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PubMed2004

The distribution of Entamoeba histolytica and Entamoeba dispar in northern, central, and southern Iran.

The present study was carried out from August 1999 through February 2002 in order to determine the prevalence of Entamoeba histolytica and Entamoeba dispar in three different climatic regions of Iran by using a PCR-RFLP method. A total of 16,592 stool samples were randomly collected from different age-groups in central, northern, and southern Iran in both urban and rural areas. The samples were examined by direct and formalin-ether concentration methods. A total of 226 samples were positive for E. histolytica/E. dispar cysts. Of these, 101 isolates were cultured and maintained successfully in Robinson's medium and were identified by the PCR-RFLP method. The study showed that 92.1% of isolates were E. dispar and 7.9% were E. histolytica or mixed infections. The ratio of E. histolytica to E. dispar was higher in southern regions (tropical and subtropical) than in the other two regions. This study demonstrated that E. dispar is the predominant species found among "cyst passers" in Iran.

Animals↗
PubMed1978

Electrophoretic isoenzyme patterns of Entamoeba histolytica and Entamoeba coli.

Cultures of 14 stocks of Entamoeba histolytica and one only of Entamoeba coli were compared by electrophoretic patterns of three enzymes: glucose-phosphate isomerase, phosphoglucomutase and L-malate: NADP+ oxidoreductase (oxaloacetate-decarboxylating). Easily distinguished patterns divided E. histolytica into three groups, whilst a distinctly different pattern for E. coli was also seen.

Electrophoresis, Starch Gel↗
PubMed2005

Entamoeba histolytica: alterations in EhRabB protein in a phagocytosis deficient mutant correlate with the Entamoeba dispar RabB sequence.

We analyzed the expression and location of EhRabB in clone L-6, a phagocytosis-deficient mutant of Entamoeba histolytica, in comparison with the wild-type clone A. Intriguingly, trophozoites of clone L-6 express more EhRabB than those of clone A. However, the majority of EhRabB-containing vesicles remained in the cytoplasm of clone L-6 during phagocytosis. To investigate molecular alterations in EhRabB of clone L-6 we compared the EhrabB gene sequences from clones L-6 and A. We also isolated, sequenced and compared the RabB protein of Entamoeba dispar. Results showed that EhrabB gene of clone L-6 is 98.2 and 94.1% identical to rabB genes of E. dispar and clone A, respectively. The rabB genes from clone A and E. dispar have 92.2% identity. Four out of five amino acids changes in RabB proteins of clone L-6 and E. dispar are shared. These changes may alter the binding of effector proteins and the specific subcellular location of EhRabB.

Amino Acid Sequence↗
PubMed2006

DNA methylation and targeting of LINE retrotransposons in Entamoeba histolytica and Entamoeba invadens.

In this study, we have isolated by affinity chromatography, using anti-m5C antibody as a ligand, a DNA encoding reverse transcriptase of LINE retrotransposon (RT LINE) in both Entamoeba invadens and Entamoeba histolytica. RT LINE transcripts were detected in E. histolytica but were absent from E. invadens. The methylation status of genomic copies of E. invadens RT LINE was confirmed by bisulfite analysis. In contrast, all the genomic copies of the E. histolytica RT LINE analyzed in this study were not methylated. Many of these genomic copies diverge from the RT LINE isolated by m5C affinity chromatography by a number of mutations that includes conversion of C to T and G to A. These mutations are reminiscent of the conversion of C to T (and G to A on the complementary DNA strand) that occurred during primate evolution in Alu elements following accelerated deamination of methylated cytosines. E. invadens and E. histolytica RT LINEs isolated by affinity chromatography were cloned in a pEhAct Neo vector, amplified in E. coli GM2163 (dam-dcm) and transformed into E. histolytica. Bisulfite analysis of transfected amoeba showed the presence of m5C in E. invadens RT LINE replicated in E. histolytica, but not in E. histolytica RT LINE or in the neomycine phosphotransferase gene, which is also carried by the pEhAct Neo vector. These results suggest the existence of a specific mechanism based on DNA methylation that controls retrotransposons in these parasites.

