Dientamoeba fragilis and dientamoebiasis: aspects of clinical parasitology and laboratory diagnosis

The flagellate Dientamoeba fragilis is a potential intestinal pathogen of humans. It is probably undervalued overall because its identification relies on permanent stains directly on fresh fecal specimens (Giemsa) or preserved in PVA or SAF (Trichrome or Iron-hematoxylin). During 1999 we processed stoll samples of 151 subjects and we searched also D. fragilis. We processed 105 cases of enteritis (82 children and 23 adults) and 46 samples of formed stools to investigate only for intestinal parasites (12 children and 34 adults). The microscopical observations were made on smears directly in saline solution, and after Dobell solution, MIF and Giemsa stained. Iron hematoxylin and trichrome stains were performed in some circumstances only. D. fragilis was observed in 11.3% of cases, 7.6% among the group of enteritis and 13.8% among the other subjects. These positive 17 cases were observed more often among adults (76.5% of cases) than among children (23.5%). In our study area D. fragilis seems more frequent than Giardia intestinalis and represents the third important intestinal parasite, following Enterobius vermicularis (children) and Strongyloides stercoralis (elders).     

SL1 RNA gene recovery from Enterobius vermicularis ancient DNA in pre-Columbian human coprolites

Enterobius vermicularis, pinworm, is one of the most common helminths worldwide, infecting nearly a billion people at all socio-economic levels. In prehistoric populations the paleoparasitological findings show a pinworm homogeneous distribution among hunter-gatherers in North America, intensified with the advent of agriculture. This same increase also occurred in the transition from nomad hunter-gatherers to sedentary farmers in South America, although E. vermicularis infection encompasses only the ancient Andean peoples, with no record among the pre-Colombian populations in the South American lowlands. However, the outline of pinworm paleoepidemiology has been supported by microscopic finding of eggs recovered from coprolites. Since molecular techniques are precise and sensitive in detecting pathogen ancient DNA (aDNA), and also could provide insights into the parasite evolutionary history, in this work we have performed a molecular paleoparasitological study of E. vermicularis. aDNA was recovered and pinworm 5S rRNA spacer sequences were determined from pre-Columbian coprolites (4110 BC-AD 900) from four different North and South American archaeological sites. The sequence analysis confirmed E. vermicularis identity and revealed a similarity among ancient and modern sequences. Moreover, polymorphisms were identified at the relative positions 160, 173 and 180, in independent coprolite samples from Tulán, San Pedro de Atacama, Chile (1080-950 BC). We also verified the presence of peculiarities (Splicing leader (SL1) RNA sequence, spliced donor site, the Sm antigen biding site, and RNA secondary structure) which characterise the SL1 RNA gene. The analysis shows that the SL1 RNA gene of contemporary pinworms was present in pre-Columbian E. vermicularis by 6110 years ago. We were successful in detecting E. vermicularis aDNA even in coprolites without direct microscopic evidence of the eggs, improving the diagnosis of helminth infections in the past and further pinworm paleoepidemiological studies.     

Detection of Dientamoeba fragilis in fresh stool specimens using PCR

Dientamoeba fragilis is a trichomonad parasite that causes human gastrointestinal disease. Currently microscopy is considered to be the gold standard for diagnosis of D. fragilis infection. However, this method is time-consuming and relatively insensitive. A PCR assay based on the small-subunit ribosomal RNA gene of D. fragilis for the specific detection of D. fragilis DNA in fresh unpreserved stool samples was developed. The D. fragilis PCR was positive in 29/31 samples with positive microscopy and did not cross-react with other protozoan parasites. The PCR protocol showed a specificity of 100% and a sensitivity of 93.5% and the entire procedure can be performed in one day.     

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.     

On the clinical importance of Dientamoeba fragilis infections in childhood

Clinical and laboratory findings among 123 paediatric patients infected by intestinal protozoa were analysed. Dientamoeba fragilis (D. f) was found in 102 cases. The other patients proved to be carriers of Giardia lamblia or of mixed infections with several protozoa. Acute and recurrent diarrhoea have been found to be the most common symptoms, whereas abdominal pain was most common in children with chronic infections. Peripheral blood eosinophilia was seen in a third of the children with dientamoebiasis. Metronidazole, oxytetracycline, doxycycline, and erythromycin were effective drugs in the treatment of D. f. infections. The therapy coincidentally led to the elimination of protozoal infections as well as the abdominal complaints. These results underline the pathogenic role of D. f. in children with gastrointestinal symptoms.     

