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Sample records for entamoeba histolytica ptpases

  1. Cloning and partial characterization of Entamoeba histolytica PTPases

    SciTech Connect

    Herrera-Rodriguez, Sara Elisa; Baylon-Pacheco, Lidia; Talamas-Rohana, Patricia; Rosales-Encina, Jose Luis . E-mail: rosales@cinvestav.mx

    2006-04-21

    Reversible protein tyrosine phosphorylation is an essential signal transduction mechanism that regulates cell growth, differentiation, mobility, metabolism, and survival. Two genes coding for protein tyrosine phophatases, designed EhPTPA and EhPTPB, were cloned from Entamoeba histolytica. EhPTPA and EhPTPB proteins showed amino acid sequence identity of 37%, both EhPTPases showed similarity with Dictyostelium discoideum and vertebrate trasmembranal PTPases. mRNA levels of EhPTPA gene are up-regulated in trophozoites recovered after 96 h of liver abscess development in the hamster model. EhPTPA protein expressed as a glutathione S-transferase fusion protein (GST::EhPTPA) showed enzymatic activity with p-nitrophenylphosphate as a substrate and was inhibited by PTPase inhibitors vanadate and molybdate. GST::EhPTPA protein selectively dephosphorylates a 130 kDa phosphotyrosine-containing protein in trophozoite cell lysates. EhPTPA gene codifies for a 43 kDa native protein. Up-regulation of EhPTPA expression suggests that EhPTPA may play an important role in the adaptive response of trophozoites during amoebic liver abscess development.

  2. Pathogenicity of entamoeba histolytica.

    PubMed

    Kagan, I G

    1975-01-01

    The pathogenicity of Entamoeba histolytica is discussed from an immunologic point of view. The evidence that there is some "trigger" mechanism which converts a commensal dwelling organism into a tissue invasive pathogen is rejected as inadequate. The number of liver abscess cases in comparison with the number of intestinal amebic infections in a population is so low that this in itself suggests that tissue invasion is a rare event in the life history of the ameba. A review is made of the experimental evidence that some type of sensitization is necessary before ameba can invade tissue. In postulating an immunologic basis for the pathogenicity of ameba, a parallel between the behavior of malignant cells in the body and an amebic infection in the gut is made. An appealing hypothesis which deserves further research effort is that an altered immune response is the basis for the pathogenic mechanism in the host. PMID:171223

  3. [Entamoeba histolytica: a standing threat].

    PubMed

    Conde-Bonfil, M C; de la Mora-Zerpa, C

    1992-01-01

    The importance and limitations of microscopic differential diagnosis of Entamoeba histolytica in asymptomatic carriers is reviewed. The possibility that some nonpathogenic strains of E. histolytica could be Entamoeba hartmanni is discussed. The imperative necessity to encourage research in epidemiology and diagnosis is emphasized. The need to know the prevalence in Mexico and distinguish E. histolytica from E. hartmanni is also discussed. This differential diagnosis is fundamental in the treatment of carriers and in epidemiological studies. Treatment must be directed only to acute and chronic patients and asymptomatic carries who prepare food. Appropriate drugs are still metronidazole and quinolines. PMID:1615351

  4. Susceptibility testing of Entamoeba histolytica

    SciTech Connect

    Cedeno, J.R.; Krogstad, D.J.

    1983-12-01

    The growth of Entamoeba histolytica in microtiter plates in vitro in a variety of environments with reduced oxygen tensions is reported. With 3% O/sub 2/, 3% CO/sub 2/, and 94% N/sub 2/, the parasite growth in microtiter plates was identical to that in screw-capped culture tubes, as measured by (/sup 3/H)thymidine incorporation and by quantitative parasite counts. There were no significant differences between the drug concentrations necessary to inhibit parasite growth by 50% based on (/sup 3/H)thymidine incorporation vs those defined by quantitative parasite counts for the 15 antimicrobial agents tested (including seven drugs used for the treatment of amebiasis). This technique provides a reproducible method to quantitate the activity of potential antiamebic agents in vitro. The isotopic method should be of particular value in defining the metabolism of the parasite and effects of antimicrobial agents on it, whereas the morphologic method may be more valuable for workers with limited resources available to them.

  5. PCR Detection of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii in Stool Samples from Sydney, Australia▿

    PubMed Central

    Fotedar, R.; Stark, D.; Beebe, N.; Marriott, D.; Ellis, J.; Harkness, J.

    2007-01-01

    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. PMID:17229864

  6. Glucose Starvation Boosts Entamoeba histolytica Virulence

    PubMed Central

    Tovy, Ayala; Hertz, Rivka; Siman-Tov, Rama; Syan, Sylvie; Faust, Daniela; Guillen, Nancy; Ankri, Serge

    2011-01-01

    The unicellular parasite, Entamoeba histolytica, is exposed to numerous adverse conditions, such as nutrient deprivation, during its life cycle stages in the human host. In the present study, we examined whether the parasite virulence could be influenced by glucose starvation (GS). The migratory behaviour of the parasite and its capability to kill mammalian cells and to lyse erythrocytes is strongly enhanced following GS. In order to gain insights into the mechanism underlying the GS boosting effects on virulence, we analyzed differences in protein expression levels in control and glucose-starved trophozoites, by quantitative proteomic analysis. We observed that upstream regulatory element 3-binding protein (URE3-BP), a transcription factor that modulates E.histolytica virulence, and the lysine-rich protein 1 (KRiP1) which is induced during liver abscess development, are upregulated by GS. We also analyzed E. histolytica membrane fractions and noticed that the Gal/GalNAc lectin light subunit LgL1 is up-regulated by GS. Surprisingly, amoebapore A (Ap-A) and cysteine proteinase A5 (CP-A5), two important E. histolytica virulence factors, were strongly down-regulated by GS. While the boosting effect of GS on E. histolytica virulence was conserved in strains silenced for Ap-A and CP-A5, it was lost in LgL1 and in KRiP1 down-regulated strains. These data emphasize the unexpected role of GS in the modulation of E.histolytica virulence and the involvement of KRiP1 and Lgl1 in this phenomenon. PMID:21829737

  7. [Report on a fungus parasitizing Entamoeba histolytica].

    PubMed

    Cao, C Q; Feng, Y S

    1989-01-01

    Infection of Entamoeba histolytica with chytridiaceous fungus Sphaerita was observed in some specimens obtained from a farmer and stained with iron-haematoxylin. The fungi were found in 78% of the cysts, mostly immature ones. Within the amoebae this parasite occurred singly, in groups, or in the form of a sporangium. It was located in the cytoplasm, the glycogen mass or the chromatoidal bars. In the same specimen, the parasitic fungus was also found in 18% of E. coli cysts; in 11% of E. nana cysts; while only one of 16 E. hartmanni cysts was parasitized. It is an interesting case of superimposed parasitism so far reported in China as well as a rare case of several species of amoebae being heavily involved with the same in the scientific literature. PMID:2548767

  8. Entamoeba histolytica: observations on metabolism based on thegenome sequence

    SciTech Connect

    Anderson, Iain J.; Loftus, Brendan J.

    2005-07-01

    The sequencing of the genome of Entamoeba histolytica has allowed a reconstruction of its metabolic pathways, many of which are unusual for a eukaryote. Based on the genome sequence, it appears that amino acids may play a larger role than previously thought in energy metabolism, with roles in both ATP synthesis and NAD regeneration. Arginine decarboxylase may be involved in survival of E. histolytica during its passage through the stomach. The usual pyrimidine synthesis pathway is absent, but a partial pyrimidine degradation pathway could be part of a novel pyrimidine synthesis pathway. Ribonucleotide reductase was not found in the E. histolytica genome, but it was found in the close relatives Entamoeba invadens and Entamoeba moshkovskii, suggesting a recent loss from E. histolytica. The usual eukaryotic glucose transporters are not present, but members of a prokaryotic monosaccharide transporter family are present.

  9. Flavodiiron Oxygen Reductase from Entamoeba histolytica

    PubMed Central

    Gonçalves, Vera L.; Vicente, João B.; Pinto, Liliana; Romão, Célia V.; Frazão, Carlos; Sarti, Paolo; Giuffrè, Alessandro; Teixeira, Miguel

    2014-01-01

    Flavodiiron proteins (FDPs) are a family of enzymes endowed with bona fide oxygen- and/or nitric-oxide reductase activity, although their substrate specificity determinants remain elusive. After a comprehensive comparison of available three-dimensional structures, particularly of FDPs with a clear preference toward either O2 or NO, two main differences were identified near the diiron active site, which led to the construction of site-directed mutants of Tyr271 and Lys53 in the oxygen reducing Entamoeba histolytica EhFdp1. The biochemical and biophysical properties of these mutants were studied by UV-visible and electron paramagnetic resonance (EPR) spectroscopies coupled to potentiometry. Their reactivity with O2 and NO was analyzed by stopped-flow absorption spectroscopy and amperometric methods. These mutations, whereas keeping the overall properties of the redox cofactors, resulted in increased NO reductase activity and faster inactivation of the enzyme in the reaction with O2, pointing to a role of the mutated residues in substrate selectivity. PMID:25151360

  10. Complement sensitivity of Entamoeba histolytica and various nonpathogenic amoeba species.

    PubMed

    Förster, B; Ebert, F; Horstmann, R D

    1994-12-01

    Culture forms of the potentially pathogenic Entamoeba histolytica were compared to those of the nonpathogenic species of E. dispar, E. hartmanni, E. coli, Endolimax nana, and E. moshkovskii regarding the sensitivity to lysis by human complement activated through the alternative pathway. E. dispar was found unique in its complement resistance; all other nonpathogenic isolates resembled E. histolytica in that they were complement sensitive. Thus, a state of complement sensitivity is not a particular property of potentially pathogenic amoebae. PMID:7716404

  11. 21 CFR 866.3220 - Entamoeba histolytica serological reagents.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Entamoeba histolytica serological reagents. 866.3220 Section 866.3220 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents §...

  12. 21 CFR 866.3220 - Entamoeba histolytica serological reagents.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Entamoeba histolytica serological reagents. 866.3220 Section 866.3220 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents §...

  13. 21 CFR 866.3220 - Entamoeba histolytica serological reagents.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Entamoeba histolytica serological reagents. 866.3220 Section 866.3220 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents §...

  14. 21 CFR 866.3220 - Entamoeba histolytica serological reagents.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Entamoeba histolytica serological reagents. 866.3220 Section 866.3220 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents §...

  15. 21 CFR 866.3220 - Entamoeba histolytica serological reagents.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Entamoeba histolytica serological reagents. 866.3220 Section 866.3220 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents §...

  16. A Sequential Model of Host Cell Killing and Phagocytosis by Entamoeba histolytica

    PubMed Central

    Sateriale, Adam; Huston, Christopher D.

    2011-01-01

    The protozoan parasite Entamoeba histolytica is responsible for invasive intestinal and extraintestinal amebiasis. The virulence of Entamoeba histolytica is strongly correlated with the parasite's capacity to effectively kill and phagocytose host cells. The process by which host cells are killed and phagocytosed follows a sequential model of adherence, cell killing, initiation of phagocytosis, and engulfment. This paper presents recent advances in the cytolytic and phagocytic processes of Entamoeba histolytica in context of the sequential model. PMID:21331284

  17. Photoacoustic spectroscopy of Entamoeba histolytica strains

    NASA Astrophysics Data System (ADS)

    Acosta-Avalos, D.; Alvarado-Gil, J. J.; Silva, E. F.; Orozco, E.; de Menezes, L. F.; Vargas, H.

