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Purpose: Dientamoeba fragilis (D. fragilis) is a common intestinal protozoan with a global distribution. In the present study, we aimed to determine genetic diversity of D. fragilis isolates with multilocus sequence typing (MLST) in the southwest of Turkey and analyse the clinical findings., Materials and Methods: The study included faecal samples from 200 individuals in Aydin, Turkey. The positivity of D. fragilis was determined with 18 S rRNA gene-based PCR assay. Six nested-PCR reactions were set to amplify partial D. fragilis housekeeping genes in the positive samples. The sequences were aligned with the references from GenBank to detect nucleotide polymorphisms and haplotypes. Additionally, the clinical findings and demographic characteristics of patients were statistically analysed between D. fragilis-infected and non-infected cases., Results: The positivity of D. fragilis was 16% (32 out of 200 cases) with 18 S rRNA based-PCR, and all were classified as "genotype 1". The analysis of six MLST loci revealed different haplotypes only at one locus; the remaining five loci exhibited no polymorphisms. The haplotypes in the present study were identical to at least one previously reported reference, except the locus "large subunit of RNA polymerase II" locus. There were no significant differences in any of the clinical findings or demographic characteristics between the infected and non-infected groups., Conclusions: Our study revealed a low genetic diversity of D. fragilis isolates from Turkey, like other countries including Italy, Denmark, the UK, Australia, and Brazil. The high degree of sequence similarity in housekeeping genes indicated the clonal distribution of D. fragilis., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Stool examination using microscopy was the traditional method for the diagnosis of intestinal parasites. Recently, the use of molecular tests to identify stool protozoa has become the main tool used in most clinical laboratories in Israel. This study aimed to evaluate the prevalence of intestinal parasites in Israel and to compare this prevalence in laboratories that use molecular tests vs a laboratory that uses microscopy. Samples collected from January to October 2021 at seven laboratories were analyzed by real-time PCR (RT-PCR) or by microscopy. The multiplex panel included the following pathogens: Giardia lamblia , Entamoeba histolytica , Cryptosporidium spp. , Cyclospora , Dientamoeba fragilis , and Blastocystis spp . Overall, 138,415 stool samples were tested by RT-PCR and 6,444 by microscopy. At least one protozoa species was identified in 28.4% of the PCR-tested samples compared to 4.6% of the microscopy-tested samples. D. fragilis was the most common PCR-identified species (29%). D. fragilis , G. lamblia , and Cryptosporidium spp . were mainly found in pediatric population, while Blastocystis spp . was most prevalent among adults ( P < 0.001). In a sub-cohort of 21,480 samples, co-infection was found in 4,113 (19.15%) samples, with Blastocystis spp . and D. fragilis being the most common (14.9%) pair. Molecular stool testing proved more sensitive compared to microscopy. D. fragilis was the most commonly detected pathogen. The above profile was identified during the COVID pandemic when traveling was highly restricted and most likely represents the locally circulating protozoa., Importance: This study sheds light on the prevalence of stool parasites in Israel. Additionally, this study indicates that the shift from microscope analysis to molecular tests improved protozoa diagnosis., Competing Interests: The authors declare no conflict of interest.

Blastocystis sp. and Dientamoeba fragilis are intestinal protists, which are common worldwide, but the pathogenic role of these organisms in gastrointestinal diseases is still controversial. This study aimed to investigate the frequency of Blastocystis sp. and D. fragilis in stool samples from adult patients with celiac disease (CD) by using conventional and molecular methods. A total of 75 patients with CD and 75 healthy individuals were included in this study. Fresh stool specimens collected from each individual were analyzed by conventional and molecular methods. The overall prevalence of Blastocystis sp. and D. fragilis was 41.3% (31/75) and 24% (18/75) in patients with CD, and 46.7% (35/75) and 13.3% (10/75) in healthy controls, respectively. There was no statistically significant difference in the prevalence of Blastocystis sp. and D. fragilis between CD patients and healthy individuals. Blastocystis sp. subtypes were identified in 20 CD and 16 control patients and the overall subtype distribution was observed as ST1 13.9%, ST2 30.6%, and ST3 55.6%. The prevalence of Blastocystis sp. and D. fragilis in adults with CD is similar to the prevalence of protozoa in healthy adults. In this study, the most prevalent Blastocystis subtype was ST3 and the most frequent allele was a34 in both CD patients and healthy individuals. No significant difference was found between the two groups in terms of the detection rates of Blastocystis sp. and D. fragilis, and it is thought that both protists may be colonisers of the intestinal microbiome., Competing Interests: Declaration of competing interest Not applicable., (Copyright © 2024. Published by Elsevier B.V.)

Dientamoeba fragilis (D. fragilis) represents a common protozoan in both high and low income countries. Despite this, epidemiological data on dientamoebiasis are still limited, and it is possible that the actual prevalence rates of D. fragilis have been underestimated due to the challenges in its detection and identification. In the present study, symptomatic patients from Rome (Central Italy) were surveyed for two years to determine D. fragilis percentage of infection and genotypes. Stool samples collection was performed over 864 patients, DNA extracted, and RT-PCR performed by the SeeGene Allplex™ Gastrointestinal Parasite Panel Assays. Seventy-nine resulted positive for D. fragilis (9.1%). Co-infections were detected in 22 isolates: 21 displayed Blastocystis sp. + D. fragilis (27.8%). Based on the sequence of a central fragment of the SSU rRNA gene, only genotype 1 was identified. These findings are among the few available data regarding genetic diversity of D. fragilis in Italy. Large-scale human and animal research are required to enhance our knowledge of prevalence, host range, genetic variability and zoonotic transmission of this little-known intestinal protozoan., Competing Interests: Declaration of Competing Interest The authors declare that no conflict of interest exists., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Dientamoeba fragilis is a ubiquitous intestinal parasite with detection in the stools that has become increasingly frequent following the advent of PCR as a routine screening tool. However, the pathogenicity of this parasite is still much debated. In order to assess the potentially pathogenic nature of this protozoan, a retrospective case-control study was carried out between January and December 2020 on patients from Toulouse University Hospital, with the aim of evaluating the potential clinical effects and changes in laboratory parameters linked to the presence and load of D. fragilis in stools. After matching age, sex and mode of care (consultation or hospitalisation), no significant difference was observed in the frequency of clinical signs between the 36 patients who tested positive for Dientamoeba fragilis PCR in their stools and the 72 control patients who were PCR negative for this protozoan. The presence of D. fragilis in the faeces was not associated with changes in laboratory parameters. Furthermore, a high digestive load of D. fragilis had no identifiable impact on clinical and laboratory parameters. Only the concomitant presence of Blastocystis sp. in stools was significantly more frequent in the D. fragilis group (uni- and multivariate analysis). Finally, this study showed no significant difference in clinical or laboratory signs between patients carrying Dientamoeba fragilis and the control group, regardless of the intestinal parasite load, suggesting that D. fragilis could be considered a commensal of the digestive tract., (© G. Tchamwa Bamini et al., published by EDP Sciences, 2024.)