Chimpanzee pinworm, Enterobius anthropopitheci (Nematoda: Oxyuridae), maintained for more than twenty years in captive chimpanzees in Japan

The chimpanzee pinworm, Enterobius anthropopitheci (Gedoelst, 1916), was found in chimpanzees, Pan troglodytes, reared in Kumamoto Primate Research Park, Sanwa Kagaku Kenkyusho Co., Ltd., Kumamoto, Japan, in 2006. Because the chimpanzees in this institution originated from chimpanzees imported from Africa before 1984, it is considered that E. anthropopitheci infection has persisted for more than 20 yr in the chimpanzees. Analysis of pinworm specimens preserved in the institution revealed that transition of predominant pinworm species occurred, responding to the change of anthelmintics used for pinworm treatment. Present dominance of E. anthropopitheci is surmised to be caused by fenbendazole, which has been adopted from 2002. Scarcity of mixed infection with E. anthropopitheci and Enterobius vermicularis suggests interspecific competition between the pinworms.     

Egg positive rate of Enterobius vermicularis among preschool children in Cheongju, Chungcheongbuk-do, Korea

In an attempt to determine the prevalence of pinworm infection, the egg positive rate of Enterobius vermicularis was examined using the adhesive cello-tape anal swab method in 1,512 preschool children sampled from a total of 20 kindergartens in Cheongju city, in November to December of 2004 (951 children from 13 kindergartens) and September to October of 2005 (561 from 7 kindergartens). The overall egg positive rate was found to be 7.9% (119/1,512); 9.3% (73/784) for boys and 6.3% (46/728) for girls, respectively. The 5-year age group evidenced the highest egg positive rate (10.9%, 47/430) among the examined age groups. As compared to those reported from previous works (ranged from 9.2 to 26.1%), the prevalence of E. vermicularis in the Cheongju city area is relatively low.     

Dientamoeba fragilis: is it really fragile? Approach to specimen handling and rapid microscopic diagnosis

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.     

Enterobius vermicularis eggs discovered in coprolites from a medieval Korean mummy

While the presence of pinworm eggs in archaeological samples has been reported by many researchers in the New World, those have been detected very scarcely in the Old World, especially in East Asian countries. In fact, many parasite species were recovered from the archeological remains in Korea, eggs of Enterobius vermicularis had not been found. Recently, a female mummy buried in the 17th century was discovered in the Joseon tomb from Dangjin-gun, Chungcheongnam-do, Korea. After rehydration process for 12 days, investigations were carried on the luminal surface of the colon. From them, 3 eggs of E. vermicularis were recovered. They were elliptical, transparent with a thin egg shell, 50.3±5.2 μm (length) and 28.2±3.9 μm (width) in size. This is the first discovery of E. vermicularis eggs in East Asia.     

Epidemiology of Dientamoeba fragilis Jepps and Dobell, 1918. 3. Further studies on Enterobius transmission through eggs]

In two new attempts to infect human subjects with pin-worms that were infested, at that time, with protozoa Dientamoeba fragilis, presence of these protoza in the intestinal canal was observed in one case for more than 84 days, and in another case for more than 31 days after contamination. The duration of latent periods fixed for Enterobius vermicularis was almost identical (51 and 50 days). New attempts implement and confirm the results of the first successful attempts to transfer the infection (Ockert, 1972b) as well as the hypothesis of the epidemiological interrelation between both parasites (Burrows and Swerdlow 1956).     

Growth of Enterobius vermicularis in a chimpanzee after anthelmintic treatment

The growth of Enterobius vermicularis in a chimpanzee was investigated by observing worms discharged in feces after administration of pyrantel pamoate. Just after the final molting, immature adult male pinworms developed to a fully mature stage in 2 wk, after passing through a stage corresponding to the so-called Enterobius gregorii, which was surmised to be a younger adult form of E. vermicularis. The frequency distribution of body length forms 2 peaks in both male and female pinworms, with a depression in the transitional forms from the immature to the fully mature stage.This depression seems to be the result of more rapid growth or lower susceptibility to the drug in this transitional stage. Pyrantel pamoate effectively eradicated mature males, but gravid females were continuously observed in the feces after treatment. The complete eradication of pinworm infection by pyrantel pamoate could be achieved by repeated treatment at intervals shorter than 2 wk. This treatment would eradicate male worms first, resulting in females producing only unfertilized eggs, from which only males might hatch.     

