Detection and genotyping of Dientamoeba fragilis from symptomatic patients: New insights from Italy into a little-known gastrointestinal protozoan.

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.

A Cross-Sectional Study on the Occurrence of the Intestinal Protist, Dientamoeba fragilis, in the Gut-Healthy Volunteers and Their Animals.

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.

First detection and molecular characterization of Dientamoeba fragilis in cattle.

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.

First report and genotyping of Dientamoeba fragilis in pet budgerigars (Melopsittacus undulatus), with zoonotic importance.

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.

Investigation of Dientamoeba fragilis and Blastocystis in patients from Turkey with ulcerative colitis and irritable bowel syndrome: Any relation with genotypes?

Blastocystis sp. and Dientamoeba fragilis are two most common protists worldwide, whose pathogenic potentials are a matter of debate since their discovery. This study aims to investigate the relationship between the activation of ulcerative colitis (UC) and irritable bowel syndrome (IBS) with these protists. A total of 100 patients (35 IBS, 35 active UC, and 30 remittent UC), diagnosed at Hacettepe University Adult Hospital (Ankara, Turkey), were screened for D. fragilis and Blastocystis sp. with microscopic examination using the methods of wet mount, trichrome staining, conventional PCR, nested PCR, real-time PCR and genotyping. Eight patients (4 IBS, 2 active, and 2 remittent UC patients) were found to be D. fragilis positive. 18S rRNA region of the parasite was amplified in four of the patients, whereas cathepsin L-like cysteine peptidase; clan Sc, family S9, serine peptidase; and clan MH, family M20 metallopeptidase in six different patients. All isolates were Genotype 1. Sequence results showed very limited diversity. A total of nine patients (3 IBS, 5 active UC, 1 remittent UC) were found to be positive for Blastocystis sp., all of which were Subtype 3. One active UC and one IBS patient were found to be positive for both parasites. No statistically significant difference was detected between the patient groups in means of parasite detection. D. fragilis was found to be related to older age (p=0,045). In our study, no significant correlation was identified between D. fragilis and Blastocystis sp., and the activation of UC and IBS. More studies are needed on the host-parasite relationship, including the role of gut microbiota, together with transcriptomic and metabolomic assessments to unveil the pathogenicity of both protists.

Investigation of Dientamoeba fragilis Frequency in Faecal Samples of Patients with Enterobius vermicularis Infection by Polymerase Chain Reaction

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, Aydin 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 yerlesimli, kamçili bir protozoondur. Parazitin Enterobius vermicularis (E. vermicularis) yumurtalariyla tasindigi hipotezi kabul görmektedir. Çalismamizda D. fragilis ve E. vermicularis birlikteligini incelemek, bulunan D. fragilis'lerin genotiplerini belirlemek ve D. fragilis tanisinda yaygin olarak kullanilan trikrom boyama ile polimeraz zincir reaksiyon (PZR) yöntemlerini karsilastirmak amaçlanmistir. Yöntemler: Çalismamizda Aydin Adnan Menderes Üniversitesi Tip Fakültesi, Parazitoloji Laboratuvari'na 1 Ekim 2017-1 Ekim 2018 tarihleri arasinda diski ve selofan lam örnegi birlikte gönderilmis toplam 391 olgu örnegi incelenmistir. Selofanli lam örneklerinde E. vermicularis saptanan tüm gönüllü olgularin (74 olgu) diski örnegi ile E. vermicularis negatif 74 olgunun diski örnegi çalisilmistir. Tüm diskilar trikrom boyama ve PZR yöntemleri ile D. fragilis varligi açisindan incelenmistir. Saptanan D. fragilis izolatlarinin 18S ribozomal RNA bölgesi sekanslanmistir. Olgularin demografik özellikleri ve klinigi degerlendirilmistir. Bulgular: Toplam 148 olgunun 42'sinde (%28,37) D. fragilis saptanmistir. D. fragilis pozitif olan 42 olgunun %66,6'sini E. vermicularis pozitif olgular olusturmus ve iki parazitin birlikteligi anlamli bulunmustur (p<0,05). Sekanslanan tüm izolatlar genotip 1 olarak saptanmistir. Klinik bulgular, yasanilan bölge ve cinsiyet ile parazit varligi arasinda anlamli bir iliski saptanamamistir (p>0,05). Sonuç: Arastirmamizda D. fragilis'in siklikla E. vermicularis ile birliktelik gösterdigi ve bu olgularda D. fragilis varligina ayrica dikkat edilmesi gerektigi vurgulanmistir. Yanlis sonuçlari engellemede, yüksek duyarliliga sahip PZR gibi yöntemlerin önemi bir kez daha görülmüstür.