    2005-06-01

    Pathogenic and non-pathogenic strains of E. histolytica are studied using photoacoustic spectroscopy. It is shown that the pathogenic strain presents a spectrum similar to that of iron sulfur proteins. The non-pathogenic strain does not show any relevant absorption at the studied wavelength range. The differences observed between the optical absorption spectra of both strains opens the possibility of using photoacoustic spectroscopy as a reliable and simple technique to identify different types of E. histolytica strains.

  18. Differential Detection of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii by a Single-Round PCR Assay

    PubMed Central

    Hamzah, Zulhainan; Petmitr, Songsak; Mungthin, Mathirut; Leelayoova, Saovanee; Chavalitshewinkoon-Petmitr, Porntip

    2006-01-01

    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. PMID:16954247

  19. Differentiation of Entamoeba histolytica and Entamoeba dispar in cyst-passers by immunoblot.

    PubMed

    Lee, M; Hong, S T

    1996-12-01

    Differentiation of invasive strains of Entamoeba histolytica according to their pathogenicity has been a topic of long debate, but now the pathogenic species only is regarded as E. histolytica while the non pathogenic species is E. dispar. The present study applied immunoblot to differentiate infections of the two species among microscopically-detected cyst-passers in Korea. The crude extract of E. histolytica separated in 5.20% gradient gels, revealed many fractions of 94, 81, 71, 50, 44, 38.5, 37.5, 29, 19, and 18 kDa when the cysteine proteinase inhibitor, E64, was supplemented. The scrum IgG antibody of 3 proven E, histolytica cases reacted with the antigenic fractions of 117, 110, 99, 68, 66, 60, 54, 52, 46, and 45 kDa. Sera of PCR confirmed 3 cases of E. dispar reacted only to the 117 kDa fraction of the E. histolytica crude extract which was regarded as non-specific. To the antigen of monoxenic E. dispar, sera of E. dispar and E. histolytica cases showed the same immunoblot reactions. The serum IgA antibody reacted with several antigenic fractions of both E. histolytica and E. dispar, but IgM and IgE antibodies showed no reaction to either antigen. Sera of 24 symptomless amebic cyst passers were screened with the E. histolytica antigen; two were found to be infected by E. histolytica and 22 were by E. dispar. The present findings suggest that in Korea most of asymptomatic cyst passers of E. histolytica are carriers of E. dispar. Immunoblot using E. histolytica antigen is a good technique for the differentiation of E. histolytica and E. dispar infections. PMID:9017910

  20. Frequency and molecular characterisation of Entamoeba histolytica, Entamoeba dispar, Entamoeba moshkovskii, and Entamoeba hartmanni in the context of water scarcity in northeastern Brazil.

    PubMed

    Calegar, Deiviane Aparecida; Nunes, Beatriz Coronato; Monteiro, Kerla Joeline Lima; Santos, Jéssica Pereira Dos; Toma, Helena Keiko; Gomes, Tais Ferreira; Lima, Marli Maria; Bóia, Márcio Neves; Carvalho-Costa, Filipe Anibal

    2016-02-01

    This study aimed to estimate the frequency, associated factors, and molecular characterisation of Entamoeba histolytica, Entamoeba dispar, Entamoeba moshkovskii, andEntamoeba hartmanni infections. We performed a survey (n = 213 subjects) to obtain parasitological, sanitation, and sociodemographic data. Faecal samples were processed through flotation and centrifugation methods.E. histolytica, E. dispar, E. moshkovskii, and E. hartmanni were identified by nested-polymerase chain reaction (PCR). The overall prevalence of infection was 22/213 (10.3%). The infection rate among subjects who drink rainwater collected from roofs in tanks was higher than the rate in subjects who drink desalinated water pumped from wells; similarly, the infection rate among subjects who practice open defecation was significantly higher than that of subjects with latrines. Out of the 22 samples positive for morphologically indistinguishableEntamoeba species, the differentiation by PCR was successful for 21. The species distribution was as follows: 57.1% to E. dispar, 23.8% to E. histolytica, 14.3% toE. histolytica and E. dispar, and 4.8% E. dispar and E. hartmanni. These data suggest a high prevalence of asymptomatic infection by the group of morphologically indistinguishable Entamoeba histolytica/dispar/moshkovskiicomplex and E. hartmanni species. In this context of water scarcity, the sanitary and socioenvironmental characteristics of the region appear to favour transmission. PMID:26841049

  1. Frequency and molecular characterisation of Entamoeba histolytica, Entamoeba dispar, Entamoeba moshkovskii, and Entamoeba hartmanni in the context of water scarcity in northeastern Brazil

    PubMed Central

    Calegar, Deiviane Aparecida; Nunes, Beatriz Coronato; Monteiro, Kerla Joeline Lima; dos Santos, Jéssica Pereira; Toma, Helena Keiko; Gomes, Tais Ferreira; Lima, Marli Maria; Bóia, Márcio Neves; Carvalho-Costa, Filipe Anibal

    2016-01-01

    This study aimed to estimate the frequency, associated factors, and molecular characterisation of Entamoeba histolytica, Entamoeba dispar, Entamoeba moshkovskii, andEntamoeba hartmanni infections. We performed a survey (n = 213 subjects) to obtain parasitological, sanitation, and sociodemographic data. Faecal samples were processed through flotation and centrifugation methods.E. histolytica, E. dispar, E. moshkovskii, and E. hartmanni were identified by nested-polymerase chain reaction (PCR). The overall prevalence of infection was 22/213 (10.3%). The infection rate among subjects who drink rainwater collected from roofs in tanks was higher than the rate in subjects who drink desalinated water pumped from wells; similarly, the infection rate among subjects who practice open defecation was significantly higher than that of subjects with latrines. Out of the 22 samples positive for morphologically indistinguishableEntamoeba species, the differentiation by PCR was successful for 21. The species distribution was as follows: 57.1% to E. dispar, 23.8% to E. histolytica, 14.3% toE. histolytica and E. dispar, and 4.8% E. dispar and E. hartmanni. These data suggest a high prevalence of asymptomatic infection by the group of morphologically indistinguishable Entamoeba histolytica/dispar/moshkovskiicomplex and E. hartmanni species. In this context of water scarcity, the sanitary and socioenvironmental characteristics of the region appear to favour transmission. PMID:26841049

  2. PREVALENCE OF Entamoeba histolytica/Entamoeba dispar IN THE CITY OF CAMPINA GRANDE, IN NORTHEASTERN BRAZIL

    PubMed Central

    Silva, Maria Teresa Nascimento; Santana, José Valfrido; Bragagnoli, Gérson; Marinho, Alexandre Magno da Nóbrega; Malagueño, Elizabeth

    2014-01-01

    There is a clear need to perform epidemiological studies to find the true prevalence of Entamoeba histolytica around the world. The evaluation of this prevalence has been hindered by the existence of two different species which are morphologically identical, but genetically different, namely E. histolytica, which causes amebiasis, and E. dispar, which is non-pathogenic. In Brazil, the E. dispar has been detected in communities in the Southeastern (SE) and Northeastern (NE) regions with poor sanitation. However, individuals infected with E. histolytica have been identified in other regions. There is an absence of reports on the prevalence of these parasites in the state of Paraíba, which also has areas with poor sanitary conditions where a high prevalence of the E. histolytica/E. dispar complex has been detected in children from urban slums. The present study evaluated the prevalence of E. histolytica and E. dispar in 1,195 asymptomatic children between two and 10 years of age, living in a sprawling urban slum in Campina Grande, in the state of Paraíba, in Northeastern Brazil. These children were examined and their feces samples were analyzed microscopically. A total of 553 children tested positive for the E. histolytica/E. dispar complex, and 456 of the positive samples were tested with the E. histolytica II® ELISA kit. All 456 samples were negative for the presence of the adhesin E. histolytica specific antigen. The evidence suggests that in this community E. histolytica is absent and E. dispar is the dominant species. PMID:25229229

  3. Amebiasis and comparison of microscopy to ELISA technique in detection of Entamoeba histolytica and Entamoeba dispar.

    PubMed Central

    Nesbitt, Rose A.; Mosha, Franklin W.; Katki, Hormuzd A.; Ashraf, Mohammad; Assenga, Charles; Lee, Clarence M.

    2004-01-01

    The analysis of records of amoebal infection in various hospitals in Kilimanjaro indicated frequent occurrence of amebiasis. The population over the age of five years had higher rate of amoebal infection compared to less than that of a five-year-old population; however, both age groups had similar patterns of amebiasis during January 1999 to June 2001. To investigate misdiagnosis of amebiasis, 226 patients (passive cases) in three hospitals and 616 individuals (active cases) from three different localities in Kilimanjaro were examined. In passive cases, the prevalences of Entamoeba histolytica and Entamoeba dispar were 1% and 7.3%, respectively. Among active cases, 1% were infected with E. histolytica, and 15% were infected with E. dispar. There were no significant differences in amoebal infection between the male and female populations. A pool of 842 stool samples was used for diagnosis of E. histolytica and E. dispar by microscopic examination or ELISA kits. The microscopic examination indicated 8.7% amoebal infection; however, using ELISA as the gold standard, the prevalence of histolytica/dispar was 0.8% and 7.4%, respectively. This study indicated that E. dispar infection was 14.5 times more prevalent than E. histolytica infection. PMID:15160983

  4. MALDI-TOF Mass Spectrometry for the Detection and Differentiation of Entamoeba histolytica and Entamoeba dispar

    PubMed Central

    Calderaro, Adriana; Piergianni, Maddalena; Buttrini, Mirko; Montecchini, Sara; Piccolo, Giovanna; Gorrini, Chiara; Rossi, Sabina; Chezzi, Carlo; Arcangeletti, Maria Cristina; Medici, Maria Cristina; De Conto, Flora

    2015-01-01

    Detection of Entamoeba histolytica and its differentiation from Entamoeba dispar is an important goal of the clinical parasitology laboratory. The aim of this study was the identification and differentiation of E. histolytica and E. dispar by MALDI-TOF MS, in order to evaluate the application of this technique in routine diagnostic practice. MALDI-TOF MS was applied to 3 amebic reference strains and to 14 strains isolated from feces that had been differentiated by molecular methods in our laboratory. Protein extracts from cultures of these strains (axenic cultures for the 3 reference strains and monoxenic cultures for the 14 field isolates) were analyzed by MALDI-TOF MS and the spectra obtained were analyzed by statistical software. Five peaks discriminating between E. histolytica and E. dispar reference strains were found by protein profile analysis: 2 peaks (8,246 and 8,303 Da) specific for E. histolytica and 3 (4,714; 5,541; 8,207 Da) for E. dispar. All clinical isolates except one showed the discriminating peaks expected for the appropriate species. For 2 fecal samples from which 2 strains (1 E. histolytica and 1 E. dispar) out of the 14 included in this study were isolated, the same discriminating peaks found in the corresponding isolated amebic strains were detected after only 12h (E. histolytica) and 24h (E. dispar) of incubation of the fecal samples in Robinson’s medium without serum. Our study shows that MALDI-TOF MS can be used to discriminate between E. histolytica and E. dispar using in vitro xenic cultures and it also could have potential for the detection of these species in clinical samples. PMID:25874612