A survey of Enterobius vermicularis infection among children on western and southern coastal islands of the Republic of Korea

To determine the status of Enterobius vermicularis infection among children living on western and southern coastal islands of the Republic of Korea, children (3-10 years) in 39 kindergartens and primary schools were examined using the cello-tape anal swab method, during July and August 2000. Of 1,661 children examined, 307 (18.5%) were found to be positive for E. vermicularis eggs. The highest infection rate (59.3%) was found in a kindergarten and a branch school of Shinyang primary school on Chujado, Jeju-do (Province). Remarkable differences in egg positive rates were observed in different localities. The egg positive rate for boys (21.3%) was significantly higher than that of girls (15.4%) (P = 0.02). However, positive rates were not significantly dependent on age. The results of the present study show that E. vermicularis infection is highly prevalent among pre-school and primary school children living on the western and southern coastal islands of the Republic of Korea, and indicate the need for efforts to control this infection.     

Enterobius vermicularis egg positive rate in a primary school in Chungchongnam-do (province) in Korea

The egg positive rate of Enterobius vermicularis was investigated among students of a primary school and a kindergarten located in the rural area of Tangjin-gun, Chungchongnam-do in December, 1998. Of the 189 examinees, 28 (14.8%) were found to be infected with E. vermicularis by the adhesive cellotape anal swab method. The infection rates ranged from 4.2% to 26.1% among school children, and the highest rate was observed in children attending kindergarten. Three months after treatment with albendazole, four (14.3%) out of 28 infected children still remained infected with E. vermicularis. Through this survey, we were able to determine that E. vermicularis infection is still prevalent among children in rural areas of Korea.     

Egg positive rate of Enterobius vermicularis of primary school children in Geoje island

The status of pinworm (Enterobius vermicularis) egg positive rate of primary school children in Geoje island was investigated by using adhesive cellotape anal swap method, in September, 2002. Total egg positive rates of E. vermicularis were 9.8% (74/754) and those of male and female were 10.8% and 8.7%, respectively. Among three schools examined, Myeongsa primary school showed the highest egg positive rate (12.6%) followed by Yeoncho [9.8% (26/266)] and Geoje [9.1% (35/385)]. As for the age groups, the 2nd grade had the highest egg positive rate (15.3%), whereas the 5th grade showed the lowest egg positive rate (2.6%). The above result led us to confirm that the egg positive rates of E. vermicularis in primary school children in Geoje island were not significantly different from the those in the whole country including urban and rural areas, showing more than 10%.     

Phylogenetic position of the trichomonad parasite of turkeys, Histomonas meleagridis (Smith) Tyzzer, inferred from small subunit rRNA sequence.

The phylogenetic position of the trichomonad, Histomonas meleagridis was determined by analysis of small subunit rRNAs. Molecular trees including all identified parabasalid sequences available in data bases were inferred by distance, parsimony, and likelihood methods. All reveal a close relationship between H. meleagridis, and Dientamoeba fragilis. Moreover, small subunit rRNAs of both amoeboid species have a reduced G + C content and increased chain length relative to other parabasalids. Finally, the rRNA genes from H. meleagridis and D. fragilis share a recent common ancestor with Tritrichomonasfoetus, which exhibits a more developed cytoskeleton. This indicates that Histomonas and Dientamoeba secondarily lost most of the typical trichomonad cytoskeletal structures and hence, do not represent primitive morphologies. A global phylogeny of parabasalids revealed significant discrepancies with morphology-based classifications, such as the polyphyly of most of the parabasalid families and classes included in our study.     