Animals↗
PubMed2005

Entamoeba histolytica and Entamoeba dispar: comparison of two PCR assays for diagnosis in a non-endemic setting.

Detection of Entamoeba histolytica, the causative agent of amoebiasis, is an important goal of the clinical parasitology laboratory. The identification of Entamoeba dispar as a morphologically identical but non-pathogenic species has highlighted the need for non-microscopic detection methods able to differentiate between the two organisms. In this study we evaluated the utility of conventional PCR and real-time PCR as methods for identification and differentiation of E. histolytica and E. dispar. The second aim of this study was to determine the relative proportions of infections caused by E. histolytica and the non-pathogenic E. dispar, allowing a picture of the epidemiological situation in a non-endemic setting to be obtained. One hundred and sixty-six clinical samples (faecal and liver abscess samples and one intestinal biopsy) belonging to 108 patients were analysed. More patients with E. dispar infection (8.3%) than patients with E. histolytica infection (5.6%) were found by both PCR assays. It is concluded that routine diagnosis of invasive amoebiasis performed by a combination of microscopy, culture and serology should be complemented with a PCR assay such as real-time PCR that offers a practical and clinically acceptable alternative for rapid and accurate diagnosis of amoebic infection in patients presenting with symptoms indicative of this disease.

Animals↗
PubMed1998

Entamoeba histolytica and Entamoeba dispar are distinct species; clinical, epidemiological and serological evidence.

The name of the causative organism of invasive amoebiasis, Entamoeba histolytica, was first introduced in 1903, even though this intestinal amoeba had been recognised since 1875. The marked disparity between the number of infected individuals and those with invasive amoebiasis resulted in a number of explanatory hypotheses being proposed. Although none of these were universally accepted, Brumpt's concept of two morphologically identical species gained increasing acceptance 50-60 years later when technology became available to investigate this anomaly. Sargeaunt spear-headed this drive by establishing the value of isoenzyme electrophoresis for studying the host-parasite relationship. From this foundation, incorporation of clinical, epidemiological and serological parameters to studies of the parasite resulted in the conclusion that a species complex comprising two morphologically identical amoebae was implicated with the disease. The two organisms have been named E. histolytica and Entamoeba dispar. The former is a pathogen and is responsible for invasive amoebiasis, while the latter is a gut commensal. Demonstration of the existence of this species complex has subsequently been confirmed by studies on the nucleic acids from several independent laboratories. The acceptance of E. histolytica and E. dispar as distinct species has had a major impact on our understanding of amoebiasis and its clinical management.

Animals↗
PubMed1997

Molecular and biochemical characterization of phosphoglucomutases from Entamoeba histolytica and Entamoeba dispar.

Entamoeba histolytica and Entamoeba dispar have only recently been defined as two separate species. E. histolytica, the pathogenic species, is the microorganism causing invasive intestinal amoebiasis and/or liver abscess, while the morphologically similar E. dispar is nonpathogenic and noninvasive. The gold standard for the distinction of the two species has been the isoenzyme electrophoresis of phosphoglucomutases (EC 5.4.2.2) and hexokinases (EC 2.7.1.1), but there had also been a controversy about the possibility of a conversion of isoenzyme patterns. In this study, we cloned the phosphoglucomutase (PGM) cDNAs from the pathogenic and the nonpathogenic species. The deduced amino acid sequences were only 2.4% different. The cDNAs were expressed in Escherichia coli under the control of a T7 RNA polymerase promoter. The recombinant polypeptides displayed strong phosphoglucomutase activity, each of the recombinant enzymes comigrated with its natural counterpart from E. histolytica and E. dispar in the starch gel electrophoresis. Our results give a biochemical interpretation of the PGM isoenzyme pattern and support the clear distinction between the two species.

Amino Acid Sequence↗
PubMed1996

Direct sequencing of the PCR amplified SSU rRNA gene of Entamoeba dispar and the design of primers for rapid differentiation from Entamoeba histolytica.