Objectives: The aim of this study was to assess the clinical significance of Dientamoeba fragilis (DF) and Blastocystis species (Bs) in human stool., Methods: Observational study of patients ≥18 years, who were tested by stool multiplex PCR for bacteria and parasites between April 2019 and March 2022. Although DF and Bs are part of the PCR kit, these results are not routinely reported to the patient or the ordering physician. The main outcomes were the incidence of symptoms during 14 days before the referral to stool PCR test, and the incidence of several clinical outcomes during 60 days after the PCR test (symptoms, referrals to further evaluation, prescription of symptomatic, or antibiotic treatment)., Results: A total of 27 918 patients were tested by stool PCR during the 3 study years. A total of 6215 (22.3%) and 5337 (19.2%) were positive for DF and Bs, respectively. The incidence of symptoms before the test was similar in those positive for Bs or DF and those with all-negative PCR (adjusted OR and 95% CI of 0.87 [0.80-0.95] and 0.82 [0.76-0.88] for Bs and DF, respectively), whereas significantly higher (2.47 [2.23-2.73]) in those positive for the other multiplex PCR assay components. During the 60 days after the test, the prevalence of any of the outcomes was similar in those positive for Bs or DF and those with negative PCR (adjusted OR and 95% CI of 0.92 [0.83-1.02] and 0.89 [0.81-0.97] for symptoms, 0.84 [0.75-0.94] and 0.93 [0.85-1.01] for referrals, 0.88 [0.75-1.03] and 0.82 [0.71-0.94] for symptomatic treatment, and 0.88 [0.75-1.02] and 0.86 [0.75-0.98] for antibiotic treatment in the Bs and DF positive individuals, respectively). The PCR cycle threshold was not associated with any of the outcomes., Discussion: Positive stool PCR for DF or Bs was not associated with any of the measured clinical outcomes., (Copyright © 2023 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

To investigate the prevalence of Blastocystis and Dientamoeba fragilis in diarrhea patients and healthy individuals in Corum, Türkiye, fecal samples from 92 diarrhea patients and 50 healthy individuals were collected and evaluated using direct microscopy and molecular methods to screen for bacteria, protozoa, and viruses. The prevalence of Blastocystis was 24.6% in total and more frequent in the healthy group (30.0%). The commonly detected STs (subtypes) were ST3 (40.0%) and ST2 (34.2%). The distribution of Blastocystis STs in the healthy and diarrheal groups did not show any difference in sex and age, but ST3 was detected more frequently in patients aged from 40 to 59 years (p < 0.05). Alleles 4 (8/12) and 2 (4/12) were present in ST1; 9 (3/5) and 12 (2/5) in ST2; 34 (9/14), 36 (3/14), and 38 (2/14) in ST3; and only allele 42 (2/2) in ST4. D. fragilis was present in 8.4% of the population. However, there was no statistically significant difference between the healthy and diarrheic groups (12.0% and 6.5%, respectively), neither with respect to age nor sex. Co-infection was 58.3% and was more frequent in healthy individuals (33.3%) than in diarrhea patients (25.0%). Blastocystis ST3 was the most common subtype detected, with D. fragilis at 33.3%. Salmonella, Shigella, or helminth eggs were not observed in all groups, but Entamoeba histolytica, Giardia intestinalis, Cryptosporidium, Rotavirus, Adenovirus, and Clostridium difficile toxin were found only in diarrhea patients. These findings support the hypothesis that Blastocystis and D. fragilis may be part of the healthy human gut microbiome., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Dientamoeba fragilis is a cosmopolitan intestinal protist colonizing the human gut with varying prevalence depending on the cohort studied and the diagnostic methods used. Its role in human health remains unclear mainly due to the very sporadic number of cross-sectional studies in gut-healthy populations. The main objective of this study was to expand knowledge of the epidemiology of D. fragilis in gut-healthy humans and their animals. A total of 296 stool samples from humans and 135 samples from 18 animal species were analyzed. Using qPCR, a prevalence of 24% was found in humans in contrast to conventional PCR (7%). In humans, several factors were found to influence the prevalence of D. fragilis . A more frequent occurrence of D. fragilis was associated with living in a village, traveling outside Europe and contact with farm animals. In addition, co-infection with Blastocystis spp. was observed in nearly half of the colonized humans. In animals, D. fragilis was detected in 13% of samples from eight species using qPCR. Our molecular phylogenies demonstrate a more frequent occurrence of Genotype 1 in gut-healthy humans and also revealed a likely a new protist species/lineage in rabbits related to D. fragilis and other related organisms.

Dientamoeba fragilis is a flagellated protozoan with amoeba-like morphology that inhabits the human gastrointestinal tract. It is endemic in a vast geography around the world, including developed countries. There are limited studies on non-human hosts of the parasite, and suitable hosts have not been clarified. The parasite has been detected in non-human primates, pigs, cats, dogs and rats. There is no study in the literature investigating and detecting the presence of this parasite in cattle. In this study, stool samples taken from 163 different cattle and calves from 11 different farms between March 2017 and May 2022 were examined for the detection of D. fragilis via PCR. Trichrome staining was performed on all PCR-positive samples. The isolates with the expected amplicon size were sequenced using the 18S ribosomal RNA region, and their genotypes were determined by BLAST analysis. Sequences were analysed with the most similar and reference sequences in the literature, forming a phylogenetic tree. We detected D. fragilis in 31 (19.01%) of the 163 stool samples. D. fragilis cysts/trophozoites were detected by trichrome staining method in six of 31 samples. All PCR products selected for molecular analysis from positive samples had the same nucleotide sequence. As a result of BLAST analysis, all sequences were determined to belong to D. fragilis genotype 1. This study determined for the first time that cattle are suitable hosts for D. fragilis. Furthermore, the parasite subtype we detected belongs to genotype 1, which is the most common type in humans, suggesting that the parasite may have a zoonotic character. Our result is important in terms of the epidemiology of the parasite, as the mode of transmission is controversial, and available data on its suitable hosts are limited., (© 2022 Wiley-VCH GmbH.)

The protozoan Dientamoeba fragilis is one of the most common parasites in the digestive system of humans worldwide. The host range and transmission routes of D. fragilis, including the role of animals, are still ambiguous with few reports from non-human primates, sheep, rodents, pigs, a cat and a dog. In this study, we used microscopic and TaqMan qPCR analyses to investigate D. fragilisin 150 faecal samples from pet budgerigars (Melopsittacus undulatus) in the Central Anatolia Region of Turkey. Dientamoeba fragilis DNA was detected in 32 samples, resulting in a mean prevalence of 21.3%. In microscopic examination, trophozoites/cysts of D. fragilis were detected in 13 of 32 qPCR-positive samples. SSU rRNA sequence analyses of the qPCR-positive isolates identified genotype 1 of D. fragilis as predominant in budgerigars. Phylogenetic analyses of the SSU rRNA gene region clustered D. fragilis genotypes, as well as other trichomonads, in separate monophyletic clusters with bootstrap values ≥79.0. Our study provides the first evidence for the natural host status of pet budgerigars for D. fragilisand contributes to the knowledge of the epidemiology of this parasite. The high prevalence of genotype 1 of D. fragilis suggests that pet budgerigars are suitable reservoirs for zoonotic transmission. Our findings contribute to an increased awareness and knowledge of D. fragilis infections in the context of a one-health approach., (© 2022 Wiley-VCH GmbH.)

Introduction: The clinical significance of Blastocystis sp. and Dientamoeba fragilis in patients with gastrointestinal symptoms is a controversial issue. Since the pathogenicity of these protists has not been fully elucidated, testing for these organisms is not routinely pursued by most laboratories and clinicians. Thus, the prevalence of these organisms and the subtypes of Blastocystis sp. in human patients in Turkey are not well characterized. This study aimed to determine the prevalence of Blastocystis sp. and D. fragilis in the diarrheic stool samples of immunodeficient and immunocompetent patients using conventional and molecular methods and to identify Blastocystis sp. subtypes using next generation sequencing., Material and Methods: Individual stool specimens were collected from 245 immunodeficient and 193 immunocompetent diarrheic patients between March 2017 and December 2019 at the Gazi University Training and Research Hospital in Ankara, Turkey. Samples were screened for Blastocystis sp. and D. fragilis by conventional and molecular methods. Molecular detection of both protists was achieved by separate qPCRs targeting a partial fragment of the SSU rRNA gene. Next generation sequencing was used to identify Blastocystis sp. subtypes., Results: The prevalence of Blastocystis sp. and D. fragilis was 16.7% and 11.9%, respectively as measured by qPCR. The prevalence of Blastocystis sp. and D. fragilis was lower in immunodeficient patients (12.7% and 10.6%, respectively) compared to immunocompetent patients (21.8% and 13.5%, respectively). Five Blastocystis sp. subtypes were identified and the following subtype distribution was observed: ST3 54.4% (n = 37), ST2 16.2% (n = 11), ST1 4.4% (n = 3), ST6 2.9% (n = 2), ST4 1.5% (n = 1), ST2/ST3 11.8% (n = 8) and ST1/ST3 8.8% (n = 6). There was no statistically significant difference in the distribution of Blastocystis sp. subtypes between immunocompetent and immunodeficient patients., Conclusion and Recommendation: Our findings demonstrated that Blastocystis sp. and D. fragilis are commonly present in immunocompetent and immunodeficient patients with diarrhea. This study is the first to use next generation sequencing to address the presence of Blastocystis sp. mixed subtypes and intra-subtype variability in clinical samples in Turkey., Competing Interests: The authors have declared that no competing interests exist.

