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PubMed2000

Entamoeba dispar, but not E. histolytica, detected in a colony of chimpanzees in Japan.

Chimpanzees (Pan troglodytes) residing in the Kumamoto Primate Research Park, Sanwa Kagaku Kenkyusho, were surveyed for the presence of intestinal parasites. Stool samples from 107 chimpanzees were examined by microscopy after formalin-ether sedimentation. Of these animals, 100 were infected with at least 1 species of ameba. The positivity rates recorded were as follows: Entamoeba coli, 88%; E. histolytica/E. dispar, 48%; E. hartmanni, 15%; Iodamoeba buetschlii, 8%; Endolimax nana, 4%; and Entamoeba chattoni, 2%. Polymerase chain reaction (PCR) analysis to distinguish between E. histolytica and E. dispar was performed on these samples. E. dispar DNA was detected in 60 of 107 samples (56%), including 9 that had been microscopically determined to be negative for E. histolytica/ E. dispar. In contrast, no E. histolytica DNA was detected in the 107 samples. Zymodeme analysis indicated that 10 isolates were E. dispar. When 104 chimpanzees were examined serologically for E. histolytica infection, 1 sample was scored as positive by indirect hemagglutination and another was found to be positive by an indirect fluorescent antibody test. However, both specimens were borderline-positive and were clearly negative in other tests, suggesting that they might be false-positives. These results demonstrate that the pathogenic E. histolytica was absent in this colony, regardless of the high degree of prevalence of other amebas. For an accurate diagnosis, PCR analysis is recommended in addition to microscopic examination.

Animals↗
PubMed2001

Entamoeba histolytica: production of nitric oxide and in situ activity of NADPH diaphorase in amebic liver abscess of hamsters.

Entamoeba histolytica trophozoites were inoculated into the liver of hamsters and serum nitrate/nitrite levels [expressed as nitric oxide (NO) production] were determined at different times during amebic liver abscess (ALA) development. We also tested the effects of NO synthase (NOS) inhibitors such as N(G)-nitro-L-arginine methyl ester (L-NAME), aminoguanidine, and dexamethasone during ALA production. Since NOS activity has been correlated with expression of reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) in tissues, we performed histochemistry studies to determine the activity of the latter in livers infected with E. histolytica trophozoites. Production of NO in serum was directly proportional to the size of ALAs, and NOS inhibitors caused low levels of NO and smaller ALAs. Our data suggest that NO does not have any lytic effect on E. histolytica trophozoites and is therefore incapable of providing protection against the amebic liver infection. In addition, NADPHd activity was detected histochemically in hepatocytes and inflammatory cells associated with focal necrosis containing trophozoites. The positive reactivity observed in these parasites may be attributable to a close biochemical similarity of NADPHd to the NADPH:flavin oxidoreductase described in E. histolytica by other investigators.

Animals↗
PubMed2001

High prevalence of infection with Entamoeba dispar, but not E. histolytica, in captive macaques.

A total of 268 nonhuman primates (20 species) kept in the Primate Research Institute, Kyoto University, Japan, were surveyed for intestinal amebas. Total positive rates as based on the presence of cysts in the stool following formalin-ether sedimentation were as follows: Entamoeba histolytica/E. dispar, 53%; E. coli, 34%; E. hartmanni, 34%; Iodamoeba buetschlii, 25%; Endolimax nana, 8%; and E. chattoni, 3%. Positive rates were higher in Old World monkeys and lower in New World monkeys. All the 141 E. histolytica/E. dispar-positive animals were Macaca monkeys. The E. histolytica/E. dispar-positive samples were analyzed by polymerase chain reaction (PCR) for identification of E. histolytica and E. dispar. E. dispar DNA was detected in 137 samples, whereas no E. histolytica DNA was seen. Zymodeme analysis and reactivity to monoclonal antibodies of cultured trophozoites also supported the presence of E. dispar and the absence of E. histolytica. When the sera of 93 macaques were examined by an indirect fluorescent antibody test, only 3 animals proved to be positive for E. histolytica, showing the lowest titer. These results demonstrate that infection with E. dispar, but not E. histolytica, is predominant in macaques.

