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PubMed2002

[Usefulness of multiplex-PCR for identification of Entamoeba histolytica and Entamoeba dispar].

We evaluated the usefulness of a multiplex-PCR method for differentiation of Entamoeba histolytica and Entamoeba dispar, which are morphologically indistinguishable species. Cultured trophozoites of E. histolytica HM-1: IMSS and E. dispar SAW were used as the positive control. Seven human fecal samples, from which E. histolytica-like cysts were detected by microscopic examination, and three intestinal protozoan parasites, Cryptosporidium parvum HNJ-1, Giardia intestinalis Portland-1, and Blastocytis hominis Nand II, were used for the evaluation of sensitivity and specificity of the PCR method. The other PCR method, which has been used for the diagnosis of amebic infections in Japan, was also performed by using the same samples for the evaluation. In comparison with the conventional PCR method, the multiplex-PCR showed 1) higher sensitivity, 2) the size of diagnostic fragments of PCR products was clearly different in both Entamoeba species, 3) it was possible to perform PCR using a single tube per sample, and then to save the amount of DNA polymerase, 4) no diagnostic amplification products were found in other intestinal protozoan parasites, and 5) E. histolytica specific fragment was amplified in all clinical samples examined. In conclusion, it is considered that the multiplex-PCR method is a useful tool for detection of both Entamoeba species DNA from fecal samples and for the distinction between E. histolytica and E. dispar.

Animals↗
PubMedDate not supplied

Entamoeba motility: dynamics of cytoplasmic streaming, locomotion and translocation of surface-bound particles, and organization of the actin cytoskeleton in Entamoeba invadens.

The dynamics of cytoplasmic streaming, retrograde translocation of externally bound particles and locomotion by Entamoeba invadens were compared. Locomoting amoebae were monopodial, exhibited fountain flow cytoplasmic streaming and translocated externally bound erythrocytes to the rear of cells. The rates of rearward flow of peripheral cytoplasmic vacuoles and of the externally bound particles were equal to the rate of cell forward locomotion. Rhodamine-phalloidin staining revealed a distinct cortical polymerized actin cytoskelton. This was least evident about the periphery of the advancing pseudopod, increased in density toward the rear of the cell and was most concentrated in the uroid. A monoclonal anti-eucaryotic actin antibody, which recognized monomeric Entamoeba actin on immunoblots, stained trophozoites by indirect immunofluorescence throughout the cytoplasm, but not in the cortical regions stained by rhodamine-phalloidin. This and other evidence implied that the antibody recognized only unpolymerized actin in Entamoeba. We propose that locomotion, cytoplasmic streaming and translocation of externally bound particles are driven by a common actin-based mechanism in Entamoeba, possibly involving retrograde cortical actin flow and recycling.

Actins↗
PubMedDate not supplied

Distribution and differential diagnosis of Entamoeba histolytica from Entamoeba dispar by the PCR-RFLP method in Central Iran.

BACKGROUND: Entamoeba histolytica and Entamoeba dispar are two morphologically indistinguishable human protozoan parasites that are genetically distinct species. The potential invasive pathogenic Entamoeba histolytica and non-invasive parasite Entamoeba dispar can be differentiated by molecular and other methods. We used polymerase chain reaction (PCR) to determine the ratio of the two species in a population in Tehran and Karaj in central Iran. MATERIALS AND METHODS: Human stool samples (n=12 148) were randomly collected in Tehran and Karaj and examined for E. histolytica/E. dispar cysts with direct and formalin-ether methods. Eighty-seven (0.7%) cases were positive, of which 49 (62.8%) isolates were successfully cultured in Robinson s medium. A pair of oligonucleotide primers designed from sequence data for genomic DNA coding the 30-KD surface antigen of E. histolytica/E. dispar was used to amplify a 374 base-pair (bp) fragment. The electrophoretic pattern of the PCR product digested with Hinfl restriction enzyme was used for differentiation of the two species. RESULTS: The restriction fragment length polymorphism (RFLP) pattern obtained from a standard E. histolytica isolate had two fragments (219 bp and 155 bp), but the standard isolate of E. dispar showed three fragments (155, 152 and 67 bp). Differential diagnosis of 49 isolates of E. histolytica/E. dispar from Tehran and Karaj using PCR-RFLP revealed that 46 (93.9%) were E. dispar while only 2 (4.1%) were E. histolytica. One person (2%) had a mixed infection and showed both patterns. CONCLUSION: The differential diagnosis of the potentially pathogenic parasite E. histolytica from the non-pathogenic E. dispar is of clinical and epidemiological importance. This study demonstrated that E. dispar is much more prevalent than E. histolytica among the cyst passers in Tehran and Karaj in Central Iran.

