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At least 19 recordsLinked to original sources
PubMed2004

Detection and genotyping of Entamoeba histolytica, Entamoeba dispar, Giardia lamblia, and Cryptosporidium parvum by oligonucleotide microarray.

Entamoeba histolytica, Giardia lamblia, and Cryptosporidium parvum are the most frequently identified protozoan parasites causing waterborne disease outbreaks. The morbidity and mortality associated with these intestinal parasitic infections warrant the development of rapid and accurate detection and genotyping methods to aid public health efforts aimed at preventing and controlling outbreaks. In this study, we describe the development of an oligonucleotide microarray capable of detecting and discriminating between E. histolytica, Entamoeba dispar, G. lamblia assemblages A and B, and C. parvum types 1 and 2 in a single assay. Unique hybridization patterns for each selected protozoan were generated by amplifying six to eight diagnostic sequences/organism by multiplex PCR; fluorescent labeling of the amplicons via primer extension; and subsequent hybridization to a set of genus-, species-, and subtype-specific covalently immobilized oligonucleotide probes. The profile-based specificity of this methodology not only permitted for the unequivocal identification of the six targeted species and subtypes, but also demonstrated its potential in identifying related species such as Cryptosporidium meleagridis and Cryptosporidium muris. In addition, sensitivity assays demonstrated lower detection limits of five trophozoites of G. lamblia. Taken together, the specificity and sensitivity of the microarray-based approach suggest that this methodology may provide a promising tool to detect and genotype protozoa from clinical and environmental samples.

Animals↗
PubMed2007

PCR detection of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii in stool samples from Sydney, Australia.

This study investigated the presence of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii in stool samples from a patient population in Sydney, Australia. Stool samples were tested by microscopy and PCR. Five patients were found with E. histolytica infections, while E. dispar and E. moshkovskii were observed in 63 (70.8%) and 55 (61.8%) patients, respectively, by PCR. This is the first study in Australia using molecular techniques to determine the presence of E. histolytica, E. dispar, and E. moshkovskii.

Adult↗
PubMed2006

Differential detection of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii by a single-round PCR assay.

A single-round PCR assay was developed for detection and differential diagnosis of the three Entamoeba species found in humans, Entamoeba moshkovskii, Entamoeba histolytica, and Entamoeba dispar, that are morphologically identical as both cysts and trophozoites. A conserved forward primer was derived from the middle of the small-subunit rRNA gene, and reverse primers were designed from signature sequences specific to each of these three Entamoeba species. PCR generates a 166-bp product with E. histolytica DNA, a 752-bp product with E. dispar DNA, and a 580-bp product with E. moshkovskii DNA. Thirty clinical specimens were examined, and the species present were successfully detected and differentiated using this assay. It was possible to detect as little as 10 pg of E. moshkovskii and E. histolytica DNA, while for E. dispar the sensitivity was about 20 pg of DNA. Testing with DNA from different pathogens, including bacteria and other protozoa, confirmed the high specificity of the assay. We propose the use of this PCR assay as an accurate, rapid, and effective diagnostic method for the detection and discrimination of these three morphologically indistinguishable Entamoeba species in both routine diagnosis of amoebiasis and epidemiological surveys.

Animals↗
PubMed2005

Altered lipid parameters in patients infected with Entamoeba histolytica, Entamoeba dispar and Giardia lamblia.

Information on the effect of parasitic infections on lipid parameters is scarce. Certain parasites induce significant changes in lipid parameters, as demonstrated by the fact that substitution of lipid/cholesterol for serum in axenic culture medium (in vitro) and in experimental models (in vivo) supports vigorous growth of Entamoeba histolytica. Thus, significant changes in lipid parameters may be induced in an infected host. Blood samples are obtained from intestinal amoebiasis patients passing E. histolytica (n=8), E. dispar (n=15) or Giardia lamblia (n=9) cysts, or diagnosed with amoebic liver abscess (ALA; n=50) and from apparently normal healthy individuals (control group; n=30). Levels of total serum cholesterol, high-density lipoprotein and low-density lipoprotein are assessed using commercial kits. E. histolytica and E. dispar isolates are differentiated by hexokinase isoenzyme electrophoresis and by enzyme-linked immunosorbent assay (ELISA; Techlab) tests. Results show that E. histolytica, E. dispar and G. lamblia cyst passers had significantly lower levels of total serum cholesterol (73.42 +/- 2.24 mg/dL), compared to levels in ALA cases (101 +/- 2.85 mg/dL) and in controls (166.26 +/- 2.02 mg/dL). Further study of a greater number of cases is needed to explore the relevance of this finding.

Adolescent↗
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↗
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↗
PubMed1999

New insights into the role of the cytoskeleton in phagocytosis of Entamoeba histolytica.

