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PubMed2005

Prevalence and genetic diversity of Entamoeba histolytica in an institution for the mentally retarded in the Philippines.

A total of 113 mentally retarded patients residing in a mental institution in Metropolitan Manila, Philippines, were screened for the presence of Entamoeba histolytica based on microscopy and polymerase chain reaction (PCR). Anti-E. histolytica antibodies were also screened in 97 serum samples collected using immunofluorescence antibody (IFA) test. Parasitological examination showed E. histolytica/Entamoeba dispar in 43 cases (38.05%), while PCR detected 74 cases (65.48%) positive for E. histolytica and 6 cases (5.30%) positive for E. dispar. Interestingly, these 6 samples were coinfected with E. histolytica. IFA test revealed that 80.41% (78/97) of the respondents possessed significant antibody titers for intestinal infection of E. histolytica. Of this number, there were 5 patients negative in IFA test but positive in PCR. The genetic diversity of E. histolytica isolates was also investigated by analyzing polymorphism in the serine-rich gene by nested PCR on DNA directly extracted from stool specimens. A combination of the nested PCR results and the AluI digestion of the PCR products examined yielded six distinct DNA banding patterns among the 74 stool isolates. An apparent clustering of E. histolytica strains was observed in patients living in different residential cottages of the institution. These results indicate the high prevalence of E. histolytica in an institution for the mentally retarded in the Philippines.

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↗
PubMed1996

A gene highly homologous to ACP1 encoding cysteine proteinase 3 in Entamoeba histolytica is present and expressed in E. dispar.

We have cloned the complete gene encoding cysteine proteinase 3 in E. histolytica as well as a cDNA encoding a highly homologous protein in E. dispar. In addition, we have demonstrated the presence and expression of the CP3 gene in various E. histolytica and E. dispar isolates. Since the expression of the gene is rather similar within both Entamoeba species, we assume that it does not constitute the proposed virulence factor of E. histolytica.

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

Differentiation of Entamoeba histolytica from E. dispar facilitated by monoclonal antibodies against a 150-kDa surface antigen.

Murine monoclonal antibodies (mAbs) were produced against an n-octyl-beta-D-glucopyranoside-extracted fraction of trophozoites of Entamoeba histolytica HM-1:IMSS. Four of the mAbs were reactive with a 150-kDa surface antigen characterized by Western-immunoblot analysis under nonreducing conditions. When the reactivity of the four mAbs with nine reference strains of E. histolytica was examined by an indirect fluorescence antibody test, two of the mAbs (EH3015 and EH3023) were found to react with all nine strains and the other two mAbs (EH3056 and EH3126) reacted with seven strains. The four mAbs did not react with any E. dispar reference strain or with other enteric protozoan parasites. The reactivity of EH3015 and EH3023 with numerous isolates of E. histolytica and E. dispar collected in our laboratories was also examined. The 2 mAbs reacted with all of the 37 E. histolytica isolates tested but did not react with any of the 33 isolates of E. dispar. These results indicate that common antigenic epitopes of E. histolytica are on the 150-kDa surface molecule and that mAbs can distinguish between E. histolytica and E. dispar.

Animals↗
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

DNA polymerase activity in encysting Entamoeba invadens.

Using an axenic encystation system of Entamoeba invadens as a model for E. histolytica encystation, we examined the level of DNA polymerase activity in E. invadens during encystation induced in vitro. We first characterized the DNA polymerase activity of trophozoites of E. invadens, comparing it with that of E. histolytica, and found that the activity of E. invadens was lower than that of E. histolytica at pH 2, 4, and 6 and was higher at pH 8 and 10. The activity of E. invadens was completely inhibited by high concentrations of K(-). Among inhibitors of mammalian DNA polymerases, aphidicolin and N-ethylmaleimide inhibited the activity, but 2',3'-dideoxythymidine-5'-triphosphate did not. Thus, the sensitivity of the E. invadens activity to salt and inhibitors of mammalian DNA polymerases was basically the same as that recorded for E. histolytica in our previous results. The level of DNA polymerase activity in cysts decreased as encystation proceeded as compared with that of trophozoites. The results indicate that encystation is accompanied by a reduced level of DNA polymerase activity, which correlates with the previous finding that nuclear division occurs during cyst maturation in the absence of DNA synthesis.

Animals↗
PubMed1999

DNA sequences corresponding to the ariel gene family of Entamoeba histolytica are not present in E. dispar.

For the identification of quantitative genetic differences between pathogenic Entamoeba histolytica and the closely related but nonpathogenic species E. dispar, a set of 68 independent probes that had previously been isolated from an E. histolytica cDNA library were hybridized to total genomic DNA of both amoeba species. Besides ehcp5, the sequence that codes for cysteine proteinase 5 and has recently been shown to be missing in E. dispar, only one of the probes exclusively reacted with E. histolytica DNA, whereas the remainder hybridized to DNA of both species. Sequence analysis revealed that the specific probe represents a copy of the multicopy ariel gene family, which has 80% sequence identity to srehp, the gene encoding a serine-rich E. histolytica membrane protein. In contrast to ariel, srehp is present in both amoeba species, suggesting that the ariel gene product might have a particular function in E. histolytica.

