eMolecular Cure\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eOR⁽ᵃ⁾ (95% CI)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003ep-value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eParomomycin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e82.8% (24/29)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e110.4 (12.0\u0026ndash;1018.5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e88.5% (23/26)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e69.0 (10.4\u0026ndash;457.9)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTinidazole\u0026thinsp;+\u0026thinsp;Albendazole\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e35.0% (14/40)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e12.4 (1.5\u0026ndash;101.6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e29.2% (7/24)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e3.7 (0.7\u0026ndash;20.4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMetronidazole/Tinidazole\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e4.2% (1/24)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eReference\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eReference\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e10.0% (2/20)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eReference\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eReference\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"9\"\u003e\u003cem\u003eOR⁽ᵃ⁾ = Odds Ratio for cure compared to metronidazole/tinidazole monotherapy (reference group).\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eEighteen patients who failed initial treatment with NM or T\u0026thinsp;+\u0026thinsp;A and remained PCR-positive for DF were subsequently treated with PAR. Among them, 72.2% (13/18) achieved clinical cure, and 82.4% (14/17) achieved molecular cure.\u003c/p\u003e\u003cp\u003eFurther, we analyzed the association between clinical and molecular cure. Among 104 treatment courses (NM, T\u0026thinsp;+\u0026thinsp;A, or PAR) with available data on both outcomes, clinical cure was observed in 46 cases, of which 45 (97.8%) also achieved molecular cure. In contrast, among 58 treatment courses resulting in clinical failure, 49 (84.5%) remained PCR-positive for DF (p\u0026thinsp;\u0026lt;\u0026thinsp;0.00001; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePatients with \u003cem\u003eBlastocystis\u003c/em\u003e spp. coinfection were eligible for inclusion, as this organism is not considered pathogenic by most experts [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Coinfection with \u003cem\u003eBlastocystis\u003c/em\u003e spp. was detected in 34 patients (33.0% of the cohort). Excluding these patients from analysis did not significantly alter the clinical or molecular cure rates (data not shown).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cem\u003eDientamoeba fragilis\u003c/em\u003e (DF) is frequently detected when molecular diagnostic testing is used as part of the routine workup for persistent gastrointestinal symptoms [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Despite ongoing debate regarding its pathogenicity, current clinical guidelines often recommend treatment in symptomatic patients with DF identified in the absence of other pathogens [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn our study, we observed significantly higher rates of both clinical and microbiological cure among symptomatic DF patients treated with paromomycin, compared to those treated with nitroimidazole-based therapy.\u003c/p\u003e\u003cp\u003eThe optimal treatment for DF remains controversial. Metronidazole has traditionally been considered the standard of care, largely due to its in vitro activity against the parasite [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, a randomized controlled trial conducted in children more than a decade ago demonstrated no clinical benefit of metronidazole over placebo [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Additionally, several retrospective studies have shown superior outcomes with paromomycin compared to metronidazole, consistent with our findings [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan additionalcitationids=\"CR19 CR20\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Although clinical response rates with metronidazole in previous studies were somewhat higher than in our cohort, paromomycin consistently outperformed it. The relatively poor performance of metronidazole in our study may reflect increasing resistance or reduced efficacy along the years..\u003c/p\u003e\u003cp\u003eInterestingly, our results also suggest that combination therapy with an imidazole and albendazole leads to improved clinical outcomes compared to imidazole monotherapy. Thus, this regimen may be considered when first-line alternatives such as paromomycin or clioquinol are not available.\u003c/p\u003e\u003cp\u003eA key finding in our study is the strong correlation between clinical cure and microbiological eradication of DF, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. This association lends support to the assumption that DF is a true pathogen. Although \u003cem\u003eD. fragilis\u003c/em\u003e has been known for over a century [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] uncertainty regarding its clinical relevance persists. Much of the debate centers on observational data indicating similar detection rates of DF in symptomatic and asymptomatic individuals [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. However, our findings, together with those from a large Finnish cohort, challenge this assumption. In the Finnish study, involving 297 episodes of DF treated with paromomycin and 84 with metronidazole, microbiological cure was significantly associated with symptom resolution (OR 5.85 [95% CI: 3.02\u0026ndash;11.32], p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSpontaneous clearance of DF has been described in previous studies [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], but our data highlight the chronic nature of symptomatic DF infections. The median symptom duration before presentation was nine months (IQR 4\u0026ndash;36), suggesting that spontaneous resolution had not occurred and reflecting the hesitancy among clinicians to refer patients due to uncertainty regarding DF's pathogenic role.