  5. Detection of Entamoeba histolytica by Recombinase Polymerase Amplification.

    PubMed

    Nair, Gayatri; Rebolledo, Mauricio; White, A Clinton; Crannell, Zachary; Richards-Kortum, R Rebecca; Pinilla, A Elizabeth; Ramírez, Juan David; López, M Consuelo; Castellanos-Gonzalez, Alejandro

    2015-09-01

    Amebiasis is an important cause of diarrheal disease worldwide and has been associated with childhood malnutrition. Traditional microscopy approaches are neither sensitive nor specific for Entamoeba histolytica. Antigen assays are more specific, but many cases are missed unless tested by molecular methods. Although polymerase chain reaction (PCR) is effective, the need for sophisticated, expensive equipment, infrastructure, and trained personnel limits its usefulness, especially in the resource-limited, endemic areas. Here, we report development of a recombinase polymerase amplification (RPA) method to detect E. histolytica specifically. Using visual detection by lateral flow (LF), the test was highly sensitive and specific and could be performed without additional equipment. The availability of this inexpensive, sensitive, and field-applicable diagnostic test could facilitate rapid diagnosis and treatment of amebiasis in endemic regions. PMID:26123960

  6. Detection of Entamoeba histolytica by Recombinase Polymerase Amplification

    PubMed Central

    Nair, Gayatri; Rebolledo, Mauricio; White, A. Clinton; Crannell, Zachary; Richards-Kortum, R. Rebecca; Pinilla, A. Elizabeth; Ramírez, Juan David; López, M. Consuelo; Castellanos-Gonzalez, Alejandro

    2015-01-01

    Amebiasis is an important cause of diarrheal disease worldwide and has been associated with childhood malnutrition. Traditional microscopy approaches are neither sensitive nor specific for Entamoeba histolytica. Antigen assays are more specific, but many cases are missed unless tested by molecular methods. Although polymerase chain reaction (PCR) is effective, the need for sophisticated, expensive equipment, infrastructure, and trained personnel limits its usefulness, especially in the resource-limited, endemic areas. Here, we report development of a recombinase polymerase amplification (RPA) method to detect E. histolytica specifically. Using visual detection by lateral flow (LF), the test was highly sensitive and specific and could be performed without additional equipment. The availability of this inexpensive, sensitive, and field-applicable diagnostic test could facilitate rapid diagnosis and treatment of amebiasis in endemic regions. PMID:26123960

  7. Differentiating Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii using nested polymerase chain reaction (PCR) in rural communities in Malaysia

    PubMed Central

    2012-01-01

    Background In this study, a total of 426 human faecal samples were examined for the presence of Entamoeba histolytica, Entamoeba dispar, Entamoeba moshkovskii infection via a combination of microscopic examination and nested polymerase chain reaction (PCR) targeting 16S ribosomal RNA of Entamoeba species. Methods Faecal sample were collected from 426 participants in five rural villages in Peninsular Malaysia. The faecal samples were processed by direct wet smear and formalin ethyl acetate concentration technique followed by iodine staining and examined via microscopy for the presence of Entamoeba species and other intestinal parasites. Microscopically positive samples for Entamoeba species cysts were further characterized using a Nested Polymerase Chain Reaction (Nested-PCR) targeting 16S-like ribosomal RNA gene. The data entry and analysis was carried out using the SPSS software (Statistical Package for the Social Sciences) program for Windows version 17 (SPSS, Chicago, IL, USA). Results Based on single faecal examination, overall prevalence of Entamoeba infection was 17.6% (75/426). Females (19.1%) were more commonly infected compared to males (15.9%). Comparison by age groups showed that adults (23.9%) had higher infection rates than children (15.3%). The PCR results showed that 52 out of 75 microscopy positive samples successfully generated species-specific amplicons. The infection with E. histolytica (75.0%; 39/52) was the most common, followed by E. dispar (30.8%; 18/52) and E. moshkovskii (5.8%; 3/52). Of these, 33 (63.5%) were shown to contain only E. histolytica, 10 (19.2%) contained E. dispar and 3 (5.8%) contained only E. moshkovskii. Mixed infection with E. histolytica and E. dispar was found in 6 (11.5%) samples. Conclusions The present study essentially emphasized the benefit of molecular techniques in discriminating the pathogenic Entamoeba species from the non-pathogenic for accurate diagnosis and better management of amoebiasis. The presence of E

  8. Entamoeba histolytica Trophozoites and Lipopeptidophosphoglycan Trigger Human Neutrophil Extracellular Traps.

    PubMed

    Ávila, Eva E; Salaiza, Norma; Pulido, Julieta; Rodríguez, Mayra C; Díaz-Godínez, César; Laclette, Juan P; Becker, Ingeborg; Carrero, Julio C

    2016-01-01

    Neutrophil defense mechanisms include phagocytosis, degranulation and the formation of extracellular traps (NET). These networks of DNA are triggered by several immune and microbial factors, representing a defense strategy to prevent microbial spread by trapping/killing pathogens. This may be important against Entamoeba histolytica, since its large size hinders its phagocytosis. The aim of this study was to determine whether E. histolytica and their lipopeptidophosphoglycan (EhLPPG) induce the formation of NETs and the outcome of their interaction with the parasite. Our data show that live amoebae and EhLPPG, but not fixed trophozoites, induced NET formation in a time and dose dependent manner, starting at 5 min of co-incubation. Although immunofluorescence studies showed that the NETs contain cathelicidin LL-37 in close proximity to amoebae, the trophozoite growth was only affected when ethylene glycol tetra-acetic acid (EGTA) was present during contact with NETs, suggesting that the activity of enzymes requiring calcium, such as DNases, may be important for amoeba survival. In conclusion, E. histolytica trophozoites and EhLPPG induce in vitro formation of human NETs, which did not affect the parasite growth unless a chelating agent was present. These results suggest that NETs may be an important factor of the innate immune response during infection with E. histolytica. PMID:27415627

  9. Entamoeba histolytica induces human neutrophils to form NETs.

    PubMed

    Ventura-Juarez, J; Campos-Esparza, Mr; Pacheco-Yepez, J; López-Blanco, J A; Adabache-Ortíz, A; Silva-Briano, M; Campos-Rodríguez, R

    2016-08-01

    Entamoeba histolytica invades the intestine and other organs during the pathogenesis of amoebiasis. In the early stages, the host organism responds with an inflammatory infiltrate composed mostly of neutrophils. It has been reported that these immune cells, activated by E. histolytica, exert a protective role by releasing proteolytic enzymes and generating reactive oxygen/nitrogen species (ROS/RNS) and antimicrobial peptides. It is now known that neutrophils also produce neutrophil extracellular traps (NETs), which are able to damage and kill pathogens. Studies have shown that intracellular protozoan pathogens, including Toxoplasma gondi, Plasmodium falciparum and Leishmania spp, induce neutrophils to release NETs and are damaged by them. However, the action of this mechanism has not been explored in relation to E. histolytica trophozoites. Through scanning electron, epifluorescence microscopy and viability assays, we show for first time that during in vitro interaction with E. histolytica trophozoites, human neutrophils released NETs that covered amoebas and reduced amoebic viability. These NETs presented histones, myeloperoxidase and decondensed chromatin. The results suggest that NETs participate in the elimination of the parasite. PMID:27138813

  10. G protein signaling in the parasite Entamoeba histolytica

    PubMed Central

    Bosch, Dustin E; Siderovski, David P

    2013-01-01

    The parasite Entamoeba histolytica causes amebic colitis and systemic amebiasis. Among the known amebic factors contributing to pathogenesis are signaling pathways involving heterotrimeric and Ras superfamily G proteins. Here, we review the current knowledge of the roles of heterotrimeric G protein subunits, Ras, Rho and Rab GTPase families in E. histolytica pathogenesis, as well as of their downstream signaling effectors and nucleotide cycle regulators. Heterotrimeric G protein signaling likely modulates amebic motility and attachment to and killing of host cells, in part through activation of an RGS-RhoGEF (regulator of G protein signaling–Rho guanine nucleotide exchange factor) effector. Rho family GTPases, as well as RhoGEFs and Rho effectors (formins and p21-activated kinases) regulate the dynamic actin cytoskeleton of E. histolytica and associated pathogenesis-related cellular processes, such as migration, invasion, phagocytosis and evasion of the host immune response by surface receptor capping. A remarkably large family of 91 Rab GTPases has multiple roles in a complex amebic vesicular trafficking system required for phagocytosis and pinocytosis and secretion of known virulence factors, such as amebapores and cysteine proteases. Although much remains to be discovered, recent studies of G protein signaling in E. histolytica have enhanced our understanding of parasitic pathogenesis and have also highlighted possible targets for pharmacological manipulation. PMID:23519208

  11. Entamoeba histolytica Trophozoites and Lipopeptidophosphoglycan Trigger Human Neutrophil Extracellular Traps

    PubMed Central

    Ávila, Eva E.; Rodríguez, Mayra C.; Díaz-Godínez, César; Laclette, Juan P.; Becker, Ingeborg; Carrero, Julio C.

    2016-01-01

    Neutrophil defense mechanisms include phagocytosis, degranulation and the formation of extracellular traps (NET). These networks of DNA are triggered by several immune and microbial factors, representing a defense strategy to prevent microbial spread by trapping/killing pathogens. This may be important against Entamoeba histolytica, since its large size hinders its phagocytosis. The aim of this study was to determine whether E. histolytica and their lipopeptidophosphoglycan (EhLPPG) induce the formation of NETs and the outcome of their interaction with the parasite. Our data show that live amoebae and EhLPPG, but not fixed trophozoites, induced NET formation in a time and dose dependent manner, starting at 5 min of co-incubation. Although immunofluorescence studies showed that the NETs contain cathelicidin LL-37 in close proximity to amoebae, the trophozoite growth was only affected when ethylene glycol tetra-acetic acid (EGTA) was present during contact with NETs, suggesting that the activity of enzymes requiring calcium, such as DNases, may be important for amoeba survival. In conclusion, E. histolytica trophozoites and EhLPPG induce in vitro formation of human NETs, which did not affect the parasite growth unless a chelating agent was present. These results suggest that NETs may be an important factor of the innate immune response during infection with E. histolytica. PMID:27415627

  12. Appearance of sialoglycoproteins in encysting cells of Entamoeba histolytica.

    PubMed Central

    Chayen, A; Avron, B; Nuchamowitz, Y; Mirelman, D

    1988-01-01

    Amoeba-bacterium cultures of Entamoeba histolytica transferred to a hypoosmotic medium depleted of nutrients changed morphologically and biochemically. The cells ejected grains of rice starch, rounded up, and formed a distinct cell wall that was resistant to detergent, bound the sialic acid-specific lectin from Limulus polyphemus, and became fluorescent with Calcofluor M2R. A subpopulation of these cells displayed more than one nucleus. All these signs are characteristic of encysting cells and were also observed in cysts obtained from a human patient. The morphological changes were accompanied by the appearance of two new glycoproteins with apparent molecular sizes of 100 and 150 kilodaltons which contained sialic acid. Sialic acid has been reported to be absent from trophozoites of Entamoeba species. The presence of this sugar residue on cyst-specific proteins parallels recently reported findings during the encystation of the related reptilian parasite Entamoeba invadens. This may indicate a basic role for sialic acid in the encystation of Entamoeba parasites. Images PMID:2893775