Animals↗
PubMed1980

A comparative study of Entamoeba histolytica (NIH :200, HK9, etc.), "E. histolytica-like" and other morphologically identical amoebae using isoenzyme electrophoresis.

Cultures of old, well documented strains of Entamoeba histolytica (NIH:200, HK9 etc.) have been compared with "E. histolytica-like" strains and, in turn, with E. moshkovskii, E. invadens and E. chattoni. They have been grouped according to their isoenzyme patterns. The "E. histolytica-like" organisms give patterns similar to E. moshkovskii and both of these are different from E. histolytica Groups I to IV. E. invadens and E. chattoni have easily distinguished characteristic isoenzyme bands. "E. polecki" is indistinguishable from E. histolytica.

Animals↗
PubMed1988

PEHPS medium: an alternative for axenic cultivation of Entamoeba histolytica and E. invadens.

Knowledge of Entamoeba histolytica biology in the last 17 years has been acquired largely as a consequence of this parasite's axenic cultivation in TPS-1 or TYI-S-33 media. Unfortunately, there are often low yields in these media, due to variability of their main components, Panmede and yeast extract. We describe a medium, PEHPS, of which the main components are extracts of ox liver, and ox and swine pancreas (EHP). 5 strains of E. histolytica and 2 of E. invadens were quickly and easily adapted to PEHPS and serially cultivated for 3 years. Yields progressively rose initially, and then became stable. Depending on the strain, average yields in the last 6 months of this study were 1.3 to 3.1 x 10(5) amoebae/ml for E. histolytica and 5.5 to 5.7 x 10(5) for E. invadens. All the 18 EHP batches tested supported vigorous amoebal growth. PEHPS had 2 additional advantages: (a) it was stable at 4 degrees C or 25 degrees C for 9 months, and at -10 degrees C for at least 2 years, and (b) it supported amoebal growth with inocula as low as one trophozoite/ml. PEHPS avoids the variability shown by TPS-1 and TYI-S-33, and could therefore be a good alternative for axenic amoebal cultivation.

Animals↗
PubMedDate not supplied

Royal Society of Tropical Medicine and Hygiene Meeting at Manson House, London, 19 February 1998. Amoebic disease. Entamoeba histolytica and E. dispar: comparison of molecules considered important for host tissue destruction.

Entamoeba histolytica and E. dispar are genetically distinct but closely related protozoan species. Both colonize the human gut but only E. histolytica is able to invade tissues and cause disease. Comparison of the 2 species may help to elucidate the specific mechanisms involved in the pathogenicity of E. histolytica. During the last few years, various amoeba molecules considered to be important for pathogenic tissue invasion have been identified and characterized, such as a galactose-inhibitable surface lectin, pore-forming peptides and cysteine proteinases. This review summarizes present knowledge about the structure and function of these molecules, with emphasis on the differences between E. histolytica and E. dispar.

Animals↗
PubMedDate not supplied

Royal Society of Tropical Medicine and Hygiene meeting at Manson House, London, 19 February 1998. Amoebic disease. Entamoeba dispar, an organism reborn.

Entamoeba dispar was described by Emile Brumpt in 1925, and was promptly dismissed as a synonym of E. histolytica by almost all his contemporaries. In the 1970s, however, evidence began to accumulate indicating that Brumpt may have been correct and E. dispar is now accepted as a distinct species. This review introduces the organism's history and briefly summarizes what we currently know about its biology. The acceptance of E. dispar has profound implications for our understanding of the epidemiology of amoebiasis and, ultimately, may give us an insight into what makes E. histolytica a pathogen.

Animals↗
PubMed2003

Effectiveness of peptone-yeast extract (P-Y) medium in the cultivation and isolation of Entamoeba histolytica/Entamoeba dispar in Turkish patients.

Amebiasis is a common protozoan infection worldwide, causing serious health problems in both children and adults. Today, almost 10% of the world population is infected with Entamoeba histolytica/Entamoeba dispar. The aims of this study were both the comparison of the reproduction rates and densities of E. histolytica/E. dispar in Robinson, Dobell-Laidlaw and P-Y culture media and isolation of E. histolytica/E. dispar from stool samples in Peptone-Yeast (P-Y) medium. Trophozoites and cysts of E. histolytica/E. dispar, maintained in Robinson medium, and stool samples of patients with amebiasis were inoculated into P-Y, Robinson and Dobell-Laidlaw culture media. Reproduction rates reached their peak levels 48 h after the inoculation in all culture media. Reproduction rates in P-Y and Robinson media were found similar; however, they were higher than the reproduction rate in Dobell-Laidlaw medium (p < 0.01); there was no statistically significant difference between the reproduction rates of P-Y and Robinson media (p > 0.05). Twelve isolates from 12 patients were cultivated in P-Y medium and checked for reproduction everyday for 7 days. Twelve of the 12 (100%) isolates were cultivated in P-Y medium, indicating that the P-Y was an effective medium for the isolation of E. histolytica/E. dispar in stool samples. According to these results, P-Y medium could be preferred in immunologic, serologic and molecular studies and, thus the definitive diagnosis of amebiasis due to its low cost and simple formula.