Prevalence and genetic characterization of Dientamoeba fragilis in asymptomatic children attending daycare centers.

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.

Dientamoeba fragilis in the North-East of Italy: Prevalence study and treatment.

Dientamoeba fragilis is an intestinal protozoan, an inhabitant of the human gastrointestinal tract, with a worldwide distribution. The reported prevalence of D. fragilis varies worldwide in different populations between 0.3% and 82.9%, and its role as a pathogen is still unclear. The parasite has been identified in the faeces of asymptomatic patients and with different acute and chronic symptoms, like abdominal pain, diarrhoea, flatulence, nausea and vomiting. The aims of this study were to evaluate the prevalence of D. fragilis in the North-East of Italy, and the clinical improvement of symptoms after recommended treatment with paromomycin or metronidazole. Furthermore, a literature review of D. fragilis prevalence studies in Italy was carried out to show the Italian situation. Of 575 enrolled people, 85 (14.8%) were positive for D. fragilis. The most prevalent symptoms were abdominal pain 28.2%, anal itching 27.1%, watery diarrhoea 18.8%, meteorism 16.5% and nausea/lack of appetite 14.1%. The high rate of anal itching was unexpected, because it wasn't a common symptom. 32 patients were co-infected with B. hominis (37.7%) and three with G. lamblia (3.5%). Our study showed paromomycin had a high efficacy for treatment of D. fragilis infections 100.0% (45/45), while caution must be used when using metronidazole 53.3% (24/40). We recommend paromomycin for empirical treatment, given its great effectiveness in our population.

Dientamoeba fragilis Infection in Patients with Digestive and Non-Digestive Symptoms: A Case-Control Study.

In most developing countries, Dientamoeba fragilis infection is an obscure protozoan infection. We aimed to determine a frequency and clinical importance of D. fragilis infection in Taif, Saudi Arabia. A 1-year case control study included patients with gastrointestinal (cases, n=114) or non-gastrointestinal symptoms (controls, n=90). The fecal samples were examined with the classical parasitological methods for intestinal protozoa, and by real time PCR for D. fragilis. The infection by D. fragilis was detected in 5.8% by PCR and in 4.4% patients by microscopy. The infection was identified more in control group (n=9) than in cases (n=3); a sole infection in 11 patients and mixed with Giardia in 1 patient. The other enteric parasites detected were Blastocystis sp. (8.3%), Giardia sp. (5.3%), Cryptosporidium sp. (2.9%), Entamoeba histolytica (1.4%), Entamoeba coli (0.9%) and Hymenolepis nana (0.4%). Our results tend to reinforce the need to increase awareness of D. fragilis infection in Saudi Arabia.

Distribution and relevance of Dientamoeba fragilis and Blastocystis species in gastroenteritis: results from a case-control study.

The actual role of Dientamoeba fragilis and Blastocystis in patients with gastrointestinal symptoms is still under debate. A multicenter case-control study was performed in The Netherlands to elucidate the clinical relevance of molecular diagnostics results in gastroenteritis (GE). Samples from this case-control study were used to perform a detailed analysis on the presence of D. fragilis and Blastocystis in relation to gastrointestinal symptoms. In the present study, a real-time PCR for Blastocystis was performed on 1374 case samples and 1026 control samples from the multicenter gastroenteritis case-control study previously tested for D. fragilis. Prevalence of both micro-organisms was highest in children under 20 years of age and lowest in the oldest age group. A significantly lower overall detection of D. fragilis and Blastocystis was found in cases (both 25.8%) as compared to controls (37.6% and 40.0%, respectively). The difference for D. fragilis was statistically significant for subjects above 20 years of age. For Blastocystis, the difference was statistically significant in all age groups, except in children less than 5 years of age. A negative relation between D. fragilis-positive cases and diarrhea was found in this study population. More GE symptoms were reported in cases without D. fragilis or Blastocystis. In the present study, prevalence of both D. fragilis and Blastocystis is lower in cases with gastroenteritic symptoms than in controls. Besides, in cases with D. fragilis or Blastocystis, no association is shown between any of the GE symptoms. Interestingly, this suggests that the presence of these protozoans may be considered characteristic of a healthy intestinal microbiome.

Dientamoeba fragilis - the most common intestinal protozoan in the Helsinki Metropolitan Area, Finland, 2007 to 2017.

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.