  13. Copro prevalence and estimated risk of Entamoeba histolytica in Diarrheic patients at Beni-Suef, Egypt.

    PubMed

    Ibrahim, Shimaa S; El-Matarawy, Olfat M; Ghieth, Marwa A; Abu Sarea, Enas Y; El-Badry, Ayman A

    2015-02-01

    Amoebiasis diagnosis is usually based on microscopy that cannot differentiate pathogenic E. histolytica from morphologically identical non-pathogenic species. 194 fecal samples were collected from diarrheic &/or dysenteric patients and examined for Entamoeba complex microscopically, E. histolytica/E. dispar coproantigen using ICT and E. histolytica coproantigen using Tech lab E. histolytica II ELISA test. Entamoeba complex trophozoites/cysts, E. histolytica/E. dispar coproantigen and E. histolytica coproantigen were detected in 22.2, 14.4 and 3.6 % of samples, respectively. Microscopy and ICT method had limited sensitivity with poor PPV (9.3 and 7.1 %, respectively) and both slightly agree with ELISA test. The prevalence of E. histolytica was low (3.6 %) in studied individuals and was 14 times lower than non-pathogenic amoebae. E. histolytica detection studied individuals was positively associated with mucoid and bloody stool, which makes them disease predictors. E. histolytica fecal ELISA assay for E. histolytica detection surpassed microscopy and E. histolytica/E. dispar ICT assay. This has highlighted the need for practical non-microscopic detection methods that can differentiate between amoeba infections to avoid unnecessary and possibly harmful therapies and to determine the true prevalence and epidemiology of E. histolytica. PMID:25542044

  14. Development of Multiplex Real-Time Polymerase Chain Reaction for Detection of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii in Clinical Specimens

    PubMed Central

    Hamzah, Zulhainan; Petmitr, Songsak; Mungthin, Mathirut; Leelayoova, Saovanee; Chavalitshewinkoon-Petmitr, Porntip

    2010-01-01

    Multiplex real-time polymerase chain reaction (PCR) was developed for differential detection of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii. Specific primers were designed for all three species, and then differentiation of E. histolytica and E. dispar was achieved simultaneously using a hybridization probe and melting curve analysis, whereas E. moshkovskii was detected with a separate probe under the same condition. This assay detected as little as 0.2 pg of E. histolytica DNA and 2 pg each for E. dispar and E. moshkovskii DNA. Thirty-five clinical samples suspected to be E. histolytica infection by microscopy were tested. The results showed 32 positive samples; four samples were E. histolytica and 28 samples were E. dispar. Interestingly, one E. dispar positive sample showed a mixed infection with E. moshkovskii. This is the first report of E. moshkovskii infection from Thailand and this assay is currently the most rapid and sensitive method to differentiate these human amoebas. PMID:20889890

  15. Identification and functional characterization of lysine methyltransferases of Entamoeba histolytica.

    PubMed

    Borbolla-Vázquez, Jessica; Orozco, Esther; Medina-Gómez, Christian; Martínez-Higuera, Aarón; Javier-Reyna, Rosario; Chávez, Bibiana; Betanzos, Abigail; Rodríguez, Mario A

    2016-07-01

    Lysine methylation of histones, a posttranslational modification catalyzed by lysine methyltransferases (HKMTs), plays an important role in the epigenetic regulation of transcription. Lysine methylation of non-histone proteins also impacts the biological function of proteins. Previously it has been shown that lysine methylation of histones of Entamoeba histolytica, the protozoan parasite that infects 50 million people worldwide each year and causing up to 100,000 deaths annually, is implicated in the epigenetic machinery of this microorganism. However, the identification and characterization of HKMTs in this parasite had not yet been determined. In this work we identified four HKMTs in E. histolytica (EhHKMT1 to EhHKMT4) that are expressed by trophozoites. Enzymatic assays indicated that all of them are able to transfer methyl groups to commercial histones. EhHKMT1, EhHKMT2 and EhHKMT4 were detected in nucleus and cytoplasm of trophozoites. In addition EhHKMT2 and EhHKMT4 were located in vesicles containing ingested cells during phagocytosis, and they co-immunoprecipitated with EhADH, a protein involved in the phagocytosis of this parasite. Results suggest that E. histolytica uses its HKMTs to regulate transcription by epigenetic mechanisms, and at least two of them could also be implicated in methylation of proteins that participate in phagocytosis. PMID:27062489

  16. Immune Response of Amebiasis and Immune Evasion by Entamoeba histolytica

    PubMed Central

    Nakada-Tsukui, Kumiko; Nozaki, Tomoyoshi

    2016-01-01

    Entamoeba histolytica is a protozoan parasite and the causative agent of amebiasis. It is estimated approximately 1% of humans are infected with E. histolytica, resulting in an estimate of 100,000 deaths annually. Clinical manifestations of amebic infection range widely from asymptomatic to severe symptoms, including dysentery and extra-intestinal abscesses. Like other infectious diseases, it is assumed that only ~20% of infected individuals develop symptoms, and genetic factors of both the parasite and humans as well as the environmental factors, e.g., microbiota, determine outcome of infection. There are multiple essential steps in amebic infection: degradation of and invasion into the mucosal layer, adherence to the intestinal epithelium, invasion into the tissues, and dissemination to other organs. While the mechanisms of invasion and destruction of the host tissues by the amebae during infection have been elucidated at the molecular levels, it remains largely uncharacterized how the parasite survive in the host by evading and attacking host immune system. Recently, the strategies for immune evasion by the parasite have been unraveled, including immunomodulation to suppress IFN-γ production, elimination of immune cells and soluble immune mediators, and metabolic alterations against reactive oxygen and nitrogen species to fend off the attack from immune system. In this review, we summarized the latest knowledge on immune reaction and immune evasion during amebiasis. PMID:27242782

  17. Immune Response of Amebiasis and Immune Evasion by Entamoeba histolytica.

    PubMed

    Nakada-Tsukui, Kumiko; Nozaki, Tomoyoshi

    2016-01-01

    Entamoeba histolytica is a protozoan parasite and the causative agent of amebiasis. It is estimated approximately 1% of humans are infected with E. histolytica, resulting in an estimate of 100,000 deaths annually. Clinical manifestations of amebic infection range widely from asymptomatic to severe symptoms, including dysentery and extra-intestinal abscesses. Like other infectious diseases, it is assumed that only ~20% of infected individuals develop symptoms, and genetic factors of both the parasite and humans as well as the environmental factors, e.g., microbiota, determine outcome of infection. There are multiple essential steps in amebic infection: degradation of and invasion into the mucosal layer, adherence to the intestinal epithelium, invasion into the tissues, and dissemination to other organs. While the mechanisms of invasion and destruction of the host tissues by the amebae during infection have been elucidated at the molecular levels, it remains largely uncharacterized how the parasite survive in the host by evading and attacking host immune system. Recently, the strategies for immune evasion by the parasite have been unraveled, including immunomodulation to suppress IFN-γ production, elimination of immune cells and soluble immune mediators, and metabolic alterations against reactive oxygen and nitrogen species to fend off the attack from immune system. In this review, we summarized the latest knowledge on immune reaction and immune evasion during amebiasis. PMID:27242782

  18. A membrane-associated neuraminidase in Entamoeba histolytica trophozoites.

    PubMed Central

    Udezulu, I A; Leitch, G J

    1987-01-01

    Trophozoites of the parasitic amoeba Entamoeba histolytica HM-1:IMSS possess a surface neuraminidase capable of liberating N-acetylneuraminic acid (NANA) from N-acetylneuramin-lactose (alpha 2----3 or alpha 2----6) or mucin in their medium. The neuraminidase was found to be membrane associated, with more than 50% of the yield being recovered in the plasma membrane fraction. The neuraminidase specific activity of the plasma membrane fraction was six times that of internal membrane fraction enzyme. The optimum pH and temperature for this enzyme were 6.7 and 37 degrees C, respectively. Neuraminidase activity was inhibited by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and the optimum Ca2+ concentration was 2 mM. The microfilament disruptor cytochalasin D (30 micrograms/ml) inhibited motility and neuraminidase activity of intact Entamoeba trophozoites. The cytochalasin D-induced loss of surface neuraminidase activity was explained in part by a redistribution of enzyme with a loss of plasma membrane enzyme and an increase in intracellular membrane enzyme. A qualitatively similar cytochalasin D effect was observed with two other membrane-associated enzymes, calcium-regulated ATPase and acid phosphatase. Membrane-associated enzyme was minimally affected by Triton X-100 and saponin. An N-acetylneuraminic acid aldolase, optimum pH, 7.4, was found in trophozoite homogenate supernatant fractions. NANA and NANA-containing compounds stimulated trophozoite-directed motility. This motility stimulation by NANA-containing compounds did not apparently require prior release of free NANA by the trophozoite surface neuraminidase. Entamoeba neuraminidase is one of a series of enzymes that may modify the mucus blanket and target cell surface and thereby play a role in the pathogenesis of amebiasis. PMID:2878886

  19. Bioconjugated fluorescent silica nanoparticles for the rapid detection of Entamoeba histolytica.

    PubMed

    Hemadi, Ahmad; Ekrami, Alireza; Oormazdi, Hormozd; Meamar, Ahmad Reza; Akhlaghi, Lame; Samarbaf-Zadeh, Ali Reza; Razmjou, Elham

    2015-05-01

    Rapid detection of Entamoeba histolytica based on fluorescent silica nanoparticle (FSNP) indirect immunofluorescence microscopy was evaluated. Silica nanoparticles were synthesized using Stöber's method, with their surface activated to covalently bind to, and immobilize, protein A. For biolabeling, FSNP was added to conjugated E. histolytica trophozoites with monoclonal anti-E. histolytica IgG1 for microscopic observation of fluorescence. Fluorescent silica nanoparticle sensitivity was determined with axenically cultured E. histolytica serially diluted to seven concentrations. Specificity was evaluated using other intestinal protozoa. Fluorescent silica nanoparticles detected E. histolytica at the lowest tested concentration with no cross-reaction with Entamoeba dispar, Entamoeba moshkovskii, Blastocystis sp., or Giardia lamblia. Visualization of E. histolytica trophozoites with anti-E. histolytica antibody labeled with fluorescein isothiocyanate (FITC) was compared with that using anti-E. histolytica antibody bioconjugated FSNP. Although FITC and FSNP produced similar results, the amount of specific antibody required for FITC to induce fluorescence of similar intensity was fivefold that for FSNP. Fluorescent silica nanoparticles delivered a rapid, simple, cost-effective, and highly sensitive and specific method of detecting E. histolytica. Further study is needed before introducing FSNP for laboratory diagnosis of amoebiasis. PMID:25697865

  20. Different Clinical Outcomes of Entamoeba histolytica in Malaysia: Does Genetic Diversity Exist?

    PubMed Central

    Anuar, Tengku Shahrul; Al-Mekhlafi, Hesham M.; Abdul Ghani, Mohamed Kamel; Azreen, Siti Nor; Salleh, Fatmah Md; Ghazali, Nuraffini; Bernadus, Mekadina

