Coproscopy and molecular screening for detection of intestinal protozoa.

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.

[Dientamoeba fragilis: is it really fragile? Approach to specimen handling and rapid microscopic diagnosis]. / Dientamoeba fragilis: è realmente fragile? Approccio al trattamento del campione e diagnosi rapida al microscopio.

Dientamoeba fragilis is a pathogenic protozoan parasite with a world-wide distribution. Interestingly, a resistant cyst stage has not been demonstrated and it is still an unsolved problem how this parasite can survive successfully outside the human host. D. fragilis was found in 2% of approximately 2500 individuals unselected who submitted stools for parasitological examination during 2001 in Padua (Italy). The goal of this study was to detect the protozoan stages and the duration of persistence of this protozoa in faeces stored in different environmental conditions. The trophozoites of D. fragilis were detected up to 60 days after the collection of the faeces stored at 4 degrees C and Giemsa stained. The laboratory detection rate of the organism is greatly enhanced by use of preservative to fix stool specimens immediately after passage. Alternatively, a microscopic observation of the collected stool has to be performed immediately after passage followed by examination of permanently-stained smears. Demonstration of the charateristic "golf-club" and "acanthopodia-like" structures in unstained fixed faecal material by direct microscopy (400x) are suitable for a rapid identification of D. fragilis.

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.

Detection of Dientamoeba fragilis by culture.

Symptoms associated with Dientamoeba fragilis include diarrhoea, abdominal pain, nausea, vomiting, epigastric pain and weight loss. A possible link between D. fragilis and irritable bowel syndrome (IBS)-like symptoms has been reported, and therefore the presence of this parasite should be excluded before making a diagnosis of IBS. Over a six-month period, 976 faecal samples were submitted to NPHS Microbiology Aberystwyth for routine microbiological analysis. All samples were also cultured for parasites using Robinson's xenic medium. Trichrome staining was undertaken whenever practicable, but many stools had insufficient material. D. fragilis was isolated from 25 (2.6%) patients, whereas Cryptosporidium spp. was detected in 16 (1.6%) patients. D. fragilis was only detected in nine (1.3%) out of 685 specimens stained with trichrome, although four of the 25 culture-positive stools had insufficient sample for staining. Parasite culture proved to be less laborious than trichrome staining and dramatically increased D. fragilis detection rate.

[3 emerging protozoal infections in The Netherlands: Cyclospora, Dientamoeba, and Microspora infections]. / Drie opkomende protozoaire infectieziekten in Nederland: Cyclospora-, Dientamoeba- en Microspora-infecties.

Increased international travelling, an increased number of patients with immunosuppression caused by HIV infection, and renewed interest in known but little studied microorganisms, resulted in a more frequent finding of certain medically important parasites. Three emerging protozoal infection, Cyclospora cayetanensis, Dientamoeba fragilis and Microspora (Enterocytozoon bieneusi and Encephalitizoon) are causative agents of diarrhoea. Encephalitozoon infections are also associated with hepatitis, hepatoconjunctivitis and nephritis. C. cayetanensis infection was diagnosed in 28 patients in the years 1992-1995 in the Academic Medical Centre in Amsterdam, half of these patients returning from a visit to Indonesia. D. fragilis has a prevalence in the Netherlands of 8% among patients with diarrhoea lasting longer than one week referred for parasitological investigation. The prevalence of E. bieneusi in HIV positive patients with diarrhoea was 8-10% in the Academic Medical Centre in the last five years. Infection with Encephalitozoon appears to be endemic in the Netherlands.

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.