Objective: Dientamoeba fragilis ( D. fragilis ) is a flagellated protozoan with an amoeba-like morphology, located in the gastrointestinal tract. The hypothesis was that the parasite was transported by Enterobius vermicularis ( E. vermicularis ) eggs. This study aimed to determine the association of D. fragilis and E. vermicularis with the genotypes of the identified strain of D. fragilis . Results of trichrome staining were compared with those of polymerase chain reaction (PCR), which is widely used in the diagnosis of D. fragilis ., Methods: A total of 391 samples were obtained. The stool and cellophane slide samples were sent together to the Parasitology Department Laboratory, Faculty of Medicine, Aydın Adnan Menderes University, between 1 October 2017 and 1 October 2018. Stool samples of all patients with E. vermicularis (n=74) and without E. vermicularis (n=74) infection were used. All samples were examined for the presence of D. fragilis by trichrome staining and PCR. The 18S ribosomal RNA region of D. fragilis isolates was sequenced. Demographic characteristics and clinical findings of the patients were evaluated., Results: D. fragilis was detected in 42 (28.37%) of 148 samples; 28 (66.6%) of them were detected in patients with E. vermicularis infection. The coexistence of two parasites was significant (p<0.05). All isolates sequenced were genotype 1. No significant relationship was found between the presence of parasites and clinical findings, living area and gender (p>0.05)., Conclusion: D. fragilis is frequently associated with E. vermicularis , so the presence of D. fragilis should be also considered in affected patients. The use of high-sensitivity molecular methods such as PCR is important in preventing false results., Amaç: Dientamoeba fragilis ( D. fragilis ), amip benzeri morfolojiye sahip, gastrointestinal yerleşimli, kamçılı bir protozoondur. Parazitin Enterobius vermicularis ( E. vermicularis ) yumurtalarıyla taşındığı hipotezi kabul görmektedir. Çalışmamızda D. fragilis ve E. vermicularis birlikteliğini incelemek, bulunan D. fragilis ’lerin genotiplerini belirlemek ve D. fragilis tanısında yaygın olarak kullanılan trikrom boyama ile polimeraz zincir reaksiyon (PZR) yöntemlerini karşılaştırmak amaçlanmıştır., Yöntemler: Çalışmamızda Aydın Adnan Menderes Üniversitesi Tıp Fakültesi, Parazitoloji Laboratuvarı’na 1 Ekim 2017-1 Ekim 2018 tarihleri arasında dışkı ve selofan lam örneği birlikte gönderilmiş toplam 391 olgu örneği incelenmiştir. Selofanlı lam örneklerinde E. vermicularis saptanan tüm gönüllü olguların (74 olgu) dışkı örneği ile E. vermicularis negatif 74 olgunun dışkı örneği çalışılmıştır. Tüm dışkılar trikrom boyama ve PZR yöntemleri ile D. fragilis varlığı açısından incelenmiştir. Saptanan D. fragilis izolatlarının 18S ribozomal RNA bölgesi sekanslanmıştır. Olguların demografik özellikleri ve kliniği değerlendirilmiştir., Bulgular: Toplam 148 olgunun 42’sinde (%28,37) D. fragilis saptanmıştır. D. fragilis pozitif olan 42 olgunun %66,6’sını E. vermicularis pozitif olgular oluşturmuş ve iki parazitin birlikteliği anlamlı bulunmuştur (p<0,05). Sekanslanan tüm izolatlar genotip 1 olarak saptanmıştır. Klinik bulgular, yaşanılan bölge ve cinsiyet ile parazit varlığı arasında anlamlı bir ilişki saptanamamıştır (p>0,05)., Sonuç: Araştırmamızda D. fragilis ’in sıklıkla E. vermicularis ile birliktelik gösterdiği ve bu olgularda D. fragilis varlığına ayrıca dikkat edilmesi gerektiği vurgulanmıştır. Yanlış sonuçları engellemede, yüksek duyarlılığa sahip PZR gibi yöntemlerin önemi bir kez daha görülmüştür.

Objectives: The intestinal parasite Dientamoeba fragilis is a common colonizer of children in Denmark. Metronidazole has been used to reduce gastrointestinal symptoms in children colonized with D fragilis. We aimed to identify gut microbiota changes associated with D fragilis carrier status and metronidazole treatment of D fragilis-positive children., Methods: The fecal microbiota of 275 fecal samples from children treated with metronidazole (n = 48) or placebo (n = 48) were characterized by ribosomal DNA sequencing. Samples collected before (T1), 2 weeks after (T2), and 8 weeks (T5) after treatment were included. Seventy fecal samples from 70 age-matched parasite-negative children served as controls., Results: The abundance of 24 bacterial genera differed significantly according to D fragilis carrier status, with Flavonifractor being remarkably more abundant in children testing negative for D fragilis. Eight bacterial genera changed significantly in abundance in children losing versus keeping D fragilis after metronidazole treatment. Of these, 7 returned to pretreatment (T1) levels at T5. Meanwhile, the abundance of Flavonifractor continued to differ at T5, whereas for Ruminococcus the abundance only remained high in children who were D fragilis-negative at T2 and T5. Increases in Hungatella, Sutterella, and Streptococcus abundances observed at T2 were specific to metronidazole exposure and hence independent of D fragilis colonization., Conclusions: This study revealed that specific bacterial genera were associated with D fragilis colonization. Metronidazole treatment had a short-term impact on the abundance of some bacterial genera, with most of these reverting to pretreatment levels 8 weeks after completed treatment., Competing Interests: The authors report no conflicts of interest., (Copyright © 2021 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition.)

In order to provide additional data on the prevalence and genetic diversity of Dientamoeba fragilis in human populations, we conducted a study in children from low-income communities in Sao Paulo State, Brazil. Fecal samples from daycare center attendees up to 6 years old (n=156) and staff members (n=18) were submitted to PCR and sequencing of D. fragilis as well as to microscopic examination for the presence of other intestinal parasites. All children assessed were asymptomatic and 10.3% (16/156) were positive for D. fragilis. No worker was found to be positive. An association between Dientamoeba and coinfection with other intestinal parasites was observed. Concerning the genetic diversity, 14 and only two isolates were genotype 1 and genotype 2, respectively. Our findings outline interesting aspects: (1) asymptomatic children as carriers of Dientamoeba in communities in which environmental conditions ensure parasite transmission and, (2) association between Dientamoeba infection in young children and coinfection with other enteric parasites, reinforcing its transmission via the fecal-oral route.