    2013-01-01

    The present study was conducted to investigate the clinical outcomes of Entamoeba histolytica infection in symptomatic and asymptomatic Orang Asli (aborigine) communities in Malaysia. Examination was performed on 500 stool samples obtained from Orang Asli communities in 3 different states using formalin-ether concentration, trichrome staining, and single-round PCR techniques. Out of 500 stool samples, single infection of E. histolytica, Entamoeba dispar, and Entamoeba moshkovskii was identified in 3.2%, 13.4%, and 1%, respectively. In addition, 10 samples had mixed infections with E. histolytica and E. dispar. Six samples containing E. dispar were also positive for E. moshkovskii, and only 2 samples had E. histolytica in association with E. dispar and E. moshkovskii. Seventeen E. histolytica-positive samples were from symptomatic subjects, whereas the remaining 11 samples came from asymptomatic subjects. These findings suggest a predominant distribution of pathogenic potential of E. histolytica strains in this community. Therefore, further studies on genotyping of E. histolytica is required, to find out association between E. histolytica genotype and the outcome of the infection. PMID:23710093

  1. Development of monoclonal antibodies specifically recognizing the cyst stage of Entamoeba histolytica.

    PubMed

    Walderich, B; Burchard, G D; Knobloch, J; Müller, L

    1998-09-01

    Protozoan cysts were isolated according to a two-step sucrose gradient procedure. Pure cysts of Entamoeba histolytica, in fixed and native states, were injected into BALB/c mice intraperitoneally for immunization. The spleens of these animals were used for fusion with AG8 mouse myeloma cells. Hybridomas were obtained and tested for the recognition of E. histolytica, E. dispar, E. coli, E. hartmanni, Endolimax nana, Jodamoeba biitschlii, and Giardia lamblia. Three monoclonal antibodies were identified that reacted only with cysts and trophozoites of E. histolytica. These can be used for differentiation and identification of E. histolytica in feces. PMID:9749623

  2. Comparison of Various Methods in the Diagnosis of Entamoeba histolytica in Stool and Serum Specimens

    PubMed Central

    Uslu, Hakan; Aktas, Osman; Uyanik, Muhammet Hamidullah

    2016-01-01

    Objective: Entamoeba histolytica is indistinguishable from Entamoeba dispar in direct microscopic examination. A definitive diagnosis of E. histolytica is important in terms of the treatment of the patient and to avoid unnecessary costs. This study’s aim is to determine the prevalence of E. histolytica and to make a comparison of the different diagnostic tests in the patients specimens defined as E. histolytica/E. dispar infection. Materials and Methods: Faecal and serum specimens of 90 patients defined as E. histolytica/E. dispar with microscopy (wet mount examination with 0.85% saline and Lugol’s iodine) were examined. Stool samples were examined by trichrome staining for trophozoites and cysts and by immunoassay methods for specific adhesin antigens (Wampole ® E. histolytica II antigen testing) and for specific serine-rich 30 kD membrane protein (Serazym® E. histolytica antigen testing). Anti-E. histolytica antibodies were investigated using a latex slide test and indirect hemagglutination methods in serum specimens. Results: Presence of E. histolytica was not confirmed in 31.1% cases with trichrome staining, 62.2% of the Wampole antigen test, 64.4%, of the Serazym antigen test, 73.3% of the indirect hemagglutination test and 75.6%. of the latex agglutination. Considering the common results from Wampole and Serazym antigen testing as a reference standard, the specificity/sensitivity is 100/53.85% for trichrome staining, 75.00/98.11% for the latex agglutination test and 78.57/96.77% for the indirect hemagglutination test. Conclusion: It has been shown that investigation of E. histolytica in stools by direct wet-smear microscopy alone can cause significant false positive results. To obtain a reliable diagnosis for E. histolytica and to avoid unnecessary treatment for this parasite, at least one more specific assay, particularly an antigen testing and microscopy, is required. PMID:27551176

  3. Antigens in electron-dense granules from Entamoeba histolytica as possible markers for pathogenicity.

    PubMed Central

    Muñoz, M L; Lamoyi, E; León, G; Tovar, R; Pérez-García, J; De La Torre, M; Murueta, E; Bernal, R M

    1990-01-01

    In vitro interaction of Entamoeba histolytica with collagen induces intracellular formation and release of electron-dense granules (EDG) and stimulation of collagenolytic activity. Purified EDG contain 1.66 U of collagenase per mg of protein. Thus, EDG may participate in tissue destruction during invasive amebiasis. Monoclonal antibodies (MAbs) L1.1 and L7.1 reacted specifically with EDG in enzyme-linked immunosorbent assay (ELISA) and immunofluorescence and immunoelectron microscopy. MAb L7.1 immunoprecipitated three polypeptides with molecular weights of 95,000, 68,000, and 28,000 from lysates of biosynthetically labeled E. histolytica. Both MAbs recognized the pathogenic E. histolytica axenic strains HM1:IMSS, HM38:IMSS, and HK-9 but failed to react in ELISA with Entamoeba moshkovskii, Entamoeba invadens, and E. histolytica-like Laredo. In addition, MAb L7.1 reacted with one E. histolytica isolate from a symptomatic patient but did not react with four of five isolates from asymptomatic patients. EDG antigens were detected by a MAb L7.1-based ELISA in E. histolytica-containing fecal samples from symptomatic, but not asymptomatic, individuals. These results suggest that the EDG antigen detected with MAb L7.1 may be differentially expressed in pathogenic and nonpathogenic E. histolytica. Images PMID:2174899

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

    PubMed

    Tachibana, H; Cheng, X J; Kobayashi, S; Matsubayashi, N; Gotoh, S; Matsubayashi, K

    2001-01-01

    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. PMID:11199843

  5. Mutation detection analysis of a region of 16S-like ribosomal RNA gene of Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii

    PubMed Central

    Parija, Subhash Chandra; Khairnar, Krishna

    2008-01-01

    Background The level of intra-species genetic variation in Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii populations in a localized geographic area, like Puducherry, India, remains unknown. Methods In the present study the existence of genetic variation in the nested multiplex polymerase chain reaction (NM-PCR) amplified region of the 16S-like ribosomal RNA genes of E. histolytica, E. dispar and E. moshkovskii was investigated by riboprinting and single strand conformation polymorphism (SSCP) analysis. Results We found that 70 stool specimens were positive for E. histolytica, 171 stool specimens were positive for E. dispar, and 37 stool specimens were positive for E. moshkovskii by NM-PCR. Ninety liver abscess pus specimens, 21 urine specimens, and 8 saliva specimens were positive for E. histolytica by NM-PCR. Riboprinting analysis detected a mutation in the PCR product of only one E. histolytica isolate from a stool specimen. However, SSCP analysis detected mutations in the PCR products of five E. histolytica isolates and three E. moshkovskii isolates from stool specimens, and one E. histolytica isolate from a saliva specimen. The mutations detected by riboprinting and SSCP analysis were confirmed by sequencing. All the nucleotide sequences showing mutations in this study have already been deposited into the NCBI GenBank database under accession numbers [GenBank: EF682200 to GenBank: EF682208]. Conclusion The present study has revealed the subsistence of mutations in the ribosomal RNA genes of E. histolytica and E. moshkovskii, which points towards the existence of intra-species genetic variation in E. histolytica and E. moshkovskii isolates infecting humans. PMID:18822136

  6. Real-time PCR assay in differentiating Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii infections in Orang Asli settlements in Malaysia

    PubMed Central

    2013-01-01

    Background Amebiasis caused by Entamoeba histolytica is the third leading cause of death worldwide. This pathogenic amoeba is morphologically indistinguishable from E. dispar and E. moshkovskii, the non-pathogenic species. Polymerase chain reaction is the current method of choice approved by World Health Organization. Real-time PCR is another attractive molecular method for diagnosis of infectious diseases as post-PCR analyses are eliminated and turnaround times are shorter. The present work aimed to compare the results of Entamoeba species identification using the real-time assay against the established nested PCR method. Methods In this study, a total of 334 human faecal samples were collected from different Orang Asli settlements. Faecal samples were processed by direct wet smear and formalin ethyl acetate concentration methods followed by iodine staining and was microscopically examined for Entamoeba species and other intestinal parasites. Microscopically positive samples were then subject to nested PCR and real-time PCR. Results The overall prevalence of Entamoeba infection was 19.5% (65/334). SK Posh Piah recorded highest Entamoeba prevalence (63.3%) while Kampung Kemensah had the lowest prevalence (3.7%) of Entamoeba. Microscopically positive samples were then tested by real-time PCR and nested PCR for the presence of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii infection. Real-time PCR showed higher Entamoeba detection (86.2%) compared to nested PCR (80%), although the McNemar test value showed no significant difference between the two methods (p = 0.221). Conclusions This study is the first in Malaysia to report the use of real-time PCR in identifying and differentiating the three Entamoeba infections. It is also proven to be more effective compared to the conventional nested PCR molecular method. PMID:23985047

  7. An enzyme-linked immunosorbent assay for the detection of Entamoeba histolytica antigens in faecal material.

    PubMed

    Grundy, M S; Voller, A; Warhurst, D

    1987-01-01

    This paper describes a method for the detection of Entamoeba histolytica antigens in stool samples using a multi-layer ELISA. The method is sensitive and specific, showing no interference with other intestinal parasites, e.g. E. coli, E. hartmanni, Endolimax nana, Iodamoeba buetschlii, Hymenolepis nana, Giardia lamblia, Trichomonas and Ascaris. The method provides a rapid and simple screening assay for E. histolytica infections and should assist in diagnosis and epidemiological studies of the disease. PMID:2895514

  8. Real-Time PCR for Detection and Differentiation of Entamoeba histolytica and Entamoeba dispar in Fecal Samples

    PubMed Central

    Blessmann, Joerg; Buss, Heidrun; Nu, Phuong A. Ton; Dinh, Binh T.; Ngo, Quynh T. Viet; Van, An Le; Abd Alla, Mohamed D.; Jackson, Terry F. H. G.; Ravdin, Jonathan I.; Tannich, Egbert

    2002-01-01

    A closed-tube, real-time PCR assay was developed for sensitive and specific detection and differentiation of the two closely related intestinal protozoan parasites Entamoeba histolytica and Entamoeba dispar directly from human feces. The assay is performed with the LightCycler system using fluorescence-labeled detection probes and primers amplifying a 310-bp fragment from the high-copy-number, ribosomal DNA-containing ameba episome. The assay was able to detect as little as 0.1 parasite per g of feces. The two pairs of primers used were specific for the respective ameba species, and results were not influenced by the presence of other Entamoeba species even when present in exceeding amounts. PCR was evaluated using several hundred stool samples from areas of amebiasis endemicity in Vietnam and South Africa, and results were compared with those of microscopy and ameba culture. PCR was found to be significantly more sensitive than microscopy or culture, as all samples positive by microscopy and 22 out of 25 (88%) samples positive by culture were also positive by PCR, but PCR revealed a considerable number of additional E. histolytica- or E. dispar-positive samples. Compared to culture and subsequent ameba differentiation by isoenzyme analysis, PCR was 100% specific for each of the two Entamoeba species. Interestingly, the comparison with PCR revealed that culture, in particular, underestimates E. histolytica infections. Given the high sensitivity and specificity of the developed PCR assay, the inability of microscopy to distinguish between the two ameba species, and the time it takes to culture and subsequently differentiate entamoebae by isoenzyme analysis, this assay is more suitable than microscopy or culture to correctly diagnose intestinal E. histolytica or E. dispar infection. PMID:12454128