Journal Article↗
PubMed2006

Differentiation of Entamoeba histolytica from Entamoeba dispar by PCR-coupled nonisotopic SSCP analysis.

Entamoeba histolytica is a pathogenic protozoan parasite, which causes amoebic colitis, dysentery and liver abscesses in humans. Since the cyst and small trophozoite stages of this parasite are indistinguishable by light microscopy from Entamoeba dispar (which is nonpathogenic), specific diagnosis is compromised. To overcome this limitation, a PCR-coupled SSCP approach, utilising a sequence difference of 4.6% in a short region ( approximately 173-174 bp) of the small subunit of nuclear ribosomal DNA, was evaluated for the differentiation of the two species of Entamoeba. Including a range of well-defined control DNA samples (n = 67) to evaluate the specificity of the PCR, 45 DNA samples representing E. histolytica and E. dispar from human faecal samples were tested by SSCP, and unequivocal delineation between the species was achieved. This SSCP approach should provide a practical tool for the specific diagnosis of E. histolytica in humans and for investigating its epidemiology.

Animals↗
PubMed2002

Effect of proteasome inhibitors on the growth, encystation, and excystation of Entamoeba histolytica and Entamoeba invadens.

The effect of three proteasome inhibitors, lactacystin, clasto-lactacystin beta-lactone, and MG-132, on the growth, encystation, and excystation of Entamoeba histolytica and Entamoeba invadens was examined. All of these drugs blocked E. histolytica growth in a concentration-dependent manner; lactacystin was most potent for the inhibition and MG-132 showed the inhibitory effect only at higher concentrations. E. invadens was more resistant to these drugs than E. histolytica. Encystation of E. invadens was also inhibited and was more sensitive to the drugs than was growth. Beta-lactone was the most potent encystation inhibitor. The inhibitory effect of lactacystin and the beta-lactone on encystation was slightly and little abrogated by the removal of the drug, respectively. Multinucleation occurred in E. histolytica trophozoites treated with these drugs, being most marked with lactacystin. In contrast, no multinucleation was observed in E. invadens treated with the drugs. Electron microscopy revealed that the treatment of E. histolytica trophozoites with lactacystin led to an increase in the number of cells with many glycogen granules in the cytoplasm. Lactacystin, beta-lactone and MG-132 had no or little effect on the excystation and metacystic development of E. invadens. These results suggest that proteasome function plays an important role for Entamoeba growth and encystation, but has no obvious effect on excystation or metacystic development.

Animals↗
PubMed2006

Comparison of the proteome profiles of Entamoeba histolytica and its close but non-pathogenic relative Entamoeba dispar.