Background: Dientamoeba fragilis in children has been associated with gastrointestinal symptoms, like abdominal pain and diarrhea. The mechanism underlying these symptoms in children with D. fragilis remains unclear. We hypothesized that concomitant microbial alterations, which have been described in other parasitic infections, may be associated with gastrointestinal symptoms in D. fragilis., Methods: In this case-control study performed in 2 centers, 19 children referred to a pediatrician because of gastrointestinal symptoms and with a positive fecal PCR for D. fragilis were included as cases. We included 19 healthy children as controls and matched for age and gender, selected from an existing cohort of 63 children. A PCR for D. fragilis was performed on fecal samples of the 19 controls to assess D. fragilis carriership in this asymptomatic group. Microbiota was analyzed with the IS-pro technique, and the intestinal microbiota composition and diversity were compared between the 2 groups., Results: Microbiota of children with D. fragilis and gastrointestinal symptoms did not significantly differ in terms of composition and diversity compared with controls, both on phylum and species level. In the asymptomatic controls, a positive fecal PCR for D. fragilis was found in 16 of 19 (84.2%)., Conclusion: Intestinal microbiota does not seem to play a key role in the presence of clinical symptoms in children with D. fragilis. The pathogenicity of D. fragilis and pathophysiologic pathways underlying the development of gastrointestinal symptoms remains yet to be clarified., Competing Interests: A.E.B. is CEO of InBiome and has the proprietary rights on the IS-pro platform technology. M.A.B. is a scientific consultant for Shire, Sucampo, Astrazeneca, Norgine, Zeria, Coloplast, Danone, Friesland Campina, Sensus, and Novalac. P.H.M.S. is scientific advisor of InBiome and has to proprietary rights on the IS-pro platform technology. The other authors have no funding or conflicts of interest to disclose., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Nucleic acid-based methods are increasingly used for screening of gastrointestinal parasites. Microscopy is still used and Swedish routine protocol consists of formalin ethyl-acetate concentration and do not include screening for trophozoites or Cryptosporidium spp. This study aimed to compare detection with the Swedish routine microscopy method to an extended method that includes screening for trophozoites and Cryptosporidium. Furthermore, we also developed a method for DNA recovery from SAF-fixed faecal samples and compared the real-time PCR detection of Giardia intestinalis, Dientamoeba fragilis , Cryptosporidium spp., Entamoeba histolytica and Entamoeba dispar from SAF-fixed and unpreserved faecal samples. PCR results were then compared with microscopy results. Methods: SAF-fixed and unpreserved faecal samples from 1000 patients at the Clinical microbiology laboratory in Region Jönköping County, Sweden, were included. Samples were analysed with routine formalin ethyl-acetate concentration, wet mounts from both concentrated and unconcentrated samples, Ziehl-Neelsen staining on patients with certain symptoms and real-time PCR. Results: We found a significant higher detection rate of parasites with the extended microscopy method compared to the Swedish routine microscopy method when SAF-fixed samples were used. The detection rate with real-time PCR in SAF-fixed samples was equal to the detection rate in unpreserved samples. There was no significant difference in detection comparing extended microscopy and real-time PCR. Conclusion: In conclusion, this study showed that the extended microscopy method increased detection of intestinal protozoa with detection of both trophozoites and Cryptosporidium spp. We also showed that SAF-fixative can be used for detection of parasite-DNA with real-time PCR.

The presence of bacterial DNA in Dientamoeba fragilis DNA extracts from culture poses a substantial challenge to sequencing the D. fragilis genome. However, elimination of bacteria from D. fragilis cultures has proven difficult in the past, presumably due to its dependence on some unknown prokaryote/s. This study explored options for removal of bacteria from D. fragilis cultures and for the generation of genome sequence data from D. fragilis. DNA was extracted from human faecal samples and xenic D. fragilis cultures. Extracts were subjected to 16S ribosomal DNA bacterial diversity profiling. Xenic D. fragilis cultures were then subject to antibiotic treatment regimens that systematically removed bacterial species depending on their membrane structure (Gram-positive or Gram-negative) and aerobic requirements. The impact of these treatments on cultures was assessed by 16S amplicon sequencing. Prior to antibiotic treatment, the cultures were dominated by Gram-negative bacteria. Addition of meropenem to cultures eliminated anaerobic Gram-negative bacteria, but it also led to protozoan death after 5 days incubation. The seeding of meropenem resistant Klebsiella pneumoniae strain KPC-2 into cultures before treatment by meropenem prevented death of D. fragilis cells beyond this 5 day period, suggesting that one or more species of Gram-negative bacteria may be an essential nutritional requirement for D. fragilis. Gram-positive cells were completely eliminated using vancomycin without affecting trophozoite growth. Finally, this study shows that genome sequencing of D. fragilis is feasible following bacterial elimination from cultures as the result of the major advances occurring in bioinformatics. We provide evidence on this fact by successfully sequencing the D. fragilis 28S large ribosomal DNA subunit gene using culture-derived DNA.

Dientamoeba fragilis (D. fragilis) is an intestinal parasite frequently detected in humans with abdominal pain and diarrhoea, but it is also commonly found in asymptomatic subjects. Hence its clinical relevance is often disputed. The introduction of polymerase chain reaction (PCR) is a versatile and sensitive diagnostic technique for the detection of intestinal parasites, and in some Western world countries PCR has almost completely replaced microscopic diagnostics. PCR has however resulted in an increase in the number of D. fragilis-positive patients. The disputed pathogenic nature of this intestinal parasite and an apparent increase in the incidence of patients with positive PCR results have renewed the discussions between clinicians and microbiologists on how to deal with an infected patient. Moreover, treatment guidelines differ throughout the world which makes it difficult for clinicians to choose an optimal therapeutic regimen.AimTo summarize and discuss the current knowledge on the pathogenicity, best diagnostic approach, treatment and follow-up of children and adults infected with D. fragilis.

BackgroundDespite the global distribution of the intestinal protozoan Dientamoeba fragilis, its clinical picture remains unclear. This results from underdiagnosis: microscopic screening methods either lack sensitivity (wet preparation) or fail to reveal Dientamoeba (formalin-fixed sample).AimIn a retrospective study setting, we characterised the clinical picture of dientamoebiasis and compared it with giardiasis. In addition, we evaluated an improved approach to formalin-fixed samples for suitability in Dientamoeba diagnostics.MethodsThis study comprised four parts: (i) a descriptive part scrutinising rates of Dientamoeba findings; (ii) a methodological part analysing an approach to detect Dientamoeba -like structures in formalin samples; (iii) a clinical part comparing demographics and symptoms between patients with dientamoebiasis (n = 352) and giardiasis (n = 272), and (iv) a therapeutic part (n = 89 patients) investigating correlation between faecal eradication and clinical improvement.ResultsThe rate of Dientamoeba findings increased 20-fold after introducing criteria for Dientamoeba -like structures in formalin-fixed samples (88.9% sensitivity and 83.3% specificity). A further increase was seen after implementing faecal PCR. Compared with patients with giardiasis, the symptoms in the Dientamoeba group lasted longer and more often included abdominal pain, cramping, faecal urgency and loose rather than watery stools. Resolved symptoms correlated with successful faecal eradication (p < 0.001).ConclusionsPreviously underdiagnosed, Dientamoeba has become the most frequently recorded pathogenic enteroparasite in Finland. This presumably results from improved diagnostics with either PCR or detection of Dientamoeba -like structures in formalin-fixed samples, an approach applicable also in resource-poor settings. Symptoms of dientamoebiasis differ slightly from those of giardiasis; patients with distressing symptoms require treatment.

Objective: To study the association between Dientamoebafragilis colonisation and faecal calprotectin to see whether the parasite is a harmless commensal or a gut pathogen., Design: Cross-sectional study of previously collected stool samples., Setting and Patients: Two hundred stool samples originated from children aged 5-19 years with chronic abdominal pain and diarrhoea, who were seen in paediatric clinics in the Netherlands and Belgium and in whom somatic gastrointestinal disorders were excluded. Another 122 samples came from a healthy community-based reference population of the same age. All stool samples were analysed with real-time PCR for the detection of D. fragilis and with an ELISA for calprotectin-a biomarker of gastrointestinal inflammation., Main Outcome Measures: Prevalence of D. fragilis colonisation and results of stool calprotectin testing., Results: D. fragilis was detected in 45% (95% CI 38% to 51%) of patients and in 71% (95% CI 63% to 79%) of healthy children. Median (IQR) concentrations of calprotectin in patients and healthy children with a positive PCR result were not different from those with a negative PCR result (40 (40-55) μg/g vs 40 (40-75) μg/g, respectively)., Conclusion: Since D. fragilis colonisation is most prevalent in healthy children and is not associated with an increase in faecal calprotectin concentration, our data do not support the inference that D. fragilis is a pathogenic parasite. Routinely testing for D. fragilis in children with chronic abdominal pain should therefore be discouraged., Competing Interests: Competing interests: PFvR received financial support from BÜHLMANN Laboratories AG (Schönenbuch, Switzerland) for other ongoing trials., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Dientamoebiasis is globally distributed and detected in a large number of subjects with diarrhea, abdominal discomfort, flatulence, fatigue and loss of appetite. The life cycle and transmission of Dientamoeba fragilis are poorly understood. Microscopic examination of permanent stained smears is traditionally employed to diagnose the infection. However, this approach is time-consuming and the success in detecting D. fragilis depends on the microscopist's experience. Hence, only a few laboratories routinely carry out tests for D. fragilis. Consequently, the prevalence of D. fragilis infection is probably underestimated. Although novel, rapid and more sensitive diagnostic tests are becoming available for detecting intestinal parasites, they also possess some limitations. The aim of this study was to emphasize the importance of performing microscopic examination of permanent stained smears from at least one fresh stool specimen after sample arrival at the laboratory, as a mandatory practice for the diagnosis of dientamoebiasis, particulary where it is not possible to perform molecular assays.