Animals↗
PubMed2002

DNA polymorphism in north indian isolates of Entamoeba histolytica detected by PCR fingerprinting.

Entamoeba histolytica infection in humans is still one of the leading causes of parasitic diseases among the developing countries, including India. It therefore becomes extremely important to characterize this parasite with the aim of preventing and controlling the amoebiasis which it causes. The present study describes for the first time the ability of random amplified polymorphic DNA (RAPD)-PCR fingerprinting strategies to subtype amoebae and to detect considerable genetic variability present among the various North Indian isolates of E. histolytica studied. The number of rapdemes generated by the application of arbitrary primers in RAPD-PCR showed a significant variation in these isolates, both in the size and number of bands, thus revealing the presence of considerable genetic polymorphism in them. Hence, this rapid and easy method could suitably be employed in carrying out significant molecular epidemiological studies and also in the large-scale epidemiological typing of this parasite.

Animals↗
PubMed1996

Differentiation of Entamoeba histolytica and E. dispar DNA from cysts present in stool specimens by polymerase chain reaction: its field application in the Philippines.

It has been established that two distinct species exist within what was originally known as Entamoeba histolytica. These are E. dispar and E. histolytica, for the nonpathogenic and pathogenic forms, respectively. Differentiation of these two organisms is of great clinical importance since they are morphologically indistinguishable and both forms can infect the human intestinal cavity to different degrees. A simple and rapid DNA-extraction method that can be used directly on formalin-fixed stool specimens has been developed. The extracted DNA was used for the identification of the species existing in the stools by polymerase chain reaction (PCR). A total of 72 randomly collected stool samples from the Philippines were analyzed. In all, 19 samples reacted with E. dispar primers, resulting in the expected 101-bp PCR products; however, none reacted with E. histolytica primers. Furthermore, sensitivity assay suggests that genomic DNA from as few as five cysts can be used as a template for PCR. These observations imply that the use of genomic DNA directly extracted from formalin-fixed stool specimens for PCR amplification is a useful tool for obtaining a sensitive and accurate diagnosis that can be applied even in epidemiology studies.

Animals↗
PubMed1997

Identification of Entamoeba histolytica and E. dispar cysts in stool by polymerase chain reaction.

An attempt to identify cysts of Entamoeba histolytica and E. dispar in human stool was conducted by polymerase chain reaction (PCR) using two sets of primers (p11 plus p12 and p13 plus p14) specific for either species of ameba. The cysts in stool specimens obtained from 12 infected individuals were concentrated, freeze-thawed, and treated with Triton X-100 before their examination by PCR. The results of PCR on the cysts were generally consistent with data obtained by PCR on ameba trophozoites hatched from the cysts, by zymodeme analysis, and by enzyme-linked immunosorbent assay (ELISA) and with clinical findings. This PCR was negative for the stool containing large numbers of cysts of either E. coli, E. hartmanni, or Giardia lamblia as well as for the stool specimens obtained from uninfected individuals. The ameba cyst in stool processed using the present method was effective for the PCR analysis even after 1 month of storage at 4 degrees C. The present PCR was sensitive enough to detect ten cysts of either of the amebae.

Animals↗
PubMed1997

First isolation and characterization in humans of Entamoeba histolytica (laboratory-made) zymodeme XX.

Isoenzyme analysis by starch-gel electrophoresis has proved to be a useful method for the biochemical differentiation of pathogenic Entamoeba histolytica and non-pathogenic E. dispar isolates. Of the known 24 zymodemes, 3 are laboratory-made and have not previously been identified in humans. Parasitology screening was carried out in a psychiatric institution. Two amebic stocks were isolated and characterized that had never previously been found in humans and that have protein patterns identical to that of the laboratory-made zymodeme XX.

Adult↗
PubMed1997

A new method for isolation and differentiation of native Entamoeba histolytica and E. dispar cysts from fecal samples.

A new method for the purification of protozoan cysts from feces was established, allowing to isolation of native cysts. The procedure consists of two sucrose-density gradients and enzymatic digestion of cellulose particles by cellulase and can be accomplished in a few hours. The cyst fractions were differentiated into Entamoeba histolytica and E. dispar using the DNA probes P145 and B133 and a dot-blot test.