The microaerophilic protist Entamoeba histolytica is a source of considerable morbidity and mortality in many countries of the world by acting as a causative agent of amoebic dysentery and liver abscess. Its close relative, Entamoeba dispar, also colonises the human colon but is non-pathogenic. In the present study, we wanted to assess if the close relatedness of these two organisms could be used to identify virulence factors in E. histolytica through a comparison of the protein expression profiles of the pathogenic and the non-pathogenic species. We applied two-dimensional polyacrylamide gel electrophoresis in order to identify proteins which are specifically expressed in E. histolytica and which therefore could constitute candidate proteins potentially involved in E. histolytica pathogenicity. The evaluation of overall protein expression profiles derived from whole cell extracts, however, revealed that, in spite of the close relatedness at the genetic level, the disparity of the proteomic profiles of these two Entamoeba species is far too great in order to pinpoint proteins whose expression might render E. histolytica pathogenic. Taken together, our study performed at the proteomic level clearly supports the notion of E. histolytica and E. dispar as two distinct species.

Animals↗
PubMed2005

Entamoeba histolytica and Entamoeba dispar: prevalence infection in a rural Mexican community.

The frequency of Entamoeba histolytica and Entamoeba dispar infection was analyzed in a rural community in the state of Morelos, Mexico, through PCR technique by using specie specific primer. The E. histolytica specie was detected in 33 of 290 analyzed stool samples (11.4%), E. dispar specie was observed in 21 samples (7.2%) and both species of Entamoeba were detected in seven samples (2.4%). So a higher E. histolytica than E. dispar frequency infection was detected (13.8 versus 9.6%). Even though in our design we did not considered the follow-up of included individuals, the absence of invasive amebiasis cases in the studied population during our stay in town was unexpected.

Animals↗
PubMed2006

Diagnostic methods for differentiation of Entamoeba histolytica and Entamoeba dispar in carriers: performance and clinical implications in a non-endemic setting.

Unpreserved faecal samples, suspected to contain Entamoeba histolytica/Entamoeba dispar cysts or trophozoites on the basis of microscopic examination, and serum samples from 416 patients were collected in a prospective study to determine whether stool antigen assays and detection of antibodies in serum are reliable methods to distinguish between carriers of E. histolytica and E. dispar in comparison to the reference test: real-time PCR. In 283 patients (68%) DNA of E. histolytica or E. dispar was amplified by real-time PCR: 6 patients with amoebic colitis (2%), 19 carriers of E. histolytica (6.7%), and 258 carriers of E. dispar (91.2%). In 133 patients (31%) no DNA of E. histolytica or E. dispar could be amplified in the stool samples. This patient group was used as control for the evaluation of diagnostic tests. Using real-time PCR as a reference test, the sensitivity and specificity of (1) the Entamoeba test for the diagnosis of E. histolytica/E. dispar carrier were 59% and 98%, (2) E. histolytica II for the diagnosis of E. histolytica carrier was 71% and 100%, and (3) serology for the diagnosis of E. histolytica infection was 83.3% and 95.2%, respectively. Applied to carriers that did not originate from an endemic country the sensitivity of serology for E. histolytica infection was 90% and specificity was 98.8%. In comparison to real-time PCR the performances of Entamoeba test and E. histolytica II lacked sensitivity for a reliable diagnosis of E. histolytica/E. dispar infection in a non-endemic setting. In carriers of E. histolytica/E. dispar from non-endemic countries the high specificity of serology can be used to establish the diagnosis of E. histolytica infection if antibodies are present.

Adolescent↗
PubMed1976

The rhabdoviruses of Entamoeba histolytica and Entamoeba invadens.

Rhabdoviruses have been described in plants, arthropods and vertebrates including man. Members of the group are of agricultural, veterinary and medical importance. The presence of a rhabdovirus in Entamoeba histolytica and Entamoeba invadens is the first record of their existence within protozoa. The morphology of this virus is described and its significance discussed, in relation to a possible lysogenic state and pathogenecity of Entamoeba species.

Animals↗
PubMedDate not supplied

A redescription of Entamoeba histolytica Schaudinn, 1903 (Emended Walker, 1911) separating it from Entamoeba dispar Brumpt, 1925.

