Diversity profiling of xenic cultures of Dientamoeba fragilis following systematic antibiotic treatment and prospects for genome sequencing.

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.

MALDI-TOF MS as a new tool for the identification of Dientamoeba fragilis.

BACKGROUND: In this study for the first time, a Dientamoeba fragilis protein profile by MALDI-TOF MS was created in order to identify specific markers for the application of this technology in the laboratory diagnosis of dientamoebiasis. In particular, one D. fragilis reference strain was used to create a reference spectrum and 14 clinical isolates to verify the reliability of the obtained results. RESULTS: While 15 peaks were found to be discriminating between the reference strain and the culture medium used, six peaks, observed in all the 14 strains tested, were considered as markers able to identify D. fragilis. CONCLUSIONS: In our hands, MALDI-TOF MS technology was demonstrated as a useful tool to be used in association with or in replacement of the real-time PCR assay for the identification of D. fragilis used in our laboratory on xenic cultures, due to its accuracy, rapidity and low cost.

Multilocus sequence typing of Dientamoeba fragilis identified a major clone with widespread geographical distribution.

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.

Dientamoeba fragilis, the Neglected Trichomonad of the Human Bowel.

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.

Detection of Dientamoeba fragilis among diarrheal patients referred to Tabriz health care centers by nested PCR.

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.

Limited intra-genetic diversity in Dientamoeba fragilis housekeeping genes.

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.

Treatment of Dientamoeba fragilis in patients with irritable bowel syndrome.

The role of Dientamoeba fragilis in irritable bowel syndrome (IBS) is incompletely known. We aimed to investigate whether eradication of D. fragilis alleviates symptoms in IBS. Twenty-five D. fragilis-positive IBS patients were treated with Metronidazole (MZ) or Tetracycline. The patients were mostly female (89%), and mean age (SD) was 35.1 (8.2) years. Microbiological response, evaluated 2 weeks post-treatment, was observed in 15 of 25 patients (60%), all by MZ. Clinical response, defined as adequate relief of symptoms, was observed in 7 of 22 patients (32%), all by MZ. In a logistic regression analysis, we found no significant association between clinical and microbiological response. This case study did not support our hypothesis of a simple association between D. fragilis and IBS. Some D. fragilis-infections were insufficiently treated by MZ. Further studies into the prevalence and effect of eradication of D. fragilis in IBS and into efficient treatments of D. fragilis are warranted.

A review of Dientamoeba fragilis carriage in humans: several reasons why this organism should be considered in the diagnosis of gastrointestinal illness.

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.

The ambiguous life of Dientamoeba fragilis: the need to investigate current hypotheses on transmission.

Dientamoeba fragilis is an inhabitant of the human bowel and is associated with gastrointestinal illness. Despite its discovery over a century ago, the details of Dientamoeba's life cycle are unclear and its mode of transmission is unknown. Several theories exist which attempt to explain how Dientamoeba may be transmitted. One theory suggests that animals are responsible for the transmission of Dientamoeba. However, reports of Dientamoeba in animals are sporadic and most are not supported by molecular evidence. Another theory suggests that Dientamoeba may be transmitted via the ova of a helminth. Given that the closest relative of Dientamoeba is transmitted via the ova of a helminth, this theory seems plausible. It has also been suggested that Dientamoeba could be transmitted directly between humans. This theory also seems plausible given that other relatives of Dientamoeba are transmitted in this way. Despite numerous investigations, Dientamoeba's mode of transmission remains unknown. This review discusses the strengths and weaknesses of theories relating to Dientamoeba's mode of transmission and, by doing so, indicates where gaps in current knowledge exist. Where information is lacking, suggestions are made as to how future research could improve our knowledge on the life cycle of Dientamoeba.

Clinical and laboratory findings in 220 children with recurrent abdominal pain.

AIM: To investigate the clinical and laboratory findings in children with recurrent abdominal pain (RAP). METHODS: Consecutive patients with RAP (Apley criteria), age 4-16 years, referred to a secondary medical centre were evaluated by a standardized history, physical examination and laboratory tests. The tests encompassed Helicobacter pylori (Hp), gastrointestinal bacterial infections, protozoa, coeliac disease, carbohydrate malabsorption, food intolerance, abdominal ultrasound and plain abdominal X-ray. More investigations were obtained if indicated. Patient characteristics were compared with surgical patients without abdominal pain (control group). RESULTS: A total of 220 consecutive patients were included (92 M, mean age 8.8 years [4.1-16.0 years]). In 88% of the patients, abnormalities were found that refer to possible causes. Especially, protozoa were present in 33% of the patients, mostly Dientamoeba fragilis, Yersinia enterocolitica in 12% and endoscopically proven infection with Hp in 11%. In 36%, a plain abdominal X-ray raised suspicion of constipation. CONCLUSION: In 220 consecutive patients with RAP, referred to secondary care, a standardized work-up yielded abnormal results in a high percentage. The clinical significance of these findings remains to be established.