Investigation of Intestinal Parasites in Living Nursing Home

Objective: Intestinal infections are common in the elderly, presented with atypical symptoms and may be the cause of mortality with a more severe clinical manifestation. The weakening of cellular and humoral immunity by aging affects the intestinal flora and increases the risk of infection in the presence of chronic diseases. The aim of this study was to investigate the presence of possible parasitic agents in the intestinal system of ≥65-year-old nursing home residents through fecal examination, and to determine the demographic features (age and gender) of this elderly group. Methods: A total of 82 stool samples were examined (100x, 40x) with saline and iodine preparations, formol-ethyl acetate concentration process, trichrome and modified Erlich Ziehl Neelsen stained preparations. Results: One or more parasitological agents were detected in 17 (20.7%) of the 82 stool samples examined. The most common agent was Blastocystis spp. (13.4%), followed by Cryptosporidium spp. (2.4%) and Dientamoeba fragilis (2.4%). Conclusion: In this study, it was determined that attention should be given to elderly population with regard to intestinal parasitic infections. Because of changes in the immune system, more opportunistic factors could be detected. More frequent screening in public areas such as nursing homes is important for preventing infections.

Epidemiological and clinical characteristics of Dientamoeba fragilis infection / Características clínicoepidemiológicas de las infecciones por Dientamoeba fragilis

INTRODUCTION: The characteristics of D. fragilis infection are described, with special focus on the clinical and epidemiological aspects. MATERIALS AND METHODS: A retrospective and descriptive study was performed, including all the patients with Dientamoeba fragilis infection who attended a specialized unit between January 2012 and December 2017. PCR was used to diagnose D. fragilis. Patients were treated with metronidazole or paromomycin and reviewed at four and eight weeks post-treatment. Cure was defined as the negativization of all parasitological tests, in absence of symptoms. RESULTS: 163 patients were diagnosed. The most frequent symptoms were abdominal pain (36.2%), chronic diarrhoea (12.3%), anal itching (10.4%), abdominal discomfort (9.2%), skin disease (8%), acute diarrhoea (4.3%) and vomiting (4.3%). Fifty patients were asymptomatic. Forty-two patients had eosinophilia in blood. Thirty-eight cases (23.3%) had a coinfection by Enterobius vermicularis. One hundred and seven patients received treatment, sixty-one of them with metronidazole and the rest with paromomycin. Ninety-nine patients (91%) were cured. The rate of cure was 100% in the paromomycin group versus 86.8% in the metronidazole group (p = 0.005; OR: 1.173 [1.057-1.302]). The absence of cure was associated with E. vermicularis coinfection (p = 0.014; OR: 6.167 [1.432-26.563] and with longer duration of the symptoms (175 [± 159SD]) versus 84 [± 88SD] days, p = 0.014) but multivariable analysis did not confirm these associations. CONCLUSION: Dientamoeba fragilis is an important and underestimated cause of gastrointestinal disease in both the autochthonous and immigrant or traveller population. More studies are needed to clarify its optimal treatment and the role played by E. vermicularis in its transmission and maintenance

INTRODUCCIÓN: Se describen las características clínicas y epidemiológicas de la infección por Dientamoeba fragilis. MATERIAL Y MÉTODOS: Se realizó un estudio retrospectivo y descriptivo de los pacientes diagnosticados de infección por D. fragilis en una unidad especializada entre 2012-2017. El diagnóstico de D. fragilis se realizó mediante PCR. Los pacientes fueron tratados con metronidazol o paromomicina y revisados a las 4 y 8 semanas tras tratamiento. Se consideró a los pacientes curados tras negativización microbiológica en ausencia de síntomas. RESULTADOS: Se analizaron 163 pacientes. Los síntomas más frecuentes fueron: dolor abdominal (36,2%), diarrea crónica (12,3%), prurito anal (10,4%), malestar abdominal (9,2%), síntomas cutáneos (8%), diarrea aguda y vómitos (4,3%, respectivamente). Cincuenta pacientes estaban asintomáticos. Cuarenta y dos pacientes presentaron eosinofilia. En 38 pacientes se observó coinfección por Enterobius vermicularis. Ciento siete pacientes recibieron tratamiento, 61 con metronidazol y el resto con paromomicina, con una curación del 91%. La tasa de curación fue del 100% en los pacientes tratados con paromomicina y del 86,8% en el grupo del metronidazol (p = 0,005; OR: 1,173 [1,057-1,302]). La no curación se asoció a la coinfección por E. vermicularis (p = 0,014; OR: 6,167 [1,432-26,563]) y con la mayor duración de los síntomas (175 [± 159 DE] versus 84 [± 88 DE] días; p = 0,014), pero el análisis multivariable no confirmó dichas asociaciones. CONCLUSIÓN: D. fragilis es causa importante y subestimada de enfermedad gastrointestinal tanto en poblaciones autóctonas como inmigrantes o viajeros. Se necesitan más estudios para aclarar su tratamiento óptimo y el papel desempeñado por E. vermicularis en su tratamiento

Comparison and Recommendations for Use of Dientamoeba fragilis Real-Time PCR 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.