Dientamoeba fragilis is a gastrointestinal trichomonad parasite whose pathogenicity is yet to be determined. The difficulty involved in microscopically diagnosing D. fragilis in feces led to the development of real-time PCR methodologies for the detection of D. fragilis in stool samples. Prevalence studies in Europe show much higher levels of infection where a laboratory-developed real-time assay is the predominant assay for the detection of Dientamoeba fragilis than in regions that use the EasyScreen assay for detection of gastrointestinal pathogens. The aim of this study was to compare a commercially available Dientamoeba fragilis assay (Genetic Signatures EasyScreen assay) to a widely used laboratory-developed real-time PCR method. Two hundred fifty fecal samples were screened using the laboratory-developed real-time assay on four real-time PCR platforms producing a number of discrepant results. Limit-of-detection studies were undertaken to attempt to resolve sensitivity for each platform tested. The presence or absence of Dientamoeba fragilis DNA in discrepant samples was shown using PCR amplicon next-generation sequencing. Eukaryotic 18S diversity profiling was conducted on discrepant samples to identify the presence or absence of additional protozoan species in samples that may be responsible for cross-reactivity seen in these samples. The results revealed the potential for multiple false-positive results when using the laboratory-developed real-time assay across multiple real-time platforms using manufacturer default settings. This report provides recommendations to resolve these issues where possible and suggestions for future prevalence studies, and it emphasizes the EasyScreen assay as the molecular method of choice as well as the need for standardization of detection assays across all nations screening for D. fragilis ., (Copyright © 2019 American Society for Microbiology.)

Objectives: Multiplex PCR assays offer highly sensitive and specific tools for the detection of enteric pathogens. This prospective study aimed at comparing the novel Roche LightMix Modular Assay Gastro Parasites (LMAGP) detecting Giardia duodenalis, Entamoeba histolytica, Cryptosporidium spp., Blastocystis hominis, and Dientamoeba fragilis with routine laboratory procedures., Methods: Stool specimens (n = 1062 from 1009 patients) were consecutively examined by LMAGP, R-Biopharm Ridascreen enzyme immunoassays (EIAs) detecting G. duodenalis or E. histolytica/dispar, and microscopy of wet mounts. Discrepant results were analysed by in-house PCR., Results: D. fragilis or B. hominis were detected by LMAGP in 131 (14.4%) and 179 (19.9%; 16 samples positive by microscopy; p < 0.0001) of 909 samples, respectively. Of 918 samples analysed for Cryptosporidium spp., six were positive by LMAGP (three could be confirmed by Kinyoun staining and one by in-house PCR). G. duodenalis was detected by LMAGP, EIA, or microscopy in 20, 16, or 9 of 1039 stool samples, respectively; all four samples missed by EIA were confirmed by in-house PCR. In total, 938 stool samples were analysed for E. histolytica/dispar. Nine of ten EIA-positive samples were negative by LMAGP but positive by in-house PCR for E. dispar. One E. histolytica infection (positive by both LMAGP and in-house PCR) was missed by EIA and microscopy. Parasites only detected by microscopy included Enterobius vermicularis eggs (n = 3) and apathogenic amoebae (n = 27)., Conclusions: The data call for routine use of multiplex PCR assays for the detection of enteric protozoan parasites in laboratory diagnostics., (Copyright © 2018 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Introduction: Intestinal parasitic infections are a major public health problem in inter-tropical areas. The aim of our study was to describe the situation in Mahajanga, Madagascar with a particular focus on two protozoa, Dientamoeba fragilis and Blastocystis sp., Methods: This was a prospective study from February to June 2015. Stool samples from symptomatic hospitalized patients and asymptomatic volunteers were submitted to microscopy and molecular assays in order to detect parasites., Results: A wide panel of intestinal parasites were identified among the 265 included subjects, protozoa being the most prevalent with 72.8% whereas the prevalence of helminths and microsporidia was of 7.9% and 4.5%, respectively. Blastocystis sp. was the most prevalent protozoa (64.5% of the entire cohort) followed by various amoebas (35.5%) and flagellates (27,5%). We only detected subtypes 1, 2 and 3 of Blastocystis sp. Among the patients positive for D. fragilis (9.4%), 23 carried genotype 1 and 1 genotype 2. For the first time, we detected in 4 human stools the DNA of a recently described protozoon, Simplicimonas similis. Interestingly, subjects living in urban areas harbored significantly more different parasitic species than subjects living in rural areas with a correlation between sanitary level of neighborhood and protozoan infection. However, there was no difference in prevalence of digestive symptoms between parasite-free and parasite-infected subjects, except for Giardia intestinalis which had more symptomatic carriers., Discussion: Our study reveals a high overall parasite prevalence, similar to what had been found in 2003 in the same city and to other prevalence studies conducted in Africa. The poor access of the population to sanitary infrastructures may explain this result. Data from our study provide valuable key for sanitation programs and prevention of fecal-related infectious diseases., Competing Interests: The authors have declared that no competing interests exist.

The flagellated protozoan Dientamoeba fragilis is one of the most commonly diagnosed parasite of the human gut, with a global distribution. Nevertheless, essential aspects of its biology remain incompletely understood or controversial, most notably life cycle, host range, transmission routes and the ability to cause disease. Molecular epidemiologic studies are also scarce, and limited by the lack of informative genotyping tools. To date, two D. fragilis genotypes (1 and 2) are recognized, with a strong predominance of genotype 1 in both humans and few animal hosts. Recent studies have shown that a very low level of genetic variability characterizes parasite isolates collected in various geographic areas and from both symptomatic and asymptomatic cases. This has raised the hypothesis D. fragilis may be a clonal organism. The recent availability of transcriptome data should greatly assist the development of markers useful to understand genetic diversity of D. fragilis at the population level., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Objective: A low prevalence of intestinal parasites has been identified in individuals with irritable bowel syndrome (IBS), but potential associations with alterations in the bacterial microbiome remain largely unexplored. We aimed to investigate the relationship between parasites and bacteria in individuals with IBS in order to identify potential trans-kingdom microbial characteristics., Design: Stool samples were collected from the Danish background population classified into IBS (n = 119), unspecific gastrointestinal (GI) symptoms (n = 114), and asymptomatic controls (n = 186) based on the Rome III criteria for IBS. Bacterial (16S) and eukaryotic (18S) ribosomal DNA was sequenced, and 18S data were merged with data from conventional parasite laboratory tests. The bacterial microbiome was analyzed according to symptom group and parasite colonization status., Results: Bacterial richness and diversity were similar for IBS and controls but higher in those with unspecific GI symptoms. A higher abundance of Bacteroides and a lower abundance of Faecalibacterium were detected in individuals with IBS and unspecific GI symptoms compared with controls. Principal component analyses indicated differences in bacterial composition related to parasite colonization rather than symptom group. Parasites were detected at the lowest frequency in the IBS group (39%) and in samples dominated by Bacteroides. Higher bacterial richness and diversity were found in parasite-positive samples from controls and those with unspecific GI symptoms but not in individuals with IBS., Conclusion: Parasite colonization, rather than bacterial composition, differed between individuals with IBS and healthy controls. Parasite colonization was associated to a rich and diverse bacterial microbiome; however, this association was altered in IBS.