Animals↗
PubMed1999

Detection of Entamoeba dispar DNA in macaque feces by polymerase chain reaction.

We used the polymerase chain reaction (PCR) to determine the prevalence of Entamoeba histolytica and E. dispar in the wild population of macaque monkeys (Macaca fuscata) in Mt. Takasaki, Oita Prefecture, Japan. Of the 101 samples collected, 41 (42.57%) were found to be positive for E. dispar. However, no E. histolytica was detected from the collected samples. The results of this survey demonstrate the high prevalence of E. dispar in macaque monkeys in the study area. Moreover, they provide additional baseline information on naturally acquired infectious agents of macaque monkeys and offer an accurate tool for detection of E. histolytica and E. dispar, which are needed for biomedical research using nonhuman primate models.

Amebiasis↗
PubMed2005

Hemolytic uremic syndrome associated with Entamoeba histolytica intestinal infection.

Amebiasis secondary to Entamoeba histolytica (E. histolytica) continues to be a major source of morbidity and mortality worldwide, mainly in developing countries. An association between amebic dysentery and hemolytic uremic syndrome (HUS) has been mentioned, but a cause-and-effect relationship has never been confirmed. We report the case of an 11-year-old healthy white female who developed severe bloody diarrhea after drinking contaminated unboiled water, developing classic HUS and requiring acute intermittent hemodialysis. The etiological study confirmed numerous cysts and hematophagous trophozoites of E. histolytica on stool smear, but without evidence of other pathogens. This report contributes to the concept that classic post-diarrheal HUS, usually related to colitis secondary to enterohemorragic Shiga-toxin producing Escherichia coli (E.coli) strains, may be associated with other pathogens, including E. histolytica. Additional information regarding mechanisms of virulence of E. histolytica and host immunological reaction is needed to clarify the temporal association of these two disorders (HUS and amebiasis).

Child↗
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

Sensitivity and specificity of a new commercial enzyme-linked immunoassay kit for detecting Entamoeba histolytica IgG antibodies in serum samples.

The study presented here was conducted to evaluate the performance of the newly available RIDASCREEN Set (R-Biopharm AG, Darmstadt, Germany) for the detection of immunoglobulin G antibodies against Entamoeba histolytica. The sensitivity and specificity of this new enzyme-linked immunosorbent assay were evaluated using a panel of sera from 239 individuals. The assay was positive for 43 of 44 patients with invasive amebiasis, including all 18 patients with amebic liver abscess, while it was negative for 190 of 195 adult controls who were either healthy individuals or patients with other parasitic diseases. The kit was found to be highly specific (97.4%) and sensitive (97.7%) for detecting antibodies against E. histolytica in humans. Although antibody titers in patients with amebic liver abscess tend to be higher on average than in patients with invasive amebiasis, it is not possible to distinguish the two forms solely based on the results of this commercial test.

Animals↗
PubMed2000

Differentiation of Entamoeba histolytica and Entamoeba dispar cysts using polymerase chain reaction on DNA isolated from faeces with spin columns.

Since Entamoeba histolytica and Entamoeba dispar were formally recognized as two different species at the World Health Organization (WHO)/Pan American Health Organization (PAHO)/United Nations Educational, Scientific and Cultural Organization (UNESCO) meeting in Mexico City in 1997, the specific differentiation of the two morphologically identical species would seem relevant in clinical diagnosis. Several polymerase chain reaction (PCR)-based methods have been described and used successfully, but methods for DNA isolation from cysts in stool samples are time-consuming and problematic due to inhibitory factors in faeces. The use of the slightly modified QIAamp tissue method (Qiagen, Germany) for DNA isolation was evaluated in 657 unpreserved faecal samples from cases of suspected Entamoeba histolytica/Entamoeba dispar infection. In only 1.7% of the cases was PCR hampered by inhibitors present in the faeces. The DNA isolation procedure was found to be rapid, simple and one that could easily be implemented in a routine diagnostic setting. In 98.8% of Entamoeba histolytica/Entamoeba dispar cyst-positive faecal samples, the true identity of the cysts could be determined using PCR specific for Entamoeba histolytica and Entamoeba dispar, respectively.