No association between abdominal pain and Dientamoeba in Dutch and Belgian children.

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.

Blastocystis prevalence and subtypes in autochthonous and immigrant patients in a referral centre for parasitic infections in Italy.

In this study we characterized the presence and subtype (ST1-ST4) of Blastocystis in patients attended at a referral center for tropical diseases in Northern Italy. We also, evaluated the organism's association with other intestinal parasites. Parasite screening was performed on 756 patients, from different geographical origins (namely, Italians, Africans, South Americans, Asian and non-Italian Europeans) in which Italians represented the largest group. Blastocystis was seen to be the most prevalent parasite in the study. Subtype 3 and 1 were the most frequently found in the Italians and Africans. Our data confirmed previous studies performed in Italy, in which ST3 proved to be the most prevalent subtype, but we highlighted also a high frequency of mixed subtypes, which were probably underestimated in former analyses. Interestingly, the mixed subtypes group was the most prevalent in all the analysed geographical areas. About half of our cases showed other co-infecting parasites and the most frequent was Dientamoeba fragilis. Our study confirms that, in Blastocystis infection, multiple subtypes and co-infecting parasites are very frequently present, in particular Dientamoeba fragilis.

Investigation of Dientamoeba fragilis Prevalence and Evaluation of Sociodemographic and Clinical Features in Patients with Gastrointestinal Symptoms.

BACKGROUND: Dientamoeba fragilis is a protozoan parasite of the human gastrointestinal tract and still controversial in association with gastrointestinal symptoms. PURPOSE: We present cross-sectional study of the prevalence of D. fragilis, and sociodemographic and clinical features in the patients with gastrointestinal symptoms. METHODS: A total of 490 fecal specimens were collected from outpatients with gastrointestinal symptoms in the Department of Parasitology, Faculty of Medicine, Ege University and Celal Bayar University, Turkey. Fecal specimens were examined with microscopy and inoculated in Robinson medium. D. fragilis-positive samples were examined for the presence of other intestinal parasites using enzyme immunoassay. Real-time PCR analysis was performed on all samples. RESULTS: Of the 490 stool specimens examined by real-time PCR, 59 patients were positive for D. fragilis infection with prevalence rate of 12.04%. Forty-four of positive patients (74.5%) were found to be infected with only D. fragilis, while 23.7% were co-infected with Blastocystis and 1.7% were co-infected with Rotavirus. No statistically significant difference was found in all the examined patients in terms of D. fragilis positivity for all sociodemographic parameters. Loose stool consistency was associated with the presence of D. fragilis, with 18.3% (P = 0.001). When the clinical symptoms of all the patients participating in this study were examined, diarrhea was statistically more significant in patients with the presence of D. fragilis (16.3%; P = 0.001). The rate of diarrhea in D. fragilis-positive patients (84.09%; P = 0.0005) was higher than that of D. fragilis-negative patients and it was statistically significant. CONCLUSION: This study is important for assessing the prevalence of D. fragilis and its association with other factors in symptomatic patients in a large sample group in Turkey, as well as investigating the relationship of identified symptoms with the D. fragilis pathogenicity. It is suggested that D. fragilis in this case is not a commensal parasite but a pathogenic parasite and that the most common clinical symptom is diarrhea.

Clinical relevance of enteropathogen co-infections in preschool children-a population-based repeated cross-sectional study.