In this study, we aimed to investigate the incidence of Dientamoeba fragilis with different diagnostic methods in patients with gastrointestinal symptoms and determine the sensitivity and specificity of existing diagnostic methods. Fecal samples collected from 101 patients with gastrointestinal complaints (especially upper abdominal pain, abdominal and pelvic pain, nausea and vomiting, gastroenteritis and colitis, unexplained fever and diarrhea) and 20 control cases from various clinics were included in the study. Samples were first examined with native-Lugol (N-L) method and cultured in Robinson medium. All 121 stool and culture samples were stained with iron hematoxylin stain (IHS) and trichrome stain (TS) methods and examined by PCR and QPCR for D.fragilis. Among 121 stool samples 13 (10.7%), 2 (1.7%), 7 (5.7%) 13 (10.7%), and 7 (5.8%), 4 (3.3%), 2 (1.7%), 3 (2.5%) of cultured samples were determined positive with IHS, TS, PCR, QPCR respectively. Fifteen of the 121 stool samples were determined as diarrheal. All diarrheal stool samples were negative with IHS and TS. One of the diarrheal stools and 6 (4.9%) of the non-diarrheal stools were positive by PCR. All of the diarrheal stools were negative. Thirteen of the non-diarrheal stool samples (10.7%) were positive by QPCR. When the QPCR method was considered as gold standard, sensitivity and specificity values were determined as 46% and 93% in IHS, 0% and 99% in TS, 54% and 100% by PCR and sensitivity and specificity values were 67% and 96% in IHS, 33% and 98% in TS, 67% and 100% by PCR among cultured stool samples. As a result, it was determined that there was a statistically significant difference between the samples of the patients and the control groups and the sensitivity and specificity of the conventional and molecular methods (IHS, TS, PCR and QPCR) determined in this study supported the results of other compared studies. It has been determined that staining methods used for the diagnosis of D.fragilis gave false positivite or negativite results. In addition, the QPCR method is more advantageous in terms of time saving for the diagnosis and initiation of the treatment and in cases where QPCR is not available, IHS and conventional PCR methods should be used together. In our opinion, this study will contribute to the results of epidemiological and scientific studies on D.fragilis in Turkey.

Background: Intestinal parasitosis is one of several health concerns about immigrants who travel from endemic to non-endemic regions. Reliable rapid sensitive diagnostic tools, for use in non-endemic regions, are urgently required to enable frequent assessment of immigrant workers in jobs where risk of local transmission is a particular concern (e.g. food-handlers). We assessed the burden of intestinal protozoa in newly arrived immigrants and those applying for renewal of work permits in Qatar (n = 735), by both microscopic examination of stool samples and by Real Time PCR methodology., Results: Prevalence was considerably higher using RT-PCR compared with coproscopy (Blastocystis hominis: 65.2 vs 7.6%; Giardia duodenalis: 14.3 vs 2.9%; Entamoeba histolytica: 1.6 vs 1.2%). Dientamoeba fragilis was sought only by RT-PCR (prevalence of 25.4%). Prevalence of G. duodenalis was significantly higher in male subjects, associated with blue collar workers and declined over time. Prevalence of B. hominis varied significantly with region of origin of subjects with highest values recorded among African immigrants. Prevalence of D. fragilis also varied with region of origin of subjects, and was lower in young female subjects and in renewal applicants compared with first-time applicants for work permits., Conclusions: We strongly recommend that, henceforth, intestinal protozoa should be screened by RT-PCR, with a particular focus on frequent assessment of immigrant food-handlers.

Pediatric diarrhea is a common cause of death among children under 5 years of age. In the current study, we investigated the frequency of intestinal parasites among 580 pediatric patients with chronic diarrhea. Parasitic protozoa (all species combined) were detected by molecular tools in 22.9% of the children and the most common parasite was Cryptosporidium spp. (15.1%). Blastocystis hominis was detected in 4.7%, Dientamoeba fragilis in 4%, Giardia duodenalis in 1.7%, and Entamoeba histolytica in 0.17%. Protozoan infections were observed among all regional groups, but prevalence was highest among Qatari subjects and during the winter season. Typing of Cryptosporidium spp. revealed a predominance of Cryptosporidium parvum in 92% of cases with mostly the IIdA20G1 subtype. Subtypes IIdA19G2, IIdA18G2, IIdA18G1, IIdA17G1, IIdA16G1, and IIdA14G1 were also detected. For Cryptosporidium hominis , IbA10G2 and IbA9G3 subtypes were identified. This study provides supplementary information for implementing prevention and control strategies to reduce the burden of these pediatric protozoan infections. Further analyses are required to better understand the local epidemiology and transmission of Cryptosporidium spp. in Qatar.

The flagellated protozoan Dientamoeba fragilis is often detected in humans with gastrointestinal symptoms, but it is also commonly found in healthy subjects. As for other intestinal protozoa, the hypothesis that genetically dissimilar parasite isolates differ in their ability to cause symptoms has also been raised for D. fragilis. To date, only two D. fragilis genotypes (1 and 2) have been described, of which genotype 1 largely predominates worldwide. However, very few markers are available for genotyping studies and therefore the extent of genetic variation among isolates remains largely unknown. Here, we performed metagenomics experiments on two D. fragilis-positive stool samples, and identified a number of candidate markers based on sequence similarity to the phylogenetically related species Trichomonas vaginalis. Markers corresponding to structural genes and to genes encoding for proteases were selected for this study, and PCR experiments confirmed their belonging to the D. fragilis genome; two previously described markers (small subunit ribosomal DNA and large subunit of RNA polymerase II) were also included. Using this panel of markers, 111 isolates of human origin were genotyped, all of which, except one, belonged to genotype 1. These isolates had been collected at different times from symptomatic and asymptomatic persons of different age groups in Italy, Denmark, Brazil and Australia. By sequencing approximately 160kb from 500 PCR products, a very low level of polymorphism was observed across all the investigated loci, suggesting the existence of a major clone of D. fragilis with a widespread geographical distribution., (Copyright © 2016 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Dientamoeba fragilis is a protozoan parasite of the human bowel, commonly reported throughout the world in association with gastrointestinal symptoms. Despite its initial discovery over 100 years ago, arguably, we know less about this peculiar organism than any other pathogenic or potentially pathogenic protozoan that infects humans. The details of its life cycle and mode of transmission are not completely known, and its potential as a human pathogen is debated within the scientific community. Recently, several major advances have been made with respect to this organism's life cycle and molecular biology. While many questions remain unanswered, these and other recent advances have given rise to some intriguing new leads, which will pave the way for future research. This review encompasses a large body of knowledge generated on various aspects of D. fragilis over the last century, together with an update on the most recent developments. This includes an update on the latest diagnostic techniques and treatments, the clinical aspects of dientamoebiasis, the development of an animal model, the description of a D. fragilis cyst stage, and the sequencing of the first D. fragilis transcriptome., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

We surveyed the prevalence of Dientamoeba fragilis, Giardia duodenalis, Entamoeba histolytica, Entamoeba dispar, and Cryptosporidium spp in individuals with and without gastrointestinal (GI) symptoms residing in and around Da Nang city, Vietnam. Fecal samples were collected from children (n = 100) and adults (n = 80) with GI symptoms and from healthy individuals (n = 88) reporting no GI symptoms. Parasite detection was performed by multiplex real-time PCR. Overall, except for G. duodenalis, we found a low prevalence (<5%) of D. fragilis and E. dispar and no detection of E. histolytica and C. spp in all participants with GI symptoms. Specifically for D. fragilis this contrasts with findings in European populations of children with GI symptoms showing prevalence up to 73%. Moreover, our results indicate that the prevalence of G. duodenalis is higher in patients with GI symptoms compared to asymptomatic individuals and this difference is most obvious in young patients., (© 2016 The Authors. APMIS published by John Wiley & Sons Ltd on behalf of Scandinavian Societies for Medical Microbiology and Pathology.)