Since 1993, strains of Entamoeba histolytica sensu lato have been assigned to 2 species on the basis of clinical, biochemical, immunological and genetic evidence: the pathogenic strains to E. histolytica sensu stricto, the non-pathogenic strains to Entamoeba dispar. Analysis of the gene encoding for the small subunit ribosomal RNA (SSU rDNA) supports the existence of 2 species. However, while 3 whole SSU rDNA sequences are available in the data bases for E. histolytica, only a partial sequence has been published for E. dispar. Here we report a SSU rDNA sequence for E. dispar. Compared to those of E. histolytica, this sequence shows 1.7% nucleotide substitutions. On the basis of our rDNA data, 2 primers were designed to produce polymerase chain reaction (PCR) amplification from both E. histolytica and E. dispar. Primer specificity for the 2 amoebae was assessed both theoretically against the data bases, and experimentally against a collection of eukaryotic and prokaryotic DNAs. The amplified stretch encompasses a polymorphic Dde I restriction site which allows, after cleavage of the fragment, E. histolytica and E. dispar to be distinguished. The reliability of this method of identification was assessed comparing the results with those based on classic isoenzyme analysis.

Animals↗
PubMed2003

Short communication: Prevalence of Entamoeba histolytica and Entamoeba dispar in northern Ghana.

Since the redescription of the potentially invasive Entamoeba histolytica, separating it from the morphologically identical non-invasive Entamoeba dispar, there is a need for the reassessment of epidemiological data on amoebiasis. In this context we conducted a descriptive survey on the presence of E. histolytica and E. dispar in a rural area in northern Ghana. We found a high prevalence (39.8%) of the E. histolytica/E. dispar complex with microscopy, but E. histolytica and E. dispar-specific DNA amplification using real-time polymerase chain reaction identified only one E. histolytica case and revealed a considerably higher prevalence of E. dispar (82.8%).

Animals↗
PubMed1996

Entamoeba histolytica and Entamoeba dispar: differences in numbers and expression of cysteine proteinase genes.

In order to identify molecules that might be responsible for the difference in pathogenicity between the two closely related protozoan parasites Entamoeba histolytica and Entamoeba dispar, we focussed on cysteine proteinases because this class of enzymes has been considered important for pathogenic tissue destruction. By screening a genomic library derived from an E. histolytica isolate, a total of six distinct genes (ehcp1-ehcp6) encoding typical prepro-forms of cysteine proteinases were identified which differed from each other by 40% to 85% of their nucleotide sequences. Three of these genes, ehcp1, ehcp2, and ehcp5, which exhibited high levels of expression, were found to be responsible for approximately 90% of cysteine proteinase transcripts, whereas the remaining three were either not or only marginally expressed. Expression of the different genes directly correlated with the level of activity of the respective enzymes in trophozoite lysates. Purification of the enzymes and N-terminal sequencing revealed that virtually all cysteine proteinase activity of E. histolytica can be attributed to three enzymes namely EhCP1, EhCP2 and EhCP5. Southern blot analysis indicated that just two of these abundantly expressed genes are missing in E. dispar. On the other hand, genes analogous to four of the six genes identified in E. histolytica were found to be present in E. dispar, but only two of these are expressed within the trophozoite stage.

Amino Acid Sequence↗
PubMed1997

Sensitivity of Entamoeba histolytica and Entamoeba dispar patient isolates to human complement.

Twenty-one Entamoeba histolytica and 56 Entamoeba dispar patient isolates were investigated for their sensitivity to the classical and alternative pathway of human complement, E. histolytica and E. dispar patient isolates were differentiated by polymerase chain reaction and hexokinase isoenzyme typing. It was found that 90.3% (+/- 12.0%) of the trophozoites of E. histolytica were lysed after 30 min by the alternative pathway of complement in the presence of 50% human serum (19 isolates showed lysis rates higher than 80%), whereas E. dispar cells were less susceptible to the alternative pathway as 68.8% (+/- 28.2%) of lysis occurred. However, 23 of the E. dispar isolates were lysed between 100 and 80% (90.9% +/- 9.1%), demonstrating that about half of the tested E. dispar isolates were highly sensitive to complement lysis. Only 11 of the E. dispar isolates were proven to be ¿resistant' to the alternative pathway of complement and were lysed less than 40%. These results are in conflict to earlier publications, describing resistance of E. dispar to complement lysis (Hamelmann et al. 1992, 1993).