  9. Serosurvey of Entamoeba Histolytica Exposure among Tepehuanos Population in Durango, Mexico

    PubMed Central

    Alvarado-Esquivel, Cosme; Hernández-Tinoco, Jesús; Francisco Sánchez-Anguiano, Luis; Ramos-Nevárez, Agar; Margarita Cerrillo-Soto, Sandra; Alberto Guido-Arreola, Carlos

    2015-01-01

    The seroepidemiology of Entamoeba histolytica infection in Tepehuanos population in Mexico is largely unknown. This study aimed to study the seroprevalence and correlates of E. histolytica antibodies in Tepehuanos in Durango, Mexico. Through a cross-sectional study, we determined the frequency of E. histolytica IgG antibodies in 156 Tepehuanos people in Durango, Mexico using an enzyme-linked immunoassay. Furthermore, we studied the association of E. histolytica seroprevalence with the socio-demographic, clinical, and behavioral characteristics of the Tepehuanos studied. Forty-four (28.2%) Tepehuanos with mean age of 31.03 ± 16.71 years old had anti- E. histolytica IgG antibodies. Multivariate analysis showed that E. histolytica exposure was positively associated with laborer occupation (Odds ratio=2.77; 95% CI: 1.15, 6.66; p=0.02), and history of lymphadenopathy (Odds ratio=4.97; 95% CI: 1.74, 14.13; p=0.002), and negatively associated with soil contact (Odds ratio=0.13; 95% CI: 0.03, 0.53; p=0.004). Other behavioral characteristics including drinking untreated water or unpasteurized milk, and consumption of unwashed raw vegetables or fruits were not associated with E. histolytica exposure. The seroprevalence of E. histolytica infection in Tepehuanos in Durango is higher thanseroprevalences reported in national surveys. The factors associated with E. histolytica seropositivity reported in the present study might aid for the planning and implementation of effective measures against E. histolytica infection. PMID:26199578

  10. Knockdown of Five Genes Encoding Uncharacterized Proteins Inhibits Entamoeba histolytica Phagocytosis of Dead Host Cells.

    PubMed

    Sateriale, Adam; Miller, Peter; Huston, Christopher D

    2016-04-01

    Entamoeba histolytica is the protozoan parasite that causes invasive amebiasis, which is endemic to many developing countries and characterized by dysentery and liver abscesses. The virulence of E. histolytica correlates with the degree of host cell engulfment, or phagocytosis, and E. histolytica phagocytosis alters amebic gene expression in a feed-forward manner that results in an increased phagocytic ability. Here, we used a streamlined RNA interference screen to silence the expression of 15 genes whose expression was upregulated in phagocytic E. histolytica trophozoites to determine whether these genes actually function in the phagocytic process. When five of these genes were silenced, amebic strains with significant decreases in the ability to phagocytose apoptotic host cells were produced. Phagocytosis of live host cells, however, was largely unchanged, and the defects were surprisingly specific for phagocytosis. Two of the five encoded proteins, which we named E. histolytica ILWEQ (EhILWEQ) and E. histolytica BAR (EhBAR), were chosen for localization via SNAP tag labeling and localized to the site of partially formed phagosomes. Therefore, both EhILWEQ and EhBAR appear to contribute to E. histolytica virulence through their function in phagocytosis, and the large proportion (5/15 [33%]) of gene-silenced strains with a reduced ability to phagocytose host cells validates the previously published microarray data set demonstrating feed-forward control of E. histolytica phagocytosis. Finally, although only limited conclusions can be drawn from studies using the virulence-deficient G3 Entamoeba strain, the relative specificity of the defects induced for phagocytosis of apoptotic cells but not healthy cells suggests that cell killing may play a rate-limiting role in the process of Entamoeba histolytica host cell engulfment. PMID:26810036

  11. Characterization of the protein tyrosine phosphatase PRL from Entamoeba histolytica.

    PubMed

    Ramírez-Tapia, Ana Lilia; Baylón-Pacheco, Lidia; Espíritu-Gordillo, Patricia; Rosales-Encina, José Luis

    2015-12-01

    Protein tyrosine phosphatase of regenerating liver (PRL) is a group of phosphatases that has not been broadly studied in protozoan parasites. In humans, PRLs are involved in metastatic cancer, the promotion of cell migration and invasion. PTPs have been increasingly recognized as important effectors of host-pathogen interactions. We characterized the only putative protein tyrosine phosphatase PRL (PTP EhPRL) in the eukaryotic human intestinal parasite Entamoeba histolytica. Here, we reported that the EhPRL protein possessed the classical HCX5R catalytic motif of PTPs and the CAAX box characteristic of the PRL family and exhibited 31-32% homology with the three human PRL isoforms. In amebae, the protein was expressed at low but detectable levels. The recombinant protein (rEhPRL) had enzymatic activity with the 3-o-methyl fluorescein phosphate (OMFP) substrate; this enzymatic activity was inhibited by the PTP inhibitor o-vanadate. Using immunofluorescence we showed that native EhPRL was localized to the cytoplasm and plasma membrane. When the trophozoites interacted with collagen, EhPRL relocalized over time to vesicle-like structures. Interaction with fibronectin increased the presence of the enzyme in the cytoplasm. Using RT-PCR, we demonstrated that EhPRL mRNA expression was upregulated when the trophozoites interacted with collagen but not with fibronectin. Trophozoites recovered from amoebic liver abscesses showed higher EhPRL mRNA expression levels than normal trophozoites. These results strongly suggest that EhPRL may play an important role in the biology and adaptive response of the parasite to the host environment during amoebic liver abscess development, thereby participating in the pathogenic mechanism. PMID:26431820

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

    PubMed

    Tachibana, H; Cheng, X J; Kobayashi, S; Fujita, Y; Udono, T

    2000-07-01

    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. PMID:10935902

  13. A zymodeme study of Entamoeba histolytica in a group of South African schoolchildren.

    PubMed

    Sargeaunt, P G; Williams, J E; Jackson, T F; Simjee, A E

    1982-01-01

    Using a biphasic culture medium, stocks of intestinal amoebae were isolated from a group of children attending school in Durban, South Africa. These were compared with stocks collected in other areas of the world already characterized using the electrophoretic patterns of four enzymes: glucose phosphate isomerase (GPI), phosphoglucomutase (PGM) L-malate: NADP+ oxido-reductase (oxalacetate-decarboxylating) (ME) and hexokinase (HK). 33% of 94 samples grew Entamoeba histolytica, only one of which gave a pattern indicative of a pathogenic stock. Entamoeba hartmanni, Dientamoeba fragilis and Entamoeba coli were also grown from some samples, increasing the total positive samples for all species isolated to 40%. PMID:6287686

  14. Analysis of the Protein Phosphotome of Entamoeba histolytica Reveals an Intricate Phosphorylation Network

    PubMed Central

    Anwar, Tamanna; Gourinath, Samudrala

    2013-01-01

    Phosphorylation is the most common mechanism for the propagation of intracellular signals. Protein phosphatases and protein kinases play a dynamic antagonistic role in protein phosphorylation. Protein phosphatases make up a significant fraction of eukaryotic proteome. In this article, we report the identification and analysis of protein phosphatases in the intracellular parasite Entamoeba histolytica. Based on an in silico analysis, we classified 250 non-redundant protein phosphatases in E. histolytica. The phosphotome of E. histolytica is 3.1% of its proteome and 1.3 times of the human phosphotome. In this extensive study, we identified 42 new putative phosphatases (39 hypothetical proteins and 3 pseudophosphatases). The presence of pseudophosphatases may have an important role in virulence of E. histolytica. A comprehensive phosphotome analysis of E. histolytica shows spectacular low similarity to human phosphatases, making them potent candidates for drug target. PMID:24236039

  15. Species-Specific Immunodetection of an Entamoeba histolytica Cyst Wall Protein.

    PubMed

    Spadafora, Lauren J; Kearney, Moira R; Siddique, Abdullah; Ali, Ibne K; Gilchrist, Carol A; Arju, Tuhinur; Hoffstrom, Benjamin; Nguyen, Felicia K; Petri, William A; Haque, Rashidul; Cangelosi, Gerard A

    2016-05-01

    Entamoeba histolytica causes intestinal disease in endemic settings throughout the world. Diagnosis of E. histolytica infection would be improved by the identification of biomarkers that are expressed by cysts of E. histolytica, but not by cysts of closely related commensal species of Entamoeba. Herein, we describe two novel monoclonal antibodies (1A4 and 1D3) produced against a spacer region of the E. histolytica Jacob2 lectin, an outer cyst wall protein. These reagents demonstrated no cross-reaction to E. dispar recombinant antigen and low picomolar molecular detection limits when paired in ELISA sandwich assays. In an immunofluorescence microscopy assay, the α-Jacob2 murine antibodies labeled cysts of three xenically cultured E. histolytica isolates but did not label cysts of three E. bangladeshi isolates. Monoclonal antibody 1A4 did not cross-react with xenic cultures of three E. dispar isolates, demonstrating specificity to E. histolytica, while monoclonal antibody 1D3 cross-reacted with two out of three E. dispar isolates. Both antibodies labeled cysts in formalin-fixed slides, a potential logistical advantage in some settings. The monoclonal antibody 1A4 was also used in an immunofluorescence microscopy assay with formalin-fixed stool specimens. Seven out of ten ELISA-positive stool specimens exhibited 1A4-labeled cyst-like objects, compared to one out of seven ELISA-negative specimens. These results demonstrate that antibodies generated against the flexible spacer of E. histolytica Jacob2 lectin recognize and bind to Jacob2 protein in whole cysts and are capable of differentiating Entamoeba species in fixed specimens. Thus, Jacob2 is a promising biomarker for use in diagnosing E. histolytica infection. PMID:27152855

  16. Species-Specific Immunodetection of an Entamoeba histolytica Cyst Wall Protein

    PubMed Central

    Kearney, Moira R.; Siddique, Abdullah; Ali, Ibne K.; Gilchrist, Carol A.; Arju, Tuhinur; Hoffstrom, Benjamin; Nguyen, Felicia K.; Petri, William A.; Haque, Rashidul; Cangelosi, Gerard A.