Entamoeba histolytica, a human parasite, crosses the natural barriers of the intestine and, in turn, spreads into the deeper organs, resulting in amoebiasis. The motility of the parasite and its ability to lyse or phagocytose human cells facilitates passage of the amoeba through the intestinal epithelium. Little is known about the uptake of material by this parasite; nevertheless, the cytoskeleton is believed to play a role in phagocytosis. Myosin IB, an actin-binding protein, localizes to the phagocytic cup and, with time, surrounds the internalized phagosome itself. The role of unconventional myosins in phagocytosis has also been demonstrated in other cell types, suggesting that this molecular mechanism is a common denominator in phagocytic events. Here, we summarize the emerging view of the role of unconventional myosins as well as other cytoskeleton-associated proteins in pseudopod formation at early stages of phagocytosis and during the late step of this process in E. histolytica.

Animals↗
PubMed1997

Transcription initiation is controlled by three core promoter elements in the hgl5 gene of the protozoan parasite Entamoeba histolytica.

Entamoeba histolytica is a single cell eukaryote that is the etiologic agent of amoebic colitis. Core promoter elements of E. histolytica protein encoding genes include a TATA-like sequence (GTATTTAAAG/C) at -30, a novel element designated GAAC (GAACT) that has a variable location between TATA and the site of transcription initiation, and a putative initiator (Inr) element (AAAAATTCA) overlying the site of transcription initiation. The presence of three separate conserved sequences in a eukaryotic core promoter is unprecedented and prompted examination of their roles in regulating transcription initiation. Alterations of all three regions in the hgl5 gene decreased reporter gene activity with the greatest effect seen by mutation of the GAAC element. Positional analysis of the TATA box demonstrated that transcription initiated consistently 30-31 bases downstream of the TATA region. Mutation of either the TATA or GAAC elements resulted in the appearance of new transcription start sites upstream of +1 in the promoter of the hgl5 gene. Mutation of the Inr element resulted in no change in the site of transcription initiation; however, in the presence of a mutated TATA and GAAC regions, the Inr element controlled the site of transcription initiation. We conclude that all three elements play a role in determining the site of transcription initiation. The variable position of the GAAC element relative to the site of transcription initiation, and the multiple transcription initiations that resulted from its mutation, indicate that the GAAC element has an important and apparently novel role in transcriptional control in E. histolytica.

Animals↗
PubMed2002

A monoclonal antibody to the amebic lipophosphoglycan-proteophosphoglycan antigens can prevent disease in human intestinal xenografts infected with Entamoeba histolytica.

Entamoeba histolytica trophozoites are covered by lipophosphoglycan-peptidoglycan molecules which may be key virulence factors. We found that pretreatment of severe combined immunodeficient mice bearing human intestinal xenografts with a monoclonal antibody to the amebic lipophosphoglycan-peptidoglycan molecules can prevent or significantly reduce the human intestinal inflammation and tissue damage that are normally seen with E. histolytica colonic infection.

Animals↗
PubMed1996

Putative serine/threonine protein kinase expressed in complement-resistant forms of Entamoeba histolytica.

Entamoeba histolytica is susceptible to complement attack in its lumen-dwelling state and develops complement resistance during pathogenic tissue invasion. As experimental evidence suggests that this change in phenotype is accompanied by a change in gene expression, we constructed a subtractive cDNA library to identify genes involved. Poly(A) + RNA from complement-sensitive trophozoites was subtracted from single stranded cDNA derived from complement-resistant ones. Transcripts enriched in the library were found to code for a putative polypeptide comprising all sequence elements characteristic for serine/threonine protein kinases. The gene contains an intron of 46 nucleotides and two polyadenylation sites. Northern-blot analyses confirmed that the gene is expressed in both tissue-derived and laboratory-grown forms of complement-resistant E. histolytica.

Amino Acid Sequence↗
PubMed1988

Phorbol esters specifically enhance the cytolytic activity of Entamoeba histolytica.

Entamoeba histolytica causes invasive amebiasis by lysis of host tissue and inflammatory cells. The in vitro cytolysis of target Chinese hamster ovary (CHO) cells by axenic E. histolytica trophozoites (strain HM1:IMSS) is a calcium- and phospholipase A-dependent event initiated by the binding to the target cell of the galactose-inhibitable surface lectin of the parasite. We utilized phorbol esters as a probe to determine whether an amebic protein kinase C has a role in the cytolytic event. The addition of phorbol 12-myristate 13-acetate (PMA) at 10(-6) or 10(-7) M resulted in a greater than twofold enhancement of amebic killing of target CHO cells over 30 min (P less than 0.01). Prior exposure of only the amebae, but not the CHO cells, to PMA produced a similar effect (P less than 0.01). The inactive analog 4-alpha-phorbol had no effect on amebic killing of CHO cells. The PMA-mediated enhancement of amebic cytolysis persisted for up to 60 min after a 5-min exposure; however, after a 30-min exposure to PMA (10(-6) M) there was no augmentation of amebic killing of CHO cells. PMA (10(-6) M) did not promote adherence of parasites to CHO cells but did enhance amebic cytolysis of previously adherent target cells (P less than 0.01). Sphingosine, a specific inhibitor of protein kinase C, abolished both the PMA-stimulated and the basal cytolytic activity of E. histolytica. PMA enhanced CHO cell cytolysis by the less virulent wild-type strain H-303:NIH (P less than or equal to 0.02) but did not augment the activity of the less virulent strain H-200:NIH or two avirulent clones of HM1 (L6 and C919). In summary, these experiments with the phorbol esters and sphingosine as probes to modulate the activity of protein kinase C indicate participation of a parasite protein kinase C in the cytolytic activity of virulent, axenic E. histolytica trophozoites and thus in the pathogenesis of amebiasis.