Animals↗
PubMed2001

Introns of Entamoeba histolytica and Entamoeba dispar.

The genome of Entamoeba histolytica is considered to possess very few intervening sequences (introns), as only 5 intron-containing genes from this protozoan parasite have been reported so far. However, while sequencing a number of genomic contigs as well as three independent genes coding for ribosomal protein L27a, we have identified 9 additional intron-containing genes of E. histolytica and the closely related species Entamoeba dispar, indicating that introns are more common in these organisms than previously suggested. The various amoeba introns are relatively short comprising between 46 and 115 nucleotides only and have a higher AT-content compared to the corresponding exon sequences. In contrast to higher eukaryotes, amoeba introns do not contain a well-conserved branch point consensus, and have extended donor and acceptor splice sites of the sequences G

Animals↗
PubMed2000

A nested, multiplex, PCR assay for the simultaneous detection and differentiation of Entamoeba histolytica and Entamoeba dispar in faeces.

The detection of and differentiation between Entamoeba histolytica and Entamoeba dispar are of great importance, both for diagnosis and for epidemiological studies. Most PCR-based methods for the discrimination of these two species employ complex procedures for DNA extraction and require different protocols for E. histolytica and E. dispar, leading to relatively high expenditure, labour costs and turnaround times. A simple, rapid, cost-effective and yet sensitive and specific multiplex PCR technique has now been developed for the simultaneous detection and differentiation of E. histolytica and E. dispar in faecal samples. The detection limit is 200 trophozoites of E. dispar or 1000 trophozoites of E. histolytica/g stool sample. The sensitivity of the assay remains practically unchanged, even in the presence of 20,000 trophozoites of the other species/g stool sample. Thus, this technique may also easily reveal mixed infections, without the danger of misdiagnosis caused by one strain displacing the other in culture.

Animals↗
PubMed2005

PCR differentiation of Entamoeba histolytica and Entamoeba dispar from patients with amoeba infection initially diagnosed by microscopy.

Amoebiasis is a notifiable disease in Sweden and 400-500 cases are reported annually to the Swedish Institute for Infectious Disease Control (SMI). The true number of patients with Entamoeba histolytica infection is unknown as diagnosis mainly relies on cyst detection by microscopy. The main purpose of this study was to estimate the proportions between E. histolytica and E. dispar in patients with amoebic infection, using established PCR technologies. Secondly, we aimed to evaluate the usefulness of ethanol as a transport medium for samples forwarded for Entamoeba-PCR. Faecal samples from 207 patients with initial diagnosis of E. histolytica/E. dispar were referred to SMI for species differentiation. The PCR analysis showed that 165 patients were positive for E. dispar, whereas only 10 patients were positive for E. histolytica. No mixed infections were observed. The remaining 32 patients were negative both by microscopy and by PCR. Ethanol fixation was evaluated on 168 paired samples (transported unfixed or fixed in ethanol). Ethanol was found to be a useful transport medium as in 8 cases only the fixed sample was PCR-positive. This study shows that few patients in Sweden are infected with E. histolytica. The ability to differentiate E. dispar from E. histolytica should reduce the number of unnecessarily treated patients.

Adolescent↗
PubMed1999

Entamoeba histolytica and Entamoeba dispar: epidemiology and comparison of diagnostic methods in a setting of nonendemicity.

Recent studies suggest that stool antigen assays are more sensitive and specific than microscopy for the diagnosis of Entamoeba histolytica infection. One hundred twelve patients presenting at 3 centers with symptoms or risk factors of E. histolytica infection were prospectively enrolled in this study to evaluate new diagnostic tests for infections with E. histolytica and Entamoeba dispar. Four ELISA-based stool antigen kits for detecting E. histolytica or E. dispar were blindly compared with stool microscopy. Amebic serology was assessed by indirect hemagglutination. When antigen assays were used as the reference standard, microscopy performed at referral centers was more specific (68.4% vs. 9.5%) but less sensitive (70.4% vs. 92.1%) than microscopy performed in community laboratories. Diagnosis with the E. histolytica test and Merlin Optimun S ELISA indicated that only 3 (4.2%) of 72 coproantigen-positive stools were positive for E. histolytica. Indirect hemagglutination was a good predictor of E. histolytica infection when titers of antibody to ameba were >/=1:512.