\u003c/p\u003e\u003cp\u003eOur study has several limitations. It was observational, non-randomized, and conducted in a single center in a single country. However, patients were referred from across the country, and our findings are consistent with reports from other regions, particularly Europe. Moreover, although not interventional, the study reflects real-world clinical practice, which may better capture treatment effectiveness in community settings.\u003c/p\u003e\u003cp\u003eIn conclusion, \u003cem\u003eD. fragilis\u003c/em\u003e should be considered in the differential diagnosis of patients with chronic gastrointestinal symptoms when no alternative etiology is found. Molecular testing is essential for accurate diagnosis. Our findings demonstrate a strong association between parasite eradication and symptom resolution, and paromomycin appears to be the most effective treatment currently available. However, randomized controlled trials are needed to validate these results and establish the optimal therapeutic approach for DF.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCompeting Interests\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthical Approval\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved by the Institutional Review Board of Sheba Medical Center . All procedures performed were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki declaration and its later amendments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthor Contributions\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eE.S and D.Y conceptualized the study and supervised the project. Data collection was conducted by [A.B and E.S, and statistical analysis was performed by A.B. The manuscript was drafted by A.B. and critically revised by all authors. All authors read and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eStark D, Al-Qassab SE, Barratt JLN, Stanley K, Roberts T, Marriott D, et al. Evaluation of multiplex tandem real-time PCR for detection of Cryptosporidium spp., Dientamoeba fragilis, Entamoeba histolytica, and Giardia intestinalis in clinical stool samples. J Clin Microbiol. 2011;49:257\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJepps MW, Dobell C. \u003cem\u003eDientamoeba fragilis\u003c/em\u003e n. g., n. sp., a new Intestinal Amoeba from Man. Parasitology. 1918;10:352\u0026ndash;67.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDientamoeba fragilis. A harmless commensal or a mild pathogen? Paediatr Child Health. 1998;3:81\u0026ndash;2.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJirků M, Kašparov\u0026aacute; A, Lhotsk\u0026aacute; Z, Oborn\u0026iacute;k M, Brožov\u0026aacute; K, Petrželkov\u0026aacute; KJ et al. A Cross-Sectional Study on the Occurrence of the Intestinal Protist, Dientamoeba fragilis, in the Gut-Healthy Volunteers and Their Animals. Int J Mol Sci. 2022;23.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWong Z-W, Faulder K, Robinson JL. Does Dientamoeba fragilis cause diarrhea? A systematic review. Parasitol Res. 2018;117:971\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eStark D, Barratt J, Chan D, Ellis JT. Dientamoeba fragilis, the Neglected Trichomonad of the Human Bowel. Clin Microbiol Rev. 2016;29:553\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ehttps://\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003c/span\u003e\u003cspan address=\"http://www.cdc.gov/parasites/dientamoeba/health_professionals/index.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNagata N, Marriott D, Harkness J, Ellis JT, Stark D. In vitro susceptibility testing of Dientamoeba fragilis. Antimicrob Agents Chemother. 2012;56:487\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eR\u0026ouml;ser D, Simonsen J, Stensvold CR, Olsen KEP, Bytzer P, Nielsen HV, 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\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSchure JMA, de Ter M, Weel JFL, van Roon EN, Faber TE. Symptoms and treatment of Dientamoeba fragilis infection in children, a retrospective study. Pediatr Infect Dis J. 2013;32:e148\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHazenberg HMJL, Mank TG, Band C, Euser SM, van Soest EJ. A prospective analysis of clinical and parasitological outcomes after treatment or a wait-and-see approach of Dientamoeba fragilis infection in an adult general practice population. Eur J Clin Microbiol Infect Dis. 2025;44:143\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBurga\u0026ntilde;a A, Abellana R, Yordanov SZ, Kazan R, P\u0026eacute;rez Ortiz AM, Ramos CC, et al. Paromomycin is superior to metronidazole in Dientamoeba fragilis treatment. Int J Parasitol Drugs Drug Resist. 2019;11:95\u0026ndash;100.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKurt O, Girginkardeşler N, Balcioğlu IC, Ozbilgin A, Ok UZ. A comparison of metronidazole and single-dose ornidazole for the treatment of dientamoebiasis. Clin Microbiol Infect. 2008;14:601\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMeltzer E, Lachish T, Schwartz E. Treatment of giardiasis after nonresponse to nitroimidazole. Emerg Infect Dis. 2014;20:1742\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRifaximin for Irritable Bowel Syndrome without Constipation. N Engl J Med. 2011;364:1467\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePeretz A, Azrad M, Ken- Dror S, Strauss M, Sagas D, Parizada M et al. The epidemiology of intestinal protozoa in the Israeli population based on molecular stool test: a nationwide study. Microbiol Spectr. 2024;12.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003evan Gestel RS, Kusters JG, Monkelbaan JF. A clinical guideline on \u003cem\u003eDientamoeba fragilis\u003c/em\u003e infections. Parasitology. 2019;146:1131\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePietil\u0026auml; J-P, H\u0026auml;kkinen TA, Pakarinen L, Ollgren J, Kantele A. Treatment of Dientamoeba fragilis: A retrospective Finnish analysis of faecal clearance and clinical cure comparing four antiprotozoal drugs. New Microbes New Infect. 2023;54:101179.