Adhesiveness↗
PubMed2005

Characterization of chaperonin 10 (Cpn10) from the intestinal human pathogen Entamoeba histolytica.

Entamoeba histolytica is the causative agent of amoebiasis, a poverty-related disease that kills an estimated 100 000 people each year. E. histolytica does not contain "standard mitochondria", but harbours mitochondrial remnant organelles called mitosomes. These organelles are characterized by the presence of mitochondrial chaperonin Cpn60, but little else is known about the functions and molecular composition of mitosomes. In this study, a gene encoding molecular chaperonin Cpn10--the functional partner of Cpn60--was cloned, and its structure and expression were characterized, as well as the cellular localization of its encoded protein. The 5' untranslated region of the gene contains all of the structural promoter elements required for transcription in this organism. The amoebic Cpn10, like Cpn60, is not significantly upregulated upon heat-shock treatment. Computer-assisted protein modelling, and specific antibodies against Cpn10 and Cpn60, suggest that both proteins interact with each other, and that they function in the same intracellular compartment. Thus, E. histolytica appears to have retained at least two of the key molecular components required for the refolding of imported mitosomal proteins.

Amino Acid Sequence↗
PubMedDate not supplied

Evidence for a vacuolar-type proton ATPase in Entamoeba histolytica.

Entamoeba histolytica is a primitive eukaryote that lacks mitochondria. Golgi and a well-developed endoplasmic reticulum. Close to half of the cell volume is occupied by pinocytic vesicles, which are in continuous turnover with the plasma membrane and perform functions that in higher eukaryotic cells are taken over by lysosomes. Similar to the latter, the amebal vesicles are acidified. We report here that bafilomycin A1, a specific inhibitor of vacuolar-type (V-) ATPases, suppressed this acidification at submicromolar concentrations; concomitantly, it inhibited pinocytosis. These results strongly suggest the presence of a V-ATPase in pinocytic vesicles of E. histolytica, and thereby support the notion that the V-ATPases in the organelles of higher eukaryotes are derived from an archaic plasma membrane-bound form.

Animals↗
PubMed1993

Antigenicity, immunogenicity and vaccine efficacy of the galactose-specific adherence protein of Entamoeba histolytica.

Entamoeba histolytica is an enteric protozoan that causes amoebic colitis and liver abscess. Human immunity to E. histolytica is apparently mediated by a serum antibody response and amoebicidal cellular mechanisms. The galactose-specific adherence protein of E. histolytica is a 260 kDa glycoprotein which mediates amoebic in vitro adherence to human colonic mucins, epithelium, and inflammatory cells. Amoebic lysis of cells is dependent upon binding by this adherence protein. Serum IgG and salivary IgA antibodies from greater than 90% of subjects with invasive amoebiasis recognize the adherence protein's 170 kDa heavy subunit. Incubation of peripheral blood mononuclear cells from antibody-positive subjects with the purified galactose-specific adherence protein induces in vitro T lymphocyte proliferation, IL-2 and gamma interferon production, and direct lymphocyte amoebicidal activity. The rationale for an adherence protein vaccine includes sIgA blockage of amoebic binding to colonic mucins, humoral IgG prevention of parasitic adherence in tissues, and development of amoebicidal cell-mediated immunity. Immunization of gerbils with purified adherence protein in Freund's adjuvant provides protection against intrahepatic challenge with the trophozoites. In summary, the galactose specific adherence protein of E. histolytica contains highly conserved B- and T-cell epitopes, and has a high degree of vaccine efficacy in the gerbil model of amoebic liver abscess.

Animals↗
PubMed2003

The cell cycle of Entamoeba histolytica.

Entamoeba histolytica, is a microaerophilic protist, which causes amoebic dysentery in humans. This unicellular organism proliferates in the human intestine as the motile trophozoite and survives the hostile environment outside the human host as the dormant quadri-nucleate cyst. Lack of organelles--such as mitochondria and Golgi bodies--and an unequal mode of cell division, led to the popular belief, that this organism preceded other eukaryotes during evolution. However, data from several laboratories have shown that, contrary to this belief, E. histolytica is remarkable in its divergence from other eukaryotes. This uniqueness is witnessed in many aspects of its biochemical pathways, cellular biology and genetic diversity. In this context, I have analysed the cell division cycle of this organism and compared it to that of other eukaryotes. Studies on E. histolytica, suggest that in its proliferative phase, this organism may accumulate polyploid cells. Thus 'checkpoints' regulating alternation of genome duplication and cell division appear to be absent in this unicellular protist. Sequence homologs of several cell cycle regulating proteins have been identified in amoeba, but their structural divergence suggests that they may not have equivalent function in this organism. The regulation of cell proliferation in E. histolytica, may be ideally suited to survival of a parasite in a complex host. Analysis of these molecular details may offer solutions for eradicating the pathogen by hitherto unknown methods.

Animals↗