    2016-01-01

    Entamoeba histolytica causes intestinal disease in endemic settings throughout the world. Diagnosis of E. histolytica infection would be improved by the identification of biomarkers that are expressed by cysts of E. histolytica, but not by cysts of closely related commensal species of Entamoeba. Herein, we describe two novel monoclonal antibodies (1A4 and 1D3) produced against a spacer region of the E. histolytica Jacob2 lectin, an outer cyst wall protein. These reagents demonstrated no cross-reaction to E. dispar recombinant antigen and low picomolar molecular detection limits when paired in ELISA sandwich assays. In an immunofluorescence microscopy assay, the α-Jacob2 murine antibodies labeled cysts of three xenically cultured E. histolytica isolates but did not label cysts of three E. bangladeshi isolates. Monoclonal antibody 1A4 did not cross-react with xenic cultures of three E. dispar isolates, demonstrating specificity to E. histolytica, while monoclonal antibody 1D3 cross-reacted with two out of three E. dispar isolates. Both antibodies labeled cysts in formalin-fixed slides, a potential logistical advantage in some settings. The monoclonal antibody 1A4 was also used in an immunofluorescence microscopy assay with formalin-fixed stool specimens. Seven out of ten ELISA-positive stool specimens exhibited 1A4-labeled cyst-like objects, compared to one out of seven ELISA-negative specimens. These results demonstrate that antibodies generated against the flexible spacer of E. histolytica Jacob2 lectin recognize and bind to Jacob2 protein in whole cysts and are capable of differentiating Entamoeba species in fixed specimens. Thus, Jacob2 is a promising biomarker for use in diagnosing E. histolytica infection. PMID:27152855

  17. Seroprevalence of Entamoeba histolytica Infection among Chinese Men Who Have Sex with Men

    PubMed Central

    Li, Xiangwei; Yang, Yu; Gao, Cong; Jin, Qi; Gao, Lei

    2013-01-01

    Background Men who have sex with men (MSM) were found to be one of the high-risk populations for Entamoeba histolytica (E. histolytica) infection. Accompanied by the prevalence of human immunodeficiency virus (HIV) among MSM, invasive amebiasis caused by E. histolytica has been paid attention to as an opportunistic parasitic infection. However, the status of E. histolytica infection among MSM has been barely studied in mainland China. Methods Seroprevalance of E. histolytica was determined using an enzyme-linked immunosorbent assay based on a cross-sectional study conducted in Beijing and Tianjin, China. Factors potentially associated with E. histolytica infection were identified by logistic regression analysis. Results A total of 602 MSM were included in the study and the laboratory data on serostatus of E. histolytica were available for 599 of them (99.5%). 246 (41.1%) and 51 (8.5%) of the study participants were E. histolytica seropositive and HIV seropositive, respectively. Univariate analyses suggested preferred anal sex behaviors were associated with E. histolytica seropositivity. In multivariate logistic regression analysis, “only has receptive anal sex” (OR: 2.03; 95% CI: 1.22 3.37), “majority receptive anal sex” (OR: 1.83; 95% CI: 1.13, 2.95), and “sadomasochistic behavior (SM)” (OR: 2.30; 95% CI: 1.04, 5.13) were found to be significantly associated with E. histolytica infection. Conclusions High seroprevalence of E. histolytica infection was observed among MSM from Beijing and Tianjin, China. Receptive anal sex behavior and SM were identified as potential predictors. Therefore, E. histolytica and HIV co-infection needs to be concerned among MSM due to their sharing the common risk behaviors. PMID:23717699

  18. Rapid Diagnosis of Intestinal Parasitic Protozoa, with a Focus on Entamoeba histolytica

    PubMed Central

    Singh, Anjana; Houpt, Eric; Petri, William A.

    2009-01-01

    Entamoeba histolytica is an invasive intestinal pathogenic parasitic protozoan that causes amebiasis. It must be distinguished from Entamoeba dispar and E. moshkovskii, nonpathogenic commensal parasites of the human gut lumen that are morphologically identical to E. histolytica. Detection of specific E. histolytica antigens in stools is a fast, sensitive technique that should be considered as the method of choice. Stool real-time PCR is a highly sensitive and specific technique but its high cost make it unsuitable for use in endemic areas where there are economic constraints. Serology is an important component of the diagnosis of intestinal and especially extraintestinal amebiasis as it is a sensitive test that complements the detection of the parasite antigens or DNA. Circulating Gal/GalNac lectin antigens can be detected in the serum of patients with untreated amoebic liver abscess. On the horizon are multiplex real-time PCR assays which permit the identification of multiple enteropathogens with high sensitivity and specificity. PMID:19584941

  19. Use of a monoclonal antibody in an enzyme immunoassay for the detection of Entamoeba histolytica in fecal specimens.

    PubMed

    Ungar, B L; Yolken, R H; Quinn, T C

    1985-05-01

    An indirect enzyme-linked immunosorbent assay (ELISA) was developed for the detection of Entamoeba histolytica in human feces, using both a monoclonal antibody and rabbit antisera. It detected from less than 1 to 57 trophozoites of 6 E. histolytica strains. Stool specimens were positive by ELISA in 18 of 22 (82%) patients with E. histolytica and in 3 of 186 (2%) of patients without demonstrable E. histolytica in their stools. The latter included one from a child living near an asymptomatic cyst carrier and another from a traveler with giardiasis who had recently taken antibiotics. One hundred eight of 183 microscopy-and ELISA-negative specimens contained other parasites including Giardia (49 specimens), Endolimax nana (24), Entamoeba coli (21), Iodamoeba butschlii (2), and Entamoeba hartmanni (1). This ELISA for E. histolytica is a simple, sensitive and specific diagnostic tool. PMID:2860814

  20. Role of the Gut Microbiota of Children in Diarrhea Due to the Protozoan Parasite Entamoeba histolytica

    PubMed Central

    Gilchrist, Carol A.; Petri, Sarah E.; Schneider, Brittany N.; Reichman, Daniel J.; Jiang, Nona; Begum, Sharmin; Watanabe, Koji; Jansen, Caroline S.; Elliott, K. Pamela; Burgess, Stacey L.; Ma, Jennie Z.; Alam, Masud; Kabir, Mamun; Haque, Rashidul; Petri, William A.

    2016-01-01

    Background. An estimated 1 million children die each year before their fifth birthday from diarrhea. Previous population-based surveys of pediatric diarrheal diseases have identified the protozoan parasite Entamoeba histolytica, the etiological agent of amebiasis, as one of the causes of moderate-to-severe diarrhea in sub-Saharan Africa and South Asia. Methods. We prospectively studied the natural history of E. histolytica colonization and diarrhea among infants in an urban slum of Dhaka, Bangladesh. Results. Approximately 80% of children were infected with E. histolytica by the age of 2 years. Fecal anti-galactose/N-acetylgalactosamine lectin immunoglobulin A was associated with protection from reinfection, while a high parasite burden and expansion of the Prevotella copri level was associated with diarrhea. Conclusions. E. histolytica infection was prevalent in this population, with most infections asymptomatic and diarrhea associated with both the amount of parasite and the composition of the microbiota. PMID:26712950

  1. Identification of Entamoeba histolytica by Molecular Method in Surface Water of Rasht City, Iran

    PubMed Central

    HEMMATI, Arash; HOOSHMAND, Elham; HOSSEINI, Mohammad Javad

    2015-01-01

    Background: This study was conducted with the aim of determining surface water contamination with cysts of Entamoeba histolytica using PCR in Rasht City, Northern Iran. Methods: In this cross-sectional study, 49 water samples including 18 rivers and 6 wetlands were collected from different regions near the city of Rasht in autumn of 2012. After filtration using 0.22 μm nitrate cellulose membrane filters, the samples were examined using microscope and PCR method. Results: In microscopic examination, four samples of the 49 samples were positive for cysts of E. (histolytica / dispar / muschkovskii). By using PCR method and molecular analysis, one sample was positive for E. histolytica. Conclusion: In the molecular analysis, contamination by E. histolytica was proved in the waters of Rasht City. Further investigations including more samples and necessary preparations must be applied to prevent contamination. PMID:25905058

  2. An improved colorimetric PCR-based method for detection and differentiation of Entamoeba histolytica and Entamoeba dispar in feces.

    PubMed Central

    Britten, D; Wilson, S M; McNerney, R; Moody, A H; Chiodini, P L; Ackers, J P

    1997-01-01

    The epidemiological implications of the recent separation of "Entamoeba histolytica" into two separate species, pathogenic E. histolytica sensu stricto and commensal E. dispar, will not become apparent without methods of distinguishing between them which are applicable to large numbers of specimens. We have modified a PCR-based method to produce such a technique which may be completed in 1 day while still identifying 10(-1) E. histolytica and 1 to 10 E. dispar trophozoites per g of feces when present separately and 10 E. histolytica and 100 E. dispar trophozoites per g in the presence of 10(6) trophozoites per g of the other species. Applied to fecal specimens from 18 patients from which E. histolytica or E. dispar had been grown and identified to the species level by hexokinase isoenzyme analysis, the method in every case yielded the correct result. Positive and negative results are easily distinguished by eye, and we are now applying this technique to a large-scale epidemiological study of amebiasis in the eastern Mediterranean region. PMID:9114390

  3. Reactivity of monoclonal antibodies to species-specific antigens of Entamoeba histolytica.

    PubMed

    Tachibana, H; Kobayashi, S; Nagakura, K; Kaneda, Y; Takeuchi, T

    1991-01-01

    Twenty monoclonal antibodies were produced against trophozoites of Entamoeba histolytica strains HK-9 and HM-1: IMSS. When reactivity to various enteric protozoa was examined by an indirect fluorescence antibody test, 15 of the monoclonal antibodies were strongly reactive with E. histolytica trophozoites. Species-specific antigens recognized by these monoclonal antibodies were located on the plasma membrane, nucleus, cytoplasm, and cytoskeletal structures of the trophozoites. Two of the remaining five monoclonals reacted strongly with trophozoites of the E. histolytica-like Laredo strain. The determinant antigen was located in the cytoplasm. The three remaining monoclonal antibodies were found to recognize cross-reactive antigens between E. histolytica and E. histolytica-like Laredo, E. hartmanni, E. coli, Dientamoeba fragilis, Giardia lamblia, and Trichomonas hominis. These three antibodies were also reactive with T. vaginalis and mammalian cells such as HeLa cells. Thus, the combined use of monoclonal antibodies seems capable of distinguishing E. histolytica and/or E. histolytica-like Laredo from other enteric protozoa. PMID:1724012

  4. In vitro amoebicidal activity of borage (Borago officinalis) extract on Entamoeba histolytica.

    PubMed

    Leos-Rivas, Catalina; Verde-Star, M Julia; Torres, Lidia Osuna; Oranday-Cardenas, Azucena; Rivas-Morales, Catalina; Barron-Gonzalez, M Porfiria; Morales-Vallarta, Mario R; Cruz-Vega, Delia E

    2011-01-01

    Borage (Borago officinalis) is a plant with nutritional value that is also used in traditional medicine to treat gastrointestinal disease. This study investigated the amoebicidal activity of a methanol extract of borage. The 50% inhibitory concentration (IC₅₀) of the extract for Entamoeba histolytica was 33 μg/mL. The 50% lethal dose of the extract for brine shrimp was greater than 1,000 μg/mL. The IC₅₀ of the extract for Vero cells was 203.9 μg/mL. These results support the use of borage to prevent diseases associated with E. histolytica infection. PMID:21476887

  5. Novel hemagglutinating, hemolytic and cytotoxic activities of the intermediate subunit of Entamoeba histolytica lectin

    PubMed Central

    Kato, Kentaro; Yahata, Kazuhide; Gopal Dhoubhadel, Bhim; Fujii, Yoshito; Tachibana, Hiroshi

    2015-01-01

    Galactose and N-acetyl-D-galactosamine (Gal/GalNAc) inhibitable lectin of Entamoeba histolytica, a common protozoan parasite, has roles in pathogenicity and induction of protective immunity in mouse models of amoebiasis. The lectin consists of heavy (Hgl), light (Lgl), and intermediate (Igl) subunits. Hgl has lectin activity and Lgl does not, but little is known about the activity of Igl. In this study, we assessed various regions of Igl for hemagglutinating activity using recombinant proteins expressed in Escherichia coli. We identified a weak hemagglutinating activity of the protein. Furthermore, we found novel hemolytic and cytotoxic activities of the lectin, which resided in the carboxy-terminal region of the protein. Antibodies against Igl inhibited the hemolytic activity of Entamoeba histolytica trophozoites. This is the first report showing hemagglutinating, hemolytic and cytotoxic activities of an amoebic molecule, Igl. PMID:26354528