Animals↗
PubMed1982

Electrophoretic isoenzyme patterns of Entamoeba histolytica and Entamoeba chattoni in a primate survey.

Stocks of Entamoeba histolytica grown in a monoxenic culture system from the feces of nonhuman primates are compared with the eleven zymodemes of E. histolytica so far demonstrated from man. In a similar fashion, Entamoeba chattoni has also been grown and identified. Both E. histolytica and E. chattoni have been demonstrated in keepers of the primate collections. Comparisons have been made using the electrophoretic patterns of three enzymes: glucosephosphate isomerase [(GPI) E.C.5.3.1.9], phosphoglucomutase [(PGM) E.C.2.7.5.1], and L-malate--NADP+ oxidoreductase (oxaloacetate-decarboxylating) [(ME) E.C.1.1.1.40]. Enzyme patterns of E. histolytica from the apes were found to be identical with three of those already demonstrated from man. The enzyme pattern of E. chattoni was distinctly different from that of any of the E. histolytica zymodemes. Other protozoa found in the single fecal sample examined from each subject are also listed.

Animals↗
PubMed1999

A DNA sequence corresponding to the gene encoding cysteine proteinase 5 in Entamoeba histolytica is present and positionally conserved but highly degenerated in Entamoeba dispar.

Cysteine proteinases of Entamoeba histolytica are considered to be one of the most important classes of molecules responsible for the parasite's ability to destroy human tissues. Interestingly, one particular cysteine proteinase, located on the surface of E. histolytica trophozoites and designated cysteine proteinase 5 (CP5), is not expressed in the closely related but nonpathogenic species Entamoeba dispar. By comparing the E. histolytica and E. dispar genomic loci containing the gene for CP5 (cp5), it was found that the position of cp5 within the genomic context is conserved between the two organisms, but that the gene is highly degenerated in E. dispar, as it contains numerous nucleotide exchanges, insertions, and deletions, resulting in multiple stop codons within the cp5 reading frame. An alignment of all available orthologous E. histolytica and E. dispar DNA sequences suggested that cp5 started to degenerate in E. dispar coincidently when the two organisms began to diverge from a common ancestor.

Animals↗
PubMed2002

The abundant polyadenylated transcript 2 DNA sequence of the pathogenic protozoan parasite Entamoeba histolytica represents a nonautonomous non-long-terminal-repeat retrotransposon-like element which is absent in the closely related nonpathogenic species Entamoeba dispar.

While comparing gene expression in the pathogenic organism Entamoeba histolytica and the closely related but nonpathogenic species Entamoeba dispar, we discovered that the E. histolytica abundant polyadenylated transcript 2 (ehapt2) and corresponding genomic copies are absent in E. dispar. Although polyadenylated, ehapt2 does not contain any overt open reading frame. Southern blot and sequence analyses revealed that about 500 copies of ehapt2 genomic elements were present in each cell and that the copies were distributed throughout the ameba genome. The various ehapt2 elements are regularly located in the vicinity of protein-encoding genes, downstream of pyrimidine-rich sequence stretches (40 to 125 bp; CT content, 79.2 to 85.5%), and are flanked by duplicated target sites of variable length. Target site duplications were obviously generated during integration of ehapt2 into the E. histolytica genome as one copy of the flanking repeat and the complete ehapt2 element are specifically absent in orthologous E. dispar genomic sequences. ehapt2 shares 3' sequences with EhRLE, a recently identified non-long-terminal-repeat (non-LTR) retrotransposon-like element of E. histolytica, which contains a conceptual open reading frame for reverse transcriptase. Thus, ehapt2 has all of the properties of nonautonomous non-LTR retrotransposons. A comparison of various E. histolytica isolates suggested that transposition of ehapt2 takes place at a very low frequency as the genomic localization of ehapt2 elements was found to be well conserved. A mobile element such as ehapt2 could be a suitable mechanism to explain the infrequent and late transition of E. histolytica from a harmless gut commensal to an invasive pathogen.

Animals↗