New advances in the in-vitro culture of Dientamoeba fragilis

Dientamoeba fragilis is an intestinal protozoan in humans that is commonly associated with diarrhoea and other gastrointestinal complaints. Studies conducted to investigate the biology of this parasite are limited by methods for in vitro cultivation. The objective of this study was to improve a biphasic culture medium, based on the Loeffler's slope, by further supplementation in order to increase the yield of trophozoites in culture. The current in vitro culture of D. fragilis is a xenic culture with a mix of bacteria. Three different liquid overlays were evaluated including Earle's balanced salt solution (EBSS), PBS and Dulbecco's modified PBS (DPBS), for their ability to support the in vitro growth of D. fragilis trophozoites. Out of these 3 overlays EBSS gave the highest increase in the trophozoite numbers. The effect of supplementation was analysed by supplementing EBSS with ascorbic acid, ferric ammonium citrate, L-cysteine, cholesterol and alpha-lipoic acid and quantification of in vitro growth by cell counts. A new liquid overlay is here described based upon EBSS supplemented with cholesterol and ferric ammonium citrate that, in conjunction with the Loeffler's slope, supports the growth of D. fragilis trophozoites in vitro. This modified overlay supported a 2-fold increase in the numbers of trophozoite in culture from all 4 D. fragilis isolates tested, when compared to a PBS overlay. These advances enable the harvest of a larger number of trophozoites needed for further studies on this parasite. © Cambridge University Press 2012

A microscopic description and ultrastructural characterisation of Dientamoeba fragilis: An emerging cause of human enteric disease

Dientamoeba fragilis is a pathogenic trichomonad found in the gastrointestinal tract of humans and is implicated as a cause of diarrhoea. Despite its discovery over a century ago, there has been no recent thorough description of this parasite by microscopy. Scanning electron microscopy, transmission electron microscopy, confocal and light microscopy were therefore used to characterise D. fragilis populations growing in xenic culture. Two different populations - smooth and ruffled cells - were identifiable by scanning electron microscopy. No flagella, pelta structures, undulating membrane or pseudocyst-like forms were present. The organelles in D. fragilis were analysed by transmission electron microscopy; like Trichomonas and Histomonas, D. fragilis contains hydrogenosomes that presumably represent the site of anaerobic respiration. The nuclear morphology of D. fragilis trophozoites grown in vitro and trophozoites from clinical isolates were also compared by confocal microscopy and light microscopy. The majority of cells grown in culture were mononucleate while most cells in permanent stained faecal smears were binucleate. The two nuclei of D. fragilis are morphologically indistinguishable and contain equivalent amounts of DNA as determined by DAPI staining. The approximate cell and nuclear volume of four isolates of D. fragilis were measured and shown to be comparable to other trichomonads. In addition, the discovery of a virus-like particle is reported, to our knowledge for the first time in D. fragilis. This study therefore provides extensive and novel details of the ultrastructure of a neglected protozoan parasite that is an emerging cause of human disease. © 2011 Australian Society for Parasitology Inc

Dientamoeba fragilis, the neglected trichomonad of the human bowel

© 2016, American Society for Microbiology. 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

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

A case-controlled study of Dientamoeba fragilis infections in children

Dientamoeba fragilis is a pathogenic protozoan parasite that is implicated as a cause of human diarrhoea. A case-controlled study was conducted to determine the clinical signs associated with D. fragilis infection in children presenting to a Sydney Hospital. Treatment options are also discussed. Stool specimens were collected from children aged 15 years or younger and analysed for the presence of D. fragilis. In total, 41 children were included in the study along with a control group. Laboratory diagnosis was performed by microscopy of permanently stained, fixed faecal smears and by real-time PCR. Gastrointestinal symptoms were present in 40/41 (98%) of these children with dientamoebiasis, with diarrhoea (71%) and abdominal pain (29%) the most common clinical signs. Chronic gastrointestinal symptoms were present in 2% of cases. The most common anti-microbial used for treatment was metronidazole (n=41), with complete resolution of symptoms and clearance of parasite occurring in 85% of cases. A treatment failure rate occurred in 15% of those treated with metronidazole. Follow-up treatment comprised of an additional course of metronidazole or iodoquinol was needed in order to achieve complete resolution of infection and symptoms in this group. This study demonstrates the pathogenic potential of D. fragilis in children and as such it is recommended that all laboratories must routinely test for this organism and treat if detected. © Cambridge University Press 2011