Dientamoeba fragilis is now considered a potentially emerging gastrointestinal pathogen in both developing and developed countries. We first report an autochthonous case of D. fragilis infection in Greece. A 49-year-old female with acute non-specific abdominal pain required emergency surgical admission for active observation and repeated assessment. To the best of our knowledge, this is the first reported case of acute unexplained abdominal pain finally attributed to D. fragilis infection using microscopic and molecular methods.

Dientamoeba fragilis is a human bowel parasite with a worldwide distribution. Dientamoeba was once described as a rare and harmless commensal though recent reports suggest it is common and potentially pathogenic. Molecular data on Dientamoeba is scarce which limits our understanding of this parasite. To address this, sequencing of the Dientamoeba transcriptome was performed. Messenger RNA was extracted from cultured Dientamoeba trophozoites originating from clinical stool specimens, and sequenced using Roche GS FLX and Illumina HiSeq technologies. In total 6,595 Dientamoeba transcripts were identified. These sequences were analysed using the BLAST2GO software suite and via BLAST comparisons to sequences available from TrichDB, GenBank, MEROPS and kinase.com. Several novel KEGG pathway maps were generated and gene ontology analysis was also performed. These results are thoroughly discussed guided by knowledge available for other related protozoa. Attention is paid to the novel biological insights afforded by this data including peptidases and kinases of Dientamoeba, as well as its metabolism, novel chemotherapeutics and possible mechanisms of pathogenicity. Currently, this work represents the largest contribution to our understanding of Dientamoeba molecular biology and also represents a major contribution to our understanding of the trichomonads generally, many of which are important pathogens of humans and animals., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Dientamoeba fragilis is an inhabitant of human gastrointestinal tract with a worldwide distribution. The first description considered this protozoan a rare and harmless commensal, since then it has struggled to gain recognition as a pathogen. Commercial multiplex real-time PCR was used to detect D. fragilis in fecal samples from hospitalized children (⩽18 years) with acute gastrointestinal disease, admitted to two hospitals of Lisbon area, with different demographic characteristics. A total of 176 children were studied, 103 (58·5%) male, 144 (81·8%) children between 0 and 5 years and 32 (18·2%) above 6 years old. The overall protozoa frequency considering the four tested microorganisms were 8·5% (15/176), and the most frequently found protozoan was D. fragilis, 6·3% (11/176). Dientamoeba fragilis frequency was higher among older children (21·9%), than younger children (2·8%), and greater in boys (6·8%) than in girls (5·5%). All positive children presented with diarrhoea associated with vomiting, fever and abdominal pain. Infection was associated with the age of children (P < 0·001), school attendance (P = 0·002) and consumption of certain foods (P = 0·014), e.g. cakes with crème and ham. The frequency of diantamoebiasis found in a cohort of hospitalized Portuguese children, with acute gastrointestinal disease, could be considered a very high value when compared with the protozoan frequency normally associated with this pathology.

Recently, Dientamoeba fragilis has emerged as a significant and common enteropathogen. The majority of patients with dientamoebiasis present with gastrointestinal complaints and chronic symptoms are common. Numerous studies have successfully demonstrated parasite clearance, coupled with complete resolution of clinical symptoms following treatment with various antiparasitic compounds. Despite this, there is very little in vitro susceptibility data available for the organism. Benzimidazoles are a class of antiparasitic drugs that are commonly used for the treatment of protozoan and helminthic infections. Susceptibility testing was undertaken on four D. fragilis clinical isolates against the following benzimidazoles: albendazole, flubendazole, mebendazole, nocodazole, triclabendazole and thiabendazole. The activities of the antiprotozoal compounds at concentrations ranging from 2 μg/mL to 500 μg/mL were determined via cell counts of D. fragilis grown in xenic culture. All tested drugs showed no efficacy. The beta-tubulin transcript was sequenced from two of the D. fragilis isolates and amino acid sequences predicted a susceptibility to benzimidazoles. This is the first study to report susceptibility profiles for benzimidazoles against D. fragilis, all of which were not active against the organism. This study also found that beta-tubulin sequences cannot be used as a reliable marker for resistance of benzimidazoles in D. fragilis., (D. Stark et al., published by EDP Sciences, 2014)

Dientamoeba fragilis is an intestinal protozoan suspected of causing gastrointestinal symptoms, and its mode of transmission is unknown, although first described almost a century ago. A hypothesis is that Enterobius vermicularis is a vector for D. fragilis, and recently, D. fragilis DNA was detected within surface-sterilized eggs of E. vermicularis. Using real-time PCR, we detected D. fragilis DNA in 18 (85%) of 21 samples of E. vermicularis eggs collected from patients harbouring D. fragilis in faeces. This finding supports the hypothesis that E. vermicularis may have an important role in the transmission of D. fragilis., (© 2013 The Authors. Pathogens and Disease published by John Wiley & Sons Ltd on behalf of the Federation of European Microbiological Societies.)

The intestinal protozoon Dientamoeba fragilis remains a clinical entity of dubious significance. While several previous studies address questions of epidemiology, only a handful have systematically employed and reported on the results from real-time polymerase chain reaction (qPCR), the best currently available diagnostic modality, and the comparison of results from different studies is, therefore, difficult. Since 2007, Statens Serum Institut (Denmark) has utilised qPCR for D. fragilis as routine diagnostic work-up for intestinal parasitosis, testing more than 22,000 samples from 2008 through 2011, and the aim of this study was to report on the results and experiences gained in the process. We demonstrate a staggeringly high proportion (43%) of investigated patients positive for D. fragilis, ranging from 12 to 71% depending on age group, showing a bimodal age distribution peaking in children and adults of parental age, as well as a clear association between exposure to children and risk of D. fragilis infection. We discuss these findings in light of the pinworm egg vector hypothesis and substantiate further our knowledge of risk factors pertaining to D. fragilis carriage.

Dientamoeba fragilis is a common intestinal parasite of unsettled clinical significance. Differences in clinical outcome of parasitic infections may reflect parasite genetic diversity, and so tools to study intra-genetic diversity that could potentially reflect differences in clinical phenotypes are warranted. Here, we show that genetic analysis of three D. fragilis housekeeping genes enables clear distinction between the two known genotypes, but that integration of housekeeping genes in multi-locus sequencing tools for D. fragilis may have limited epidemiological and clinical value due to no further added genetic resolution., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Dientamoeba fragilis is a protozoan parasite of the human large intestine which is implicated as a cause of gastrointestinal diseases. The diagnosis of this parasite in direct smear preparations is difficult due to the lack of a cyst stage. The permanent staining method is generally used for diagnosis of D. fragilis, but the technique is laborious and time consuming. The purpose of this study was to evaluate the performance of PCR for detection of D. fragilis in clinical specimen of health care center in Tabriz, northwest of Iran. Stool samples of 1000 patients were collected from different laboratories and were immediately examined via wet mount and permanent staining methods. All positive samples and 55 randomly selected negative samples were studied by PCR technique. Using direct smear examination, no positive sample was found among 1000 stool samples, whereas 21 (2.1%) positive and 26 suspicious cases were reported in stained smears. PCR screening indicated that from 21 positive cases, 17 were positive by primary PCR, whereas nested PCR detected all 21 positive cases as well as 3 new positive samples from the suspicious cases (overall 24 (2.4%) positive samples), yet all negative cases remained negative through both stages of PCR amplifications. In comparison with nested PCR (if considered as gold standard), primary PCR showed 81% sensitivity and 100% specificity and those of microscopy was determined to be 87.5% and 100%, respectively. Considering the favorable sensitivity and specificity of nested PCR and its other advantages such as relative simplicity and speed this technique is proposed for rapid diagnosis of D. fragilis in clinical samples.