Blastocystis hominis and Dientamoeba fragilis in patients fulfilling irritable bowel syndrome criteria.

Studies have suggested a possible role for Blastocystis hominis and Dientamoeba fragilis in the etiology of irritable bowel syndrome (IBS). We studied the prevalence of B. hominis and D. fragilis in patients with IBS-diarrhea (IBS-D). Three hundred and thirty patients were enrolled, 171 (52%) with IBS-D and 159 (48%) were controls, respectively. Stool microscopy, culture, and polymerase chain reaction (PCR) for B. hominis and D. fragilis were done. B. hominis was positive by stool microscopy in 49% (83/171) of IBS compared to 24% (27/159) in control (p < 0.001). B. hominis culture was positive in 53% (90/171) in IBS compared to 16% (25/159) in control (p < 0.001). B. hominis PCR was positive in 44% (75/171) in IBS compared to 21% (33/159) in control (p < 0.001). D. fragilis microscopy was positive in 3.5% (6/171) in IBS-D compared to 0.6% (1/159) in control (p = 0.123). D. fragilis culture was positive in 4% (7/171) in IBS compared to 1.3% (2/159) in control (p = 0.176). D. fragilis PCR was positive in 4% (6/171) in IBS-D compared to 0% (0/159) in control (p = 0.030). B. hominis is common, while D. fragilis was less prevalent in our patients with IBS-D. B. hominis and D. fragilis culture had a better yield compared to stool microscopy and PCR.

Genetic diversity of Dientamoeba fragilis isolates of irritable bowel syndrome patients by high-resolution melting-curve (HRM) analysis.

Dientamoeba fragilis is a parasite that has been recognized as a causative agent of gastrointestinal symptoms. The search for genetic variation in D. fragilis based on the small-subunit (SSU) rRNA gene using restriction fragment length polymorphism was found not useful for molecular epidemiology. In this study, genetic variability of different clinical isolates of D. fragilis was explored by high-resolution melting curve (HRM) following polymerase chain reaction (PCR) in a one-step closed-tube method. Thirty fecal samples from irritable bowel syndrome (IBS) patients having D. fragilis trophozoites and negative for other organisms were involved in this study. According to the type of diarrhea, eight patients had acute, 14 patients had chronic intermittent, and eight patients had diarrhea alternating with constipation. HRM proved that four profiles (subtypes) were present as detecting by scanning mutation. One of these profiles (profile 1) was predominant (50%). Profile 2 was present on 20%. Profiles 3 and 4 were present on 16.7% and 13.4%, respectively. No mixed profiles were detected among the samples. The melting curves characterized by T(m)1=77.17+/-0.29 degrees C in profile 1, T(m)1=77.37+/-1.45 degrees C in profile 2, T(m)1=74.24+/-0.08 degrees C and T(m)2=79.64+/-0.09 degrees C in profile 3, and T(m)1=75.51 +/- 0.09 degrees C and T(m)=79.42 +/- 0.09 degrees C in profile 4. The relation between these profiles and types of diarrhea proved that the majority of patients having profile 1 (73.4%) and profile 4 (75%) had chronic intermittent diarrhea. All of the patients having profile 2 had acute diarrhea while all of the patients having profile 3 had diarrhea alternating with constipation. Although profile 1 was detected among all types of diarrhea, it was corresponding to 11/14 of patients with chronic intermittent diarrhea. All the differences were clinically and statistically significant. In conclusion, HRM following PCR was proved as a wide variation on D. fragilis genotypes that could be related to the characters of diarrhea among IBS patients. As the differences in HRM reflect different sequences of SSU RNA gene, thus, another study for identifying the sequences of these isolates (profiles) will be done and published later.

Intragenomic variation in the internal transcribed spacer 1 region of Dientamoeba fragilis as a molecular epidemiological marker.

Dientamoeba fragilis is a parasite that has been recognized to be a causative agent of gastrointestinal symptoms. Because in most studies only some infected persons experience symptoms, it is possible that D. fragilis is a heterogeneous species with variants that display similar morphologies but different pathogenicities. The search for genetic variation in D. fragilis was based on the small-subunit rRNA gene, which was not found to be useful for molecular epidemiology. In this report, we describe the isolation and characterization of additional rRNA gene cluster sequences, the internal transcribed spacer 1 (ITS-1)-5.8S rRNA gene-ITS-2 region. For comparative purposes, we also isolated the ITS-1-5.8S rRNA gene-ITS-2 region of Histomonas meleagridis, a protozoan parasite of birds and a close relative of D. fragilis. This region was found to be highly variable, and 11 different alleles of the ITS-1 sequence could be identified. Variation in the ITS-1 region was found to be intragenomic, with up to four different alleles in a single isolate. So-called C profiles were produced from the ITS-1 repertoire of single isolates. Analysis of the C profiles of isolates from nonrelated patients identified several clearly distinguishable strains of D. fragilis. Within families, it was shown that members can be infected with the same or different strains of D. fragilis. In conclusion, the ITS-1 region can serve as a molecular epidemiological tool for the subtyping of D. fragilis directly from feces. This may serve as a means of studying the transmission, geographical distribution, and relationships between strains and the pathogenicity of this parasite.