OBJECTIVES: This study aimed to (i) determine risk factors for enteropathogen co-infections, (ii) determine whether enteropathogen co-infections influence gastroenteritis risk, and (iii) determine whether enteropathogen co-infection occurred randomly in preschool children. METHODS: A monthly-repeated cross-sectional survey in Dutch children aged 0-48 months was conducted during October 2012 to October 2014. A total of 981 stool samples were collected along with questionnaires collecting data on gastrointestinal symptoms and potential risk factors; 822 samples were successfully tested for 19 enteropathogens using real-time multiplex PCRs. Logistic regression analysis assessed co-infections in relation to gastroenteritis and potential risk factors. RESULTS: In all, 598/822 (72.7%) stool samples tested positive for at least one enteropathogen, of which 290 (48.5%) were positive for two or more enteropathogens. Risk factors for two or more enteropathogen co-infections were young age (<12 months, OR 1.9, 95% CI 1.1-3.3; 13-36 months, OR 1.7, 95% CI 1.1-2.5, versus 37-48 months), day-care attendance (OR 1.8, 95% CI 1.3-2.5), households with three or more children versus those with one child (OR 1.7, 95% CI 1.1-2.8). Stool samples collected in spring less often had two or more enteropathogens versus summer (OR 0.4, 95% CI 0.2-0.7). Food allergy was a risk factor for three or more enteropathogen co-infections (OR 3.2, 95% CI 1.1-8.9). The frequency of co-infection was higher than expected for norovirus GI/norovirus GII, Clostridium difficile/norovirus GI, C. difficile/rotavirus, astrovirus/Dientamoeba fragilis, atypical enteropathogenic Escherichia coli/adenovirus, typical enteropathogenic E. coli/adenovirus, and enteroaggregative E. coli/astrovirus. No co-infection was associated with increased gastroenteritis risk. CONCLUSIONS: Risk factors for enteropathogen co-infections were identified and specific enteropathogens co-occurred significantly more often than expected by chance. Enteropathogen co-infections were not associated with increased gastroenteritis risk, calling into question their clinical relevance in preschool children.

Epidemiological and clinical characteristics of Dientamoeba fragilis infection.

INTRODUCTION: The characteristics of D. fragilis infection are described, with special focus on the clinical and epidemiological aspects. MATERIALS AND METHODS: A retrospective and descriptive study was performed, including all the patients with Dientamoeba fragilis infection who attended a specialized unit between January 2012 and December 2017. PCR was used to diagnose D. fragilis. Patients were treated with metronidazole or paromomycin and reviewed at four and eight weeks post-treatment. Cure was defined as the negativization of all parasitological tests, in absence of symptoms. RESULTS: 163 patients were diagnosed. The most frequent symptoms were abdominal pain (36.2%), chronic diarrhoea (12.3%), anal itching (10.4%), abdominal discomfort (9.2%), skin disease (8%), acute diarrhoea (4.3%) and vomiting (4.3%). Fifty patients were asymptomatic. Forty-two patients had eosinophilia in blood. Thirty-eight cases (23.3%) had a coinfection by Enterobius vermicularis. One hundred and seven patients received treatment, sixty-one of them with metronidazole and the rest with paromomycin. Ninety-nine patients (91%) were cured. The rate of cure was 100% in the paromomycin group versus 86.8% in the metronidazole group (p=0.005; OR: 1.173 [1.057-1.302]). The absence of cure was associated with E. vermicularis coinfection (p=0.014; OR: 6.167 [1.432-26.563] and with longer duration of the symptoms (175 [±159SD]) versus 84 [±88SD] days, p=0.014) but multivariable analysis did not confirm these associations. CONCLUSION: Dientamoeba fragilis is an important and underestimated cause of gastrointestinal disease in both the autochthonous and immigrant or traveller population. More studies are needed to clarify its optimal treatment and the role played by E. vermicularis in its transmission and maintenance.

Clinical and Epidemiological Characteristics of Patients with Dientamoeba fragilis Infection.

Dientamoeba fragilis is an intestinal protozoan, usually considered nonpathogenic. However, in the last years, there has been an attempt to clarify its possible pathogenic role. We aim to evaluate the clinical and epidemiological characteristics of D. fragilis-infected patients. Adults with D. fragilis detection in feces who attended the Vall d'Hebron University Hospital (Barcelona, Spain) were evaluated retrospectively from April 2009 to March 2014. We classified the patients in asymptomatic, symptomatic without other causes except infection of D. fragilis, and symptomatic with another cause. Among symptomatic patients, treatment response was evaluated. One hundred eight patients were included. Sixty-three percent of the patients were immigrants, 29.6% were autochthonous, and 7.4% were travelers. Forty-nine (45.3%) patients presented symptoms, and eosinophilia was observed in 26 (24.1%) patients. Overall, 59 (54.7%) patients were asymptomatic, 15 (13.8%) presented symptoms which were attributable to other causes, and 34 (31.5%) patients presented symptoms with no other causes. In this last group, 29 patients received specific treatment and 65.5% of them presented a complete resolution of the symptoms. The group of symptomatic patients with no other cause had more proportion of women, more proportion of autochthonous people, and were older compared with the group of asymptomatic patients. Dientamoeba fragilis infection should be considered as pathogenic when other causes are ruled out.