Comparison of two methods (microscopy and enzyme-linked immunosorbent assay) for the diagnosis of amebiasis

Diagnosis of amebiasis is usually performed on a clinical basis alone in most endemic countries having limited economic resources. This epidemiological study was conducted using modern diagnostic tests for amebiasis in the southeastern region of Turkey, an endemic area for amebiasis. The population of this study included patients with symptomatic diarrhea/dysentery attending both Yuzuncu Yil University, Van and Harran University, Sanliurfa, Turkey. A total of 380 stool specimens were collected and examined for Entamoeba by light microscopy (fresh, lugol, and trichrome staining) and stool antigen detection based- enzyme-linked immunosorbent assay (EIA) test (TechLab Entamoeba histolytica II). 24% (91/380) of stool specimens were positive for E. histolytica/Entamoeba dispar trophozoites/cysts microscopically using trichrome staining. 13% (51/380) of the stool specimens were found to be positive for E. histolytica by the EIA test, including 15% (14/91) of microscopy (+) stool specimens and 13% (37/289) of microscopy (-) stool specimens. Enteric parasites were common in these populations with 66% (251/380) of the study population harboring more than one parasite. In addition to the 13% (51/380) of patients determined to have E. histolytica by EIA, eighty-six patients (22.6%) had Blastocystis hominis, 54 (14.2%) Entamoeba coli, 44 (11.5%) Giardia lamblia, 16 (4.2%) Chilomastix mesnili, 15 (3.9%) Iodamoeba bütschlii, 12 (3.1%) Hymenolepis nana, 9 (2.3%) Endolimax nana, 9 (2.3%) Dientamoeba fragilis, and 8 (2.1%) had Ascaris lumbricoides. We concluded that E. histolytica infection was found in 13% of the patients presenting with diarrhea in Van and Sanliurfa Turkey.     

Prevalence of Enterobius vermicularis among preschool children in Gimhae-si, Gyeongsangnam-do, Korea

The present study was performed to determine the prevalence of Enterobius vermicularis among preschool children in Gimhae-si, Korea. A total of 6,921 preschool children in 76 kindergartens were examined using the cellotape perianal swab method. The overall egg positive rate (EPR) was 10.5%. The EPR in boys was higher than that in girls (adjusted odds ratio [AOR]: 1.5, P<0.001), and it was higher in rural than in urban children (AOR: 1.2, P=0.022). The present study confirmed that the prevalence of E. vermicularis infection is fairly high among preschool children in Gimhae-si. Therefore, systematic control and preventive measures should be adopted to reduce morbidity associated with this nematode infection.     

Giardiasis and other intestinal parasitic infections in a Manitoba residential school for the mentally retarded.

A case of severely symptomatic giardiasis in a school for the mentally retarded prompted an epidemiologic survey of the institution. The rate of parasitic infection in the children were just under 50%. Multiple infections were common and one child harboured five different protozoa. The yield included known pathogens (Giardia lamblia, Metorchis conjunctus and Diphyllobothrium sp.), protozoa of potential pathogenicity (Dientamoeba fragilis) and other protozoa, the significance of which has yet to be determined. The prevalence of G. lamblia in the index ward was significantly higher than in a control ward matched for age and mobility of the children. The epidemiologic data suggested person-to-person transmission of G. lamblia within the institution. Recommendations for the control of protozoal intestinal infections in custodial institutions are presented.     

Prevalence of Enterobius vermicularis Infection among Preschool Children in Kindergartens of Taipei City, Taiwan in 2008

The prevalence of Enterobius vermicularis infection among preschool children was reported to be low based on a 5-year screening program in Taipei City, Taiwan. The Taipei City government intended to terminate the E. vermicularis screening program among preschool children. Thus, we were entrusted with confirming whether pinworm infections among preschool children in Taipei City had truly declined. From each of 12 administrative districts 2-3 kindergartens were randomly selected for investigation. In total, 4,349 children were examined, of which 2,537 were boys and 1,812 were girls. The cellophane tape adhered to a glass slide was used, and all examinations were done by certified medical technologists. Results indicated that the overall prevalence rate of pinworm infections was 0.62% (27/4,349). Although the infection rate was higher among boys (0.67%, 17/2,537) than in girls (0.55%, 10/1,812), no significant difference was found (χ² = 0.399, P = 0.62). According to the administrative district, the infection rate ranged from no positive cases of E. vermicularis infection in the Xinyi, Zhongzhen, and Wanhua Districts (0%; 0/299, 0/165, and 0/358, respectively), to 0.26% (1/131) in Songshan District, with the highest rate of 1.88% (7/373) in Wenshan District. Because the overall infection rate (0.62%, 27/4,349) in the present study was unchanged compared to that (0.40%, 197/49,541) previously reported in 2005, we propose that regular pinworm screening and treatment programs should be continued in some parts of Taipei City.