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eClemente L, Pasut M, Carlet R, Ruscio M, Fontana F. Dientamoeba fragilis in the North-East of Italy: Prevalence study and treatment. Parasitol Int. 2021;80:102227.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003evan Hellemond JJ, Molhoek N, Koelewijn R, Wismans PJ, van Genderen PJJ. Is paromomycin the drug of choice for eradication of Dientamoeba fragilis in adults? Int J Parasitol Drugs Drug Resist. 2012;2:162\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMen\u0026eacute;ndez C, Fern\u0026aacute;ndez-Suarez J, Boga Ribeiro JA, Rodr\u0026iacute;guez-P\u0026eacute;rez M, V\u0026aacute;zquez F, Gonzalez-Sotorrios N, et al. Epidemiological and clinical characteristics of Dientamoeba fragilis infection. Enferm Infecc Microbiol Clin. 2019;37:290\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHoltman GA, Kranenberg JJ, Blanker MH, Ott A, Lisman-van Leeuwen Y, Berger MY. Dientamoeba fragilis colonization is not associated with gastrointestinal symptoms in children at primary care level. Fam Pract. 2017;34:25\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Supplementary Material","content":"\u003cp\u003eThe Supplementary Appendix file is not available with this version.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"infection","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"infe","sideBox":"Learn more about [Infection](http://link.springer.com/journal/15010)","snPcode":"15010","submissionUrl":"https://submission.nature.com/new-submission/15010/3","title":"Infection","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Gastrointestinal symptoms, Paromomycin, Metronidazole, Tinidazole, Albendazole, Antiprotozoal therapy, Molecular diagnosis, PCR, Clinical cure, D. fragilis pathogenicity","lastPublishedDoi":"10.21203/rs.3.rs-6814590/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6814590/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003ePurpose\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe clinical significance and optimal treatment of \u003cem\u003eDientamoeba fragilis\u003c/em\u003e (DF) infection remain controversial, despite its frequent detection in patients with chronic gastrointestinal symptoms. This study aimed to evaluate clinical and molecular responses to various antiparasitic regimens and to assess the association between symptom resolution and parasite eradication.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn this prospective, single-center observational study conducted between January 2019 and June 2023, we included 105 symptomatic patients with a positive stool PCR for DF. Patients were treated with paromomycin (PAR), nitroimidazole monotherapy (NM; metronidazole/tinidazole), or a combination of tinidazole and albendazole (T\u0026thinsp;+\u0026thinsp;A). Clinical and molecular outcomes were assessed one month post-treatment using a structured symptom questionnaire and repeat PCR testing.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOf 96 patients with follow-up, 73 underwent repeat PCR testing. PAR was associated with significantly higher clinical and molecular cure rates (82.8% and 88.5%, respectively) compared to T\u0026thinsp;+\u0026thinsp;A (35.0% and 29.2%) and NM (4.2% and 10.0%). Combination therapy with T\u0026thinsp;+\u0026thinsp;A was superior to NM in clinical response but not in molecular clearance. A strong correlation was observed between clinical cure and DF eradication (p\u0026thinsp;\u0026lt;\u0026thinsp;0.00001), reinforcing the pathogenic role of DF. Median symptom duration before referral was 9 months, indicating the chronic nature of untreated infection.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusions\u003c/b\u003e\u003c/p\u003e\u003cp\u003eDF should be considered in patients with unexplained chronic gastrointestinal symptoms. Molecular testing is recommended for accurate diagnosis. Paromomycin appears to be the most effective treatment, with a strong association between clinical recovery and microbiological cure. Randomized controlled trials are warranted to further define optimal management strategies for DF infection.\u003c/p\u003e","manuscriptTitle":"Treatment Outcomes in Symptomatic Dientamoeba fragilis Infection: A Prospective Clinical and Molecular Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-23 05:51:07","doi":"10.21203/rs.3.rs-6814590/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-15T13:29:03+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"153925115976965425582085745656791924418","date":"2025-11-12T23:21:40+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-11T19:28:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"48381198862916418063252807559877426955","date":"2025-10-10T07:01:12+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-19T16:31:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"256444372778854341227220804369632308856","date":"2025-07-16T13:13:06+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-16T09:14:03+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-04T12:28:43+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-04T07:42:28+00:00","index":"","fulltext":""},{"type":"submitted","content":"Infection","date":"2025-06-03T21:22:27+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"infection","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"infe","sideBox":"Learn more about [Infection](http://link.springer.com/journal/15010)","snPcode":"15010","submissionUrl":"https://submission.nature.com/new-submission/15010/3","title":"Infection","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d90db40d-8197-4875-8d85-cbcba95e1867","owner":[],"postedDate":"July 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-02T16:03:52+00:00","versionOfRecord":{"articleIdentity":"rs-6814590","link":"https://doi.org/10.1007/s15010-026-02746-6","journal":{"identity":"infection","isVorOnly":false,"title":"Infection"},"publishedOn":"2026-02-23 15:57:55","publishedOnDateReadable":"February 23rd, 2026"},"versionCreatedAt":"2025-07-23 05:51:07","video":"","vorDoi":"10.1007/s15010-026-02746-6","vorDoiUrl":"https://doi.org/10.1007/s15010-026-02746-6","workflowStages":[]},"version":"v1","identity":"rs-6814590","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6814590","identity":"rs-6814590","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}