In recent years, interest in Dientamoeba fragilis on the part of biologists, clinicians and parasitologists has increased considerably due to the need to clarify the many unresolved issues related to the parasite and the associated infection. This paper aims to provide a summary of the state of knowledge of D. fragilis in its different aspects, and, through the analysis of the literature and the most recent data, the present review aims to record the reasons why this protozoan, although little known, should not be underestimated in the diagnosis of human parasitic infections also in Italy.

With no evidence of a cyst stage, the mode of transmission of Dientamoeba fragilis, an intestinal protozoon of common occurrence and suggested pathogenicity, is incompletely known. Numerous studies have suggested that eggs of intestinal nematodes, primarily Enterobius vermicularis (pinworm), can serve as vectors for D. fragilis, although attempts to culture D. fragilis from pinworm eggs have been unsuccessful and data from epidemiological studies on D. fragilis/pinworm co-infection have been conflicting. The aim of this study was to investigate whether we could detect D. fragilis DNA from pinworm eggs collected from routine diagnostic samples (cellophane tape) and surface-sterilised by hypochlorite. DNA was extracted from individual eggs and tested by PCR using D. fragilis- and E. vermicularis-specific primers; amplicons were sequenced for confirmation. In cellophane tape samples from 64 patients with unknown D. fragilis status we detected D. fragilis DNA in 12/238 (5%) eggs, and in a patient known to harbour D. fragilis we detected D. fragilis DNA in 39/99 (39%) eggs. The finding of D. fragilis DNA within eggs of E. vermicularis strongly supports the hypothesis of D. fragilis-transmission by pinworm and has implications for antimicrobial intervention as well as control and public health measures., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Dientamoeba fragilis is a common intestinal parasite in humans. Transmission routes and natural host range are unknown. To determine whether pigs are hosts, we analyzed 152 fecal samples by microscopy and molecular methods. We confirmed that pigs are a natural host and harbor genotypes found in humans, suggesting zoonotic potential.

Dientamoeba fragilis is a commonly encountered trichomonad which has been implicated as a cause of gastrointestinal disease in humans. Despite the frequency of reports recording infections with this parasite, little research has been undertaken in terms of antimicrobial susceptibility. The aim of this study was to evaluate the susceptibility of D. fragilis to several commonly used antiparasitic agents: diloxanide furoate, furazolidone, iodoquinol, metronidazole, nitazoxanide, ornidazole, paromomycin, secnidazole, ronidazole, tetracycline, and tinidazole. Antibiotic susceptibility testing was performed on four clinical strains of D. fragilis, designated A, E, M, and V, respectively. Molecular testing followed, and all strains were determined to be genotype 1. The activities of antiprotozoal compounds at concentrations ranging from 2 μg/ml to 500 μg/ml were determined via cell counts of D. fragilis trophozoites grown in dixenic culture. Minimum lethal concentrations (MLCs) were as follows: ornidazole, 8 to 16 μg/ml; ronidazole, 8 to 16 μg/ml; tinidazole, 31 μg/ml; metronidazole, 31 μg/ml; secnidazole, 31 to 63 μg/ml; nitazoxanide, 63 μg/ml; tetracycline, 250 μg/ml; furazolidone, 250 to 500 μg/ml; iodoquinol, 500 μg/ml; paromomycin, 500 μg/ml; and diloxanide furoate, >500 μg/ml. This is the first study to report the profiles of susceptibility to a wide range of commonly used treatments for clinical isolates of D. fragilis. Our study indicated 5-nitroimidazole derivatives to be the most active compounds in vitro against D. fragilis.

In recent years, the isolation of parasitic DNA from faecal samples and PCR techniques, have been improved and simplified. Moreover, the introduction of real-time PCR has made it possible to multiplex different targets into one reaction. These new technical possibilities make it feasible to introduce PCR with its unsurpassed sensitivity and specificity in a routine laboratory setting for the diagnosis of intestinal parasites. Detection rates of the parasitic infections included in the PCR are increased significantly compared with microscopy. Molecular diagnostics, especially in children, reveal a possible cause of the gastrointestinal complaints in many more cases compared with conventional methods. Usually in GP patients no other pathogenic parasites are detected using microscopy and in the returning travellers additional parasites are found with microscopy in a minority of cases only. Multiplex real-time PCR offers a highly sensitive and specific diagnostic alternative for labour intensive microscopy in clinical laboratory practice. Additional diagnostic methods for the detection of parasitic infections that are not included as PCR target can be limited to a selected group of patients.

The aim of this study was to describe the first development and evaluation of a multiplex tandem PCR (MT-PCR) assay for the detection and identification of 4 common pathogenic protozoan parasites, Cryptosporidium spp., Dientamoeba fragilis, Entamoeba histolytica, and Giardia intestinalis, from human clinical samples. A total of 472 fecal samples submitted to the Department of Microbiology at St. Vincent's Hospital were included in the study. The MT-PCR assay was compared to four real-time PCR (RT-PCR) assays and microscopy by a traditional modified iron hematoxylin stain. The MT-PCR detected 28 G. intestinalis, 26 D. fragilis, 11 E. histolytica, and 9 Cryptosporidium sp. isolates. Detection and identification of the fecal protozoa by MT-PCR demonstrated 100% correlation with the RT-PCR results, and compared to RT-PCR, MT-PCR exhibited 100% sensitivity and specificity, while traditional microscopy of stained fixed fecal smears exhibited sensitivities and specificities of 56% and 100% for Cryptosporidium spp., 38% and 99% for D. fragilis, 47% and 97% for E. histolytica, and 50% and 100% for G. intestinalis. No cross-reactivity was detected in 100 stool samples containing various other bacterial, viral, and protozoan species. The MT-PCR assay was able to provide rapid, sensitive, and specific simultaneous detection and identification of the four most important diarrhea-causing protozoan parasites that infect humans. This study also highlights the lack of sensitivity demonstrated by microscopy, and thus, molecular methods such as MT-PCR must be considered the diagnostic methods of choice for enteric protozoan parasites.

Dientamoeba fragilis is a protozoan that inhabits the human gut. It is approximately 100 years since Dientamoeba's discovery and first description when it was described as a rare and harmless commensal. Since then it has struggled to gain recognition as a pathogen despite the evidence supporting its pathogenicity. Dientamoeba remains neglected, probably due to the misconceptions that it is uncommon and non-pathogenic. Usually, carriage of Dientamoeba is associated with symptoms such as abdominal pain and diarrhea. Moreover, antimicrobial therapy followed by resolution of symptoms coincides with the eradication of Dientamoeba. This manuscript reviews the scientific literature relating to Dientamoeba's prevalence and pathogenicity. While much of the evidence supporting its pathogenicity is only circumstantial, it is apparent that most researchers agree that Dientamoeba is pathogenic. Therefore, in symptomatic patients who harbor Dientamoeba and no other pathogen, Dientamoeba should be considered as the etiological agent and treated as such., (© 2011 Landes Bioscience)

Dientamoeba fragilis is a pathogen of the human gastrointestinal tract that is a common cause of diarrhoea. A paucity of knowledge on the in vitro cultivation and cryopreservation of Dientamoeba has meant that few studies have been conducted to investigate its biology. The objective of this study was to define, for the first time, in vitro culture conditions able to support the long-term in vitro growth of Dientamoeba. Also, we aimed to define a suitable method for cryopreserving viable Dientamoeba trophozoites. A modified BD medium, TYGM-9, Loeffler's slope medium, Robinson's medium, Medium 199, Trichosel and a Tritrichomonas fetus medium were compared, using cell counts, for their ability to support the growth of D. fragilis at various temperatures and atmospheric conditions. Loeffler's slope medium supported significantly better growth compared to other media. A temperature of 42°C and a microaerophilic atmosphere were also optimum for Dientamoeba growth. To our knowledge, this is the first study to describe and compare different culture media and conditions for the growth of clinical isolates of D. fragilis. This new technology will aid the development of diagnostics for dientamoebiasis as well as facilitate large-scale sequencing projects that will fast track molecular studies on D. fragilis.