Animals↗
PubMed1997

Virulent and avirulent Entamoeba histolytica and E. dispar differ in their cell surface phosphorylated glycolipids.

Virulent strains of Entamoeba histolytica have been reported to produce a mixture of phosphoglycoconjugates that share some structural features with the lipophosphoglycans (LPGs) of Leishmania. Purification of these glycoconjugates is essential to their precise structural characterization. In this study we have extracted 'LPG-like' molecules from various virulent E. histolytica strains and purified on the basis of charge differences, 2 apparently related glycoconjugates a 'LPG' and a 'lipophosphopeptidoglycan (LPPG)'. In marked contrast to the abundance of these 'LPG' and 'LPPG' molecules in the virulent strains, avirulent E. histolytica and E. dispar strains produce either very low, or no detectable levels of LPG, and either low levels or modified forms of 'LPPG'. Monospecific polyclonal antibodies prepared against that 'LPG' of the virulent strain HM-1:1MSS c16 identified epitopes shared between both the 'LPG' and the 'LPPG' of this and other virulent strains, using Western blot analysis. Flow cytometric analysis of a range of strains using these antibodies identified a surface distribution of these molecules and confirmed a correlation between surface exposure of epitopes bound by these antibodies and parasite virulence.

Animals↗
PubMed1998

Regulation of expression of ribosomal protein L-21 genes of Entamoeba histolytica and E. dispar is at the post-transcriptional level.

Two genes, EhgLE3 and Ehg34, encoding the ribosomal protein L21 (rp-L21) were identified and characterized from Entamoeba histolytica. Their coding regions are highly conserved, but their flanking regions differ significantly. Analogous genes (EdgLE3 and Edg34) were characterized in E. dispar. The two rp-L21 copies are transcribed at similar levels in the two parasites. However, their relative binding to the polyribosomal complex during active translation is different. In E. histolytica, binding of EhgLE3 transcripts to the polyribosomes is significantly higher in comparison with that of Ehg34 transcripts, whereas in E. dispar the binding pattern is inverse. The importance of each of the rp-L21 flanking regions to gene translation was investigated by constructing hybrid plasmids containing the CAT reporter gene flanked by rp-L21 flanking regions. The plasmids were stably transfected into E. histolytica and E. dispar, and CAT mRNA and enzymatic activity levels were determined. All plasmids promoted transcription of CAT. Yet, in E. histolytica, high levels of CAT activity were observed only when gLE3 upstream regions flanked CAT. In contrast, in E. dispar, high levels of CAT activity were observed when g34 upstream regions flanked CAT. The downstream regions showed no significant effect on CAT translation.

Animals↗
PubMed1964

LOCALIZATION OF CYTOPLASMIC NUCLEIC ACID DURING GROWTH AND ENCYSTMENT OF ENTAMOEBA INVADENS.

With a modified color-translating ultraviolet microscope, the distribution of material showing an absorption maximum at 265 mmicro was studied in samples from whole cultures of Entamoeba invadens at intervals during growth and from cysts allowed to mature under controlled conditions. Absorption by the cytoplasm in general gradually increased as trophozoites approached the period of maximum encystment. In late trophozoites and precystic forms, the absorbing material was concentrated into small bodies which coalesced to form large crystalloids of very high specific absorption. Maximum crystallization occurred in early cysts, where cytochemical tests have shown the large crystalloids to be ribonucleoprotein. Electron micrographs show that the crystalloids are composed of particles 200 to 300 A in diameter. During cyst maturation the amount of absorbing material per cyst is not visibly reduced, but the large bodies fragment into smaller units until finally there is only a very high diffuse absorption over the entire cyst. From these and other results the hypothesis is advanced that the large crystalloids ("chromatoid bodies") are a manifestation of a special parasite-host adaptive mechanism; ribonucleoprotein is synthesized under favorable conditions, crystallized in the resistant cyst stage, and dispersed in the newly excysted amebae thereby enabling them to establish themselves in a new host by a period of quick growth.

Bacteriological Techniques↗
PubMed2000

Entamoeba histolytica/Entamoeba dispar infections in human immunodeficiency virus-infected patients in the United States.

We describe the incidence of and laboratory and clinical characteristics associated with Entamoeba histolytica/Entamoeba dispar infection diagnosed in human immunodeficiency virus (HIV)-infected persons enrolled in the Adult and Adolescent Spectrum of HIV Disease Project. From 1 January 1990 to 1 January 1998 (82, 518 person-years of follow-up), 111 patients (98% men) were diagnosed with E. histolytica/E. dispar infection. Among HIV-infected patients in the United States, the incidence of diagnosed E. histolytica disease is low (13.5 cases per 10,000 person-years [95% confidence interval, 7.7-22.2], with diagnosis most common in those patients exposed to HIV through male-male sex.

AIDS-Related Opportunistic Infections↗