Explaining the low incidence of invasive disease (10%) in humans infected with Entamoeba histolytica has occupied the attention of generations of both clinical and nonclinical investigators. One possible explanation would be the existence of two morphologically identical species-one an invasive pathogen, the other noninvasive. This was first proposed by Brumpt in 1925, but his explanation was virtually ignored until 1978 when the first of several publications appeared suggesting that E. histolytica did indeed consist of two species. We have reexamined Brumpt's claim in light of recent biochemical, immunological and genetic studies and conclude that the data derived from these investigations provide unequivocal evidence supporting his hypothesis. With this in mind, we redescribe the invasive parasite retaining the name Entamoeba histolytica Schaudinn, 1903 (Emended Walker, 1911), and set it apart from the noninvasive parasite described by Brumpt, Entamoeba dispar Brumpt, 1925.

Animals↗
PubMedDate not supplied

YI-S, a casein-free medium for axenic cultivation of Entamoeba histolytica, related Entamoeba, Giardia intestinalis and Trichomonas vaginalis.

Pancreatic digests of casein are major ingredients of media used in the axenic cultivation of lumen-dwelling parasitic protozoa, especially Entamoeba, Giardia, and trichomonads. The digest used almost exclusively in the development of these media, Medo-Peptone (Trypticase BBL), has not been available since 1981. Moreover, none of dozens of similar type digests tested since then in our laboratory has proved equal to Medo-Peptone, and in the last two years it has become increasingly difficult to obtain new batches which will support even modest growth of Entamoeba histolytica. In response to this problem we have developed a casein-free medium, YI-S, consisting of a nutrient broth, vitamin mixture and serum. We recommend it as a replacement for the casein-dependent medium TYI-S-33, currently the most widely used for axenic culture of Entamoeba histolytica and other lumen-dwellers.

Animals↗
PubMed2006

Comparison of a stool antigen detection kit and PCR for diagnosis of Entamoeba histolytica and Entamoeba dispar infections in asymptomatic cyst passers in Iran.

The present study was conducted to compare stool antigen detection with PCR for the diagnosis of Entamoeba sp. infection in asymptomatic cyst passers from Iran. Entamoeba dispar and, in one case, E. moshkovskii were the Entamoeba spp. found in the amebic cyst passers. There was a 100% correlation between the results from the TechLab E. histolytica II stool antigen kit and those from nested PCR. We concluded that E. dispar is much more common in asymptomatic cyst passers in Iran and that antigen detection and PCR are comparable diagnostic modalities.

Animals↗
PubMed2005

Differences in morphology of phagosomes and kinetics of acidification and degradation in phagosomes between the pathogenic Entamoeba histolytica and the non-pathogenic Entamoeba dispar.

Phagocytosis plays an important role in the pathogenicity of the intestinal protozoan parasite Entamoeba histolytica. We compared the morphology of phagosomes and the kinetics of phagosome maturation using conventional light and electron microscopy and live imaging with video microscopy between the virulent E. histolytica and the closely-related, but non-virulent E. dispar species. Electron micrographs showed that axenically cultivated trophozoites of the two Entamoeba species revealed morphological differences in the number of bacteria contained in a single phagosome and the size of phagosomes. Video microscopy using pH-sensitive fluorescein isothiocynate-conjugated yeasts showed that phagosome acidification occurs within 2 min and persists for >12 h in both species. The acidity of phagosomes significantly differed between two species (4.58 +/- 0.36 or 5.83 +/- 0.38 in E. histolytica or E. dispar, respectively), which correlated well with the differences in the kinetics of degradation of promastigotes of GFP-expressing Leishmania amazonensis. The acidification of phagosomes was significantly inhibited by a myosin inhibitor, whereas it was only marginally inhibited by microtubules or actin inhibitors. A specific inhibitor of vacuolar ATPase, concanamycin A, interrupted both the acidification and degradation in phagosomes in both species, suggesting the ubiquitous role of vacuolar ATPase in the acidification and degradation in Entamoeba. In contrast, inhibitors against microtubules or cysteine proteases (CP) showed distinct effects on degradation in phagosomes between these two species. Although depolymerization of microtubules severely inhibited degradation in phagosomes of E. histolytica, it did not affect degradation in E. dispar. Similarly, the inhibition of CP significantly reduced degradation in phagosomes of E. histolytica, but not in E. dispar. These data suggest the presence of biochemical or functional differences in the involvement of microtubules and proteases in phagosome maturation and degradation between the two species.