Dientamoebiasis: clinical importance and recent advances.

Dientamoeba fragilis, an unusual single-celled parasite that was described first in 1918, is found worldwide in the gastrointestinal tract of humans. D. fragilis has emerged from obscurity recently because it is now recognized as a common cause of chronic diarrhoea and is treatable with drugs. Recent molecular studies have described D. fragilis as having two genotypes. Diagnostic tests, based on conventional and real-time PCR, have been developed that will provide a rapid, sensitive and specific diagnosis of D. fragilis. These tests will also aid the elucidation of the host distribution and the life cycle of this pathogen.

Dientamoeba fragilis is more prevalent than Giardia duodenalis in children and adults attending a day care centre in Central Italy.

Giardia duodenalis is a well recognised enteropathogen, while Dientamoeba fragilis is rarely detected and consequently it is not recognised as an important human pathogen. In 2002-2003, a survey has been carried out on enteroparasites in faecal samples of outpatients attending a day care centre in the town of Perugia (Central Italy). To improve the detection level, at least three samples from each patient were collected at different days and within two hours from defecation. The coproparasitological examination has been carried out by direct microscopic examination, faecal concentration, and Giemsa and modified Ziehl-Nielsen stainings of faecal smears. The genotypes of Giardia duodenalis isolates were determined by PCR of the beta-giardin gene. Of 1,989 enrolled people (966 children, 1,023 adults), 165 persons (8.3%; 153 adults, 15.0%; 12 children, 1.2%), were positive for parasites, but only 1 12 adults (73.2% of those infected) and eight children (66.7% of those infected) harboured D. fragilis and G. duodenalis. Both the Assemblages A and B were detected in 18 G. duodenalis isolates examined at the beta-giardin gene. The higher prevalence of D. fragilis infections than that of G. duodenalis is probably related to the method used, a procedure, which is rarely followed in laboratories for the diagnosis of enteric parasites. These epidemiological data suggest that when faecal samples are examined after a period of time and without Giemsa staining, most D. fragilis infections goes undetected.

Prospective study of the prevalence, genotyping, and clinical relevance of Dientamoeba fragilis infections in an Australian population.

A prospective study was conducted over a 30-month period, in which fecal specimens from 6,750 patients were submitted to the Department of Microbiology at St. Vincent's Hospital, Sydney, Australia. Trophozoites of Dientamoeba fragilis were detected in 60 (0.9%) patients by permanent staining, and confirmation was performed by PCR. Gastrointestinal symptoms were present in all patients, with diarrhea and abdominal pain the most common symptoms. Thirty-two percent of patients presented with chronic symptoms. The average age of infected patients was 39.8 years. No correlation was found between D. fragilis and Enterobius vermicularis, a proposed vector of transmission for D. fragilis. The genetic diversity of 50 D. fragilis isolates was examined by PCR, and the PCR products were analyzed for the presence of restriction fragment length polymorphisms. These results showed no variation in the small-subunit rRNA gene and demonstrated a single genotype for all Australian isolates. This study shows the potential pathogenic properties of D. fragilis and the need for all laboratories to routinely test for this organism.

Dientamoeba fragilis: the unflagellated human flagellate.

Dientamoeba fragilis is a pathogenic protozoan parasite with a world-wide distribution. Although originally described as an amoeboid organism, it has been reclassified as a flagellate, on the basis of a number of electron microscopic and immunological findings. Except for its lack of a flagellum, D. fragilis closely resembles Histomonas and Trichomonas. Interestingly, a resistant cyst stage has not been demonstrated and it is unlikely that its trophozoites can survive successfully outside the human host. As a consequence of its higher than anticipated coincidence of infection with Enterobius vermicularis, transmission may occur via ova of this pinworm. D. fragilis infection may be acute or chronic, and has been reported in both children and adults. The most common clinical symptoms include abdominal pain, persistent diarrhoea, loss of appetite, weight loss and flatulence. Occasionally, eosinophilia, urticaria and pruritus have been described. Demonstration of the characteristic nuclear structure of D. fragilis, needed for a definitive diagnosis, cannot be achieved in unstained faecal material; therefore, permanently stained smears are essential. Treatment is recommended in symptomatic cases, and iodoquinol, tetracycline and metronidazole have been used successfully.