  6. Seroepidemiology of Entamoeba histolytica Infection in General Population in Rural Durango, Mexico

    PubMed Central

    Alvarado-Esquivel, Cosme; Hernandez-Tinoco, Jesus; Sanchez-Anguiano, Luis Francisco

    2015-01-01

    Background The seroepidemiology of Entamoeba histolytica in Mexico has been scantily studied. The aim of the study was to determine the seroprevalence and correlates of E. histolytica antibodies in adults in rural areas in Durango, Mexico. Methods Through a cross-sectional study, E. histolytica IgG antibodies were determined in 282 adults living in rural Durango, Mexico using an enzyme-linked immunoassay. In addition, seroprevalence association with the socio-demographic, housing conditions, and behavioral characteristics of the subjects studied was investigated. Results One hundred and eighteen (41.8%) of the 282 rural subjects had anti-E. histolytica IgG antibodies. Multivariate analysis showed that E. histolytica exposure was positively associated with source of drinking water (OR = 2.73; 95% CI: 1.33 - 5.58; P = 0.005), and poor education of the head of the family (OR = 1.53; 95% CI: 1.03 - 2.27; P = 0.03). In contrast, E. histolytica exposure was negatively associated with consumption of unpasteurized cow milk (OR = 0.55; 95% CI: 0.31 - 0.96; P = 0.03), and crowding at home (OR = 0.33; 95% CI: 0.17 - 0.64; P = 0.0009). Conclusions The seroprevalence of E. histolytica infection found in adults in rural Durango is high compared with those reported in other Mexican populations. The correlates of E. histolytica seropositivity found in the present study may be useful for the planning of optimal preventive measures against E. histolytica infection. PMID:25883706

  7. Double-blind test of metronidazole and tinidazole in the treatment of asymptomatic Entamoeba histolytica and Entamoeba hartmanni carriers.

    PubMed

    Spillmann, R; Ayala, S C; Sanchez, C E

    1976-07-01

    One hundred and fifteen persons with asymptomatic Entamoeba histolytica or E. hartmanni infection, or both, were given metronidazole (750 mg three times daily for 5 days), tinidazole (1 g twice daily on 2 consecutive days), or a starch placebo. Three post-treatment stools were examined in the 2 weeks following initiation of treatment. Cysts of E. histolytica reappeared in the stools of 37% of 30 given metronidazole, 62% of 34 given tinidazole, and 70% of 31 given placebo. Cysts of E. hartmanni reappeared in the stools of 46% of 24 given metronidazole, 69% of 16 given tinidazole, and 90% of 10 given placebo. Rapid absorption and short duration of treatment make both drugs ineffective for the treatment of ameba carriers. PMID:183554

  8. Detection of beta-tubulin in the cytoplasm of the interphasic Entamoeba histolytica trophozoites.

    PubMed

    Gómez-Conde, Eduardo; Vargas-Mejía, Miguel Ángel; Díaz-Orea, María Alicia; Hernández-Rivas, Rosaura; Cárdenas-Perea, María Elena; Guerrero-González, Tayde; González-Barrios, Juan Antonio; Montiel-Jarquín, Álvaro José

    2016-08-01

    It is known that the microtubules (MT) of Entamoeba histolytica trophozoites form an intranuclear mitotic spindle. However, electron microscopy studies and the employment of anti-beta-tubulin (β-tubulin) antibodies have not exhibited these cytoskeletal structures in the cytoplasm of these parasites. The purpose of this work was to detect β-tubulin in the cytoplasm of interphasic E. histolytica trophozoites. Activated or non-activated HMI-IMSS-strain E. histolytica trophozoites were used and cultured for 72 h at 37 °C in TYI-S-33 medium, and then these were incubated with the anti-β-tubulin antibody of E. histolytica. The anti-β-tubulin antibody reacted with the intranuclear mitotic spindle of E. histolytica-activated trophozoites as control. In contrast, in non-activated interphasic parasites, anti-β-tubulin antibody reacted with diverse puntiform structures in the cytoplasm and with ring-shaped structures localized in the cytoplasm, cellular membrane and endocytic stomas. In this work, for the first time, the presence of β-tubulin is shown in the cytoplasm of E. histolytica trophozoites. PMID:27156446

  9. RNA interference in Entamoeba histolytica: implications for parasite biology and gene silencing

    PubMed Central

    Zhang, Hanbang; Pompey, Justine M; Singh, Upinder

    2011-01-01

    Entamoeba histolytica is a major health threat to people in developing countries, where it causes invasive diarrhea and liver abscesses. The study of this important human pathogen has been hindered by a lack of tools for genetic manipulation. Recently, a number of genetic approaches based on variations of the RNAi method have been successfully developed and cloning of endogenous small-interfering RNAs from E. histolytica revealed an abundant population of small RNAs with an unusual 5′-polyphosphate structure. However, little is known about the implications of these findings to amebic biology or the mechanisms of gene silencing in this organism. In this article we review the literature relevant to RNAi in E. histolytica, discuss its implications for advances in gene silencing in this organism and outline potential future directions towards understanding the repertoire of RNAi and its impact on the biology of this deep-branching eukaryotic parasite. PMID:21162639

  10. Ultrastructural changes on Entamoeba histolytica HM1-IMSS caused by the flavan-3-ol, (-)-epicatechin.

    PubMed

    Soto, Jacqueline; Gómez, Consuelo; Calzada, Fernando; Ramírez, María Esther

    2010-04-01

    The flavan-3-ol, (-)-epicatechin has been previously identified as the most important antiamoebic compound among the extracts from two medicinal plants: Rubus coriifolius and Geranium mexicanum. Here we report the effects of epicatechin on Entamoeba histolytica morphology, analyzed by electronic microscopy. E. histolytica trophozoites were incubated for 48 h at 37 degrees C in the presence of 1.9 microg/mL epicatechin and processed for electronic microscopy analysis. Epicatechin induced nuclear and cytoplasmic changes in the treated trophozoites. These morphological alterations are identical to the cellular changes experienced by E. histolytica trophozoites undergoing programmed cell death (PCD), suggesting that epicatechin could be an alternative compound to treat amoebiasis. PMID:19918717

  11. The epidemiology of Entamoeba histolytica in Mexico City. A pilot survey I.

    PubMed

    Sargeaunt, P G; Williams, J E; Kumate, J; Jimenez, E

    1980-01-01

    Stocks of intestinal amoebae isolated from hospital patients in Mexico City and grown in monoxenic culture were compared among themselves and with those already described (SARGEAUNT & WILLIAMS, 1979), using the electrophoretic patterns of four enzymes: glucose phosphate isomerase (GPI), phosphoglucomutase (PGM), L-malate:NADP+ oxido-reductase (oxalacetate-decarboxylating) (ME) and hexokinase (HK). New isoenzyme groups (SARGEAUNT & WILLIAMS, 1979) of all the amoebae, including Entamoeba histolytica have been demonstrated. Amongst these have been found seven more groups of E. histolytica, two new groups of E. hartmanni, one new group of Dientamoeba fragilis and one new group of E. coli. Of the seven new groups of E. histolytica three are known to originate from patients with clinical amoebiasis whilst the remainder are from asymptomatic subjects. Only 11.2% of the 125 isolations were associated with clinical amoebiasis, and these are clearly distinguished from the isolations from asymptomatic patients by their electrophoretic isoenzyme pattern. PMID:6259779

  12. Risk factors for Entamoeba histolytica infection in an agricultural community in Hanam province, Vietnam

    PubMed Central

    2011-01-01

    Background Entamoeba histolytica is an important protozoan intestinal infection in resource-poor settings, including Vietnam. The study objective was to assess risk factors of E. histolytica infection in a community in Vietnam, where wastewater and human excreta are used in agriculture. A case-control study was conducted among residents of Hanam province, Northern Vietnam. Cases (n = 46) infected with E. histolytica and non-infected controls (n = 138) were identified in a cross-sectional survey among 794 randomly selected individuals and matched for age, sex and place of residence. Potential risk factors including exposure to human and animal excreta and household wastewater were assessed with a questionnaire. Results People from households with an average socio-economic status had a much higher risk of E. histolytica infection (odds ratio [OR]=4.3, 95% confidence interval [CI]: 1.3-14.0) compared with those from households with a good socioeconomic status. Those individuals who never or rarely used soap for hand washing had a 3.4 times higher risk for infection (OR=3.4, 95% CI: 1.1-10.0), compared to those who used always soap. In contrast, none of the factors related to use of human or animal excreta was statistically significant associated with E. histolytica infection. People having close contact with domestic animals presented a greater risk of E. histolytica infection (OR = 5.9, 95% CI: 1.8-19.0) than those without animal contact. E. histolytica infection was not associated with direct contact with Nhue river water, pond water and household's sanitary conditions, type of latrine or water source used. Conclusions Our study suggests that in settings where human and animal excreta and Nhue River water are intensively used in agriculture, socio-economic and personal hygiene factors determine infection with E. histolytica, rather than exposure to human and animal excreta in agricultural activities. PMID:21663665

  13. Effect of the sesquiterpene lactone incomptine A in the energy metabolism of Entamoeba histolytica.

    PubMed

    Velázquez-Domínguez, José; Marchat, Laurence A; López-Camarillo, Cesar; Mendoza-Hernández, Guillermo; Sánchez-Espíndola, Esther; Calzada, Fernando; Ortega-Hernández, Alfredo; Sánchez-Monroy, Virginia; Ramírez-Moreno, Esther

    2013-11-01

    Entamoeba histolytica is the causative agent of human amoebiasis, which mainly affects developing countries. Although several drugs are effective against E. histolytica trophozoites, the control of amoebiasis requires the development of new and better alternative therapies. Medicinal plants have been the source of new molecules with remarkable antiprotozoal activity. Incomptine A isolated from Decachaeta incompta leaves, is a sesquiterpene lactone of the heliangolide type which has the major in vitro activity against E. histolytica trophozoites. However the molecular mechanisms involved in its antiprotozoal activity are still unknown. Using a proteomic approach based on two-dimensional gel electrophoresis and mass spectrometry (ESI-MS/MS) analysis, we evidenced that 21 E. histolytica proteins were differentially expressed in response to incomptine A treatment. Notably, three glycolytic enzymes, namely enolase, pyruvate:ferredoxin oxidoreductase and fructose-1,6-biphosphate aldolase, were down-regulated. Moreover, ultrastructural analysis of trophozoites through electronic microscopy showed an increased number of glycogen granules. Taken together, our data suggested that incomptine A could affect E. histolytica growth through alteration of its energy metabolism. PMID:23994114

  14. Protection of gerbils from amebic liver abscess by immunization with a recombinant Entamoeba histolytica antigen.

    PubMed Central

    Zhang, T; Cieslak, P R; Stanley, S L

    1994-01-01

    Amebiasis, infection by the intestinal protozoan parasite Entamoeba histolytica, is a leading parasitic cause of death. As a step in the development of a recombinant antigen vaccine to prevent E. histolytica infection, we looked at the ability of a recombinant version of the serine-rich E. histolytica protein (SREHP) to elicit a protective immune response against invasive amebic disease. Gerbils, a standard model for amebic liver abscess, were