Actins↗
PubMed1997

Entamoeba histolytica and Entamoeba dispar infection in children in Bangladesh.

The prevalence of infection by the invasive parasite Entamoeba histolytica and the noninvasive parasite Entamoeba dispar was determined in 2000 children in Bangladesh. Antigen detection identified more cases of E. histolytica-E. dispar infection than did culture or microscopy. Microscopic identification of E. histolytica-E. dispar complex infection in stool did not equate with the diagnosis of amebic dysentery because most amebic infections in this population were due to E. dispar: Urban children with diarrhea had a 4.2% prevalence of E. histolytica infection and a 6.5% prevalence of E. dispar infection; rural asymptomatic children had a 1.0% prevalence of E. histolytica infection and a 7.0% prevalence of E. dispar infection. Shigella dysenteriae and Shigella flexneri infections were more frequent in children who also had Entamoeba infection, a potentially important consideration for the empiric treatment of dysentery in this population.

Adolescent↗
PubMedDate not supplied

Entamoeba histolytica Schaudinn, 1903 and Entamoeba dispar Brumpt, 1925: differences in their cell surfaces and in the bacteria-containing vacuoles.

Entamoeba histolytica Schaudinn, 1903 and Entamoeba dispar Brumpt. 1925 are two of eight species of Entamoeba that sometimes inhabit the human colon. The former is an invasive organism capable of causing life-threatening intestinal and extra-intestinal disease: the latter appears not to be invasive. Because the two species, when viewed by light microscopy appear morphologically similar, they were long regarded as a single species. However, recent biochemical. immunological, and genetic studies provided convincing evidence that they belong to separate species. Our ultrastructural studies revealed distinct differences in at least two features of the trophozoites. 1) The cell surfaces of the trophozoites of each species differ with regard to structures exposed on the surface, and the distribution and arrangement of intra-membranous proteins. 2) The phagocytosis of bacteria differs in respect to the formation of the phagocytic vacuoles. Loose vacuoles containing several bacteria were seen in E. histolytica whereas tight vacuoles containing a single bacterium were observed in E. dispar. Furthermore, bacteria were found only within vacuoles in E. histolytica; in E. dispar, bacteria were found within vacuoles and some were found free in the cytoplasm.

Animals↗
PubMed2003

Detection and differentiation of Entamoeba histolytica and Entamoeba dispar isolates in clinical samples by PCR and enzyme-linked immunosorbent assay.

Differential diagnosis of Entamoeba histolytica (pathogenic) and Entamoeba dispar (nonpathogenic), which are two morphologically identical species of amebae, is essential both for treatment decision and public health knowledge. The study reported here was designed to choose a reference differentiation technique. Stool samples (n = 95) were tested by microscopy, TechLab enzyme-linked immunosorbent assays (ELISAs), and an in-house PCR. The target for the PCR amplification was a small region (135 bp) of the SSU rRNA selected to increase the sensitivity of the test. Sixty-eight specimens tested positive by PCR: 2 for E. histolytica and 66 for E. dispar. For detection of E. dispar, ELISA performance was lower than that of microscopy in this reference context, while PCR was much more sensitive than microscopy. Given the low proportion of E. histolytica cases, test performance for this species is difficult to assess. However, for differentiation, PCR performed well on simulated samples, while ELISA gave a discordant result for one of the two samples PCR positive for E. histolytica during the study. This report also confirms that E. dispar infection is significantly higher among travelers and underlines the possibility of acquiring E. histolytica infection in regions that are not areas of endemicity. Because of its lower sensitivity, the interest of ELISA for Entamoeba detection and differentiation in stools seems questionable in nontropical regions. On the other hand, results suggest that PCR should be useful as a reference test for sensitive differentiation of both species and to contribute to physicians' decision in treatment of E. histolytica- or E. dispar-infected patients.

Animals↗
PubMed2002

Amebic infections due to the Entamoeba histolytica-Entamoeba dispar complex: a study of the incidence in a remote rural area of Ecuador.

An epidemiologic field study was conducted in the village of Borbòn in Esmeraldas province in northern Ecuador to compare different parasitologic methods in the diagnosis of infection with the Entamoeba histolytica/Entamoeba dispar complex. The results of two stool antigen detection assays (the Prospect Entamoeba histolytica microplate assay and the E. histolytica II assay) were compared with isoenzyme characterization of the amebic isolates. Nearly all (176 of 178, 98.9%) subjects were positive for intestinal parasites on direct microscopic examination, and cysts and/or vegetative forms morphologically consistent with the E. histolytica/E. dispar complex were recorded in 48 of 178 cases (27%). Culture in Robinson's medium was positive for amebic stocks in 89 (50%) of the 178 samples tested. Of the 37 isolates successfully stabilized, cloned, and characterized by zymodeme analysis, seven (18.9%) showed isoenzyme patterns of E. histolytica, whereas 26 (70.3%) showed patterns of E. dispar. The remaining four strains were identified as Entamoeba coli (three isolates; 8.1%) and Dientamoeba fragilis (one strain; 2.7%).The immunochromatographic tests showed different degrees of sensitivity and specificity when compared with isoenzyme characterization as the reference technique. The microplate assay, which does not discriminate between E. histolytica and E.dispar, showed a sensitivity of 54.5% and a specificity of 94% for both these amebic species. In contrast, the second-generation E. histolytica II test had a sensitivity of 14.3% and a specificity of 98.4% for E. histolytica sensu stricto. Our survey clearly demonstrated that more specific and sensitive diagnostic tests, such as stool antigen detection assays and isoenzyme analysis, are needed to establish the actual worldwide distribution of E. histolytica and E. dispar.

Adolescent↗
PubMed2004

In vitro activity of antiamoebic drugs against clinical isolates of Entamoeba histolytica and Entamoeba dispar.

BACKGROUND: Amoebiasis is a major public health problem in tropical and subtropical countries. Although a number of antiamoebic agents are used for its treatment, yet the susceptibility data on clinical isolates of Entamoeba histolytica and Entamoeba dispar are not available. Therefore, the present study was aimed to assess the in vitro susceptibility of clinical isolates of E. histolytica and E. dispar to metronidazole, chloroquine, emetine and tinidazole. METHODS: A total of 45 clinical isolates (15 E. histolytica and 30 E. dispar) were maintained in polyxenic cultures followed by monoxenic cultures. In vitro drug sensitivity (IC50) of clinical isolates and standard reference strain of E. histolytica (HM1: IMSS) was assessed by nitro blue tetrazolium (NBT) reduction assay after exposure to various concentrations of each drug. RESULTS: The results showed that all clinical isolates had a higher IC50 compared to reference strain to all the four drugs. E. histolytica isolates appeared to be more susceptible [IC50 (microm) 13.2,26.3,31.2 and 12.4] compared to E. dispar isolates [IC50(microm) 15.6,28.9,32.8 and 13.2] and the reference strain of E. histolytica [IC50 (microm) 9.5, 15.5, 29.9 and 10.2] to the metronidazole, chloroquine, emetine and tinidazole respectively. CONCLUSIONS: The results indicate that till date, Entamoeba isolates in India do not seem to be resistant to the commonly used antiamoebic drugs.

Journal Article↗