Animals↗
PubMed2003

Evaluation of dot-ELISA for serological diagnosis of amebiasis.

We improved the dot enzyme-linked immunosorbent assay (dot-ELISA) reported by Itoh and Sato, and assessed the usefulness of this test for the diagnosis of amebiasis. The sensitivity of dot-ELISA was compared with that of plate ELISA, the indirect hemagglutination test (IHA), and the indirect fluorescent antibody test (IFA) for the diagnosis of amebiasis. Of 37 serum samples from patients with documented amebiasis, 36 (97.3%) were positive by dot-ELISA. There was consistency among the results of dot-ELISA, plate ELISA, and IFA, although the positive rate of IHA was lower than that of the others (78.4%; 29 of 37 cases were positive). The specificities of dot-ELISA and plate ELISA were assessed using a total of 68 sera, collected from 38 patients infected with seven different parasites other than Entamoeba histolytica, 10 patients showing diarrhea or liver abscess without parasitic infection, and 20 healthy individuals. The two assays showed no false-positive results. There were no differences in sensitivity and specificity between dot-ELISA and plate ELISA. However, the dot-ELISA technique seems to be more feasible for clinical application than plate ELISA techniques, because the assay does not require any specific equipment.

Animals↗
PubMed1985

Entamoeba histolytica: purification of cathepsin B.

A cytotoxic cysteine proteinase with a molecular weight of 16,000 was isolated from axenically grown trophozoites of Entamoeba histolytica. The enzyme was purified from frozen-thawed strain HM-1 by ion-exchange chromatography on DEAE-cellulose, organomercurial agarose affinity chromatography, and size-exclusion chromatography. The purified enzyme had proteinase activity that could be demonstrated on azocasein (pH 5), hemoglobin (pH 5), or carbobenzoxy-L-arginyl--L-arginyl-7-amino-4-trifluoromethylcoumarin++ + (Z-arg-arg-AFC), a substrate specific for cathepsin B. Enzyme activity was stable to high pH, but not to 40 C for 1 hr or 56 C for 0.5 hr. As typical of cysteine proteinases, inhibition of activity on Z-arg-arg-AFC by p-chloromercuribenzoate or mercury was reversed by free sulfhydryl groups. Both the proteinase and cytotoxic activities of the purified amoebal cathepsin B were inhibited by leupeptin and serum and activated by free sulfhydryl groups, supporting the hypothesis that both activities are characteristics of amoebal cathepsin B. Virulent strains of E. histolytica (HM-1 and Rahman) had significantly more cathepsin B activity per milligram protein than less virulent strains (HK-9, Laredo, and Huff). The correlation between higher levels of cathepsin B activity in strains with greater virulence could indicate a role for amoebal cathepsin B in the pathogenesis of amoebiasis.

Cathepsin B↗
PubMed1986

Entamoeba histolytica: effect of growth conditions and bacterial associates on isoenzyme patterns and virulence.

In xenic culture, isolates of Entamoeba histolytica from asymptomatic carriers are characterized, with rare exception, by possession of a nonpathogenic zymodeme. During the process of axenizing such an isolate, strain CDC:0784:4, a change in the pattern of the isoenzymes from nonpathogenic zymodeme I to pathogenic zymodeme II was observed 40 days after the amebae had been transferred from a medium for xenic cultivation to one used for axenic cultivation, but before axenization of the amebae had actually occurred. Axenization was accomplished by feeding the amebae lethally irradiated bacteria while suppressing and finally eradicating with antibiotics the bacterial flora accompanying the amebae in the original xenic culture. The change in zymodeme was accompanied by a change in virulence as evidenced by the ability of the amebae to produce hepatic abscesses in hamsters and to destroy monolayers of tissue culture cells. Two explanations are offered for the observed changes in zymodeme and virulence: a zymodeme is not a stable inherent property of the ameba. Alternatively, the original isolate consisted of two zymodeme populations and the conditions of growth selected for one or the other of the populations. In either case, our results suggest that the finding of a particular zymodeme in a culture of E. histolytica isolated from an asymptomatic carrier of the parasite cannot be used to predict a clinical condition or serve as a basis for the recommendation of therapy.

Amebiasis↗