Activity of benzimidazoles against Dientamoeba fragilis (Trichomonadida, Monocercomonadidae) in vitro and correlation of beta-tubulin sequences as an indicator of resistance

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

Newly defined conditions for the in vitro cultivation and cryopreservation of Dientamoeba fragilis: New techniques set to fast track molecular studies on this organism

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 42C 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. © Cambridge University Press 2010

Radiocaesium transfer and radiation exposure of frogs in Fukushima Prefecture

The International Commission on Radiological Protection has proposed an environmental assessment framework. This includes ionising radiation exposure assessment for different frog life-stages, but radiocaesium transfer parameters are unavailable. We collate data from the Fukushima Prefecture (contaminated by the Fukushima accident) and estimate radiocaesium concentration ratio (CR ) values for tadpoles and adult frogs, presenting the largest available amphibian CR dataset. In total, 513 adult frogs and 2540 tadpoles were analysed in 62 and 59 composite samples respectively. Results suggest that equilibrium was reached between water and amphibian radiocaesium activity concentrations circa one-year after the accident. Radiocaesium transfer to tadpoles was higher than to adult frogs. Dose rates were estimated for different life-stages and species in both the aquatic and terrestrial environment. Estimated dose rates to adults and tadpoles were typically similar because external exposure dominated for both organisms; frogspawn dose rates were estimated to be orders of magnitude lower than other life-stages. For the two sites assessed, which were outside of the most contaminated areas of the Fukushima Prefecture, estimated dose rates were below those anticipated to present a risk to wildlife populations; it is likely that dose rates in more contaminated areas were in excess of some effects benchmark values

Trends in suicide in Scotland 1981 – 1999: age, method and geography

BACKGROUND: Male suicide rates continued to increase in Scotland when rates in England and Wales declined. Female rates decreased, but at a slower rate than in England and Wales. Previous work has suggested higher than average rates in some rural areas of Scotland. This paper describes trends in suicide and undetermined death in Scotland by age, gender, geographical area and method for 1981 – 1999. METHODS: Deaths from suicide and undetermined cause in Scotland from 1981 – 1999 were identified using the records of the General Registrar Office. The deaths of people not resident in Scotland were excluded from the analysis. Death rates were calculated by area of residence, age group, gender, and method. Standardised Mortality Ratios (SMRs) and 95% confidence intervals were calculated for rates by geographical area. RESULTS: Male rates of death by suicide and undetermined death increased by 35% between 1981 – 1985 and 1996 – 1999. The largest increases were in the youngest age groups. All age female rates decreased by 7% in the same period, although there were increases in younger female age groups. The commonest methods of suicide in men were hanging, self-poisoning and car exhaust fumes. Hanging in males increased by 96.8% from 45 per million to 89 per million, compared to a 30.7% increase for self-poisoning deaths. In females, the commonest method of suicide was self-poisoning. Female hanging death rates increased in the time period. Male SMRs for 1981 – 1999 were significantly elevated in Western Isles (SMR 138, 95% CI 112 – 171), Highland (135, CI 125 – 147), and Greater Glasgow (120, CI 115 – 125). The female SMR was significantly high only in Greater Glasgow (120, CI 112 – 128). CONCLUSION: All age suicide rates increased in men and decreased in women in Scotland in 1981 – 1999. Previous findings of higher than expected male rates in some rural areas were supported. Rates were also high in Greater Glasgow, one of the most deprived areas of Scotland. There were changes in the methods used, with an increase in hanging deaths in men, and a smaller increase in hanging in women. Altered choice of method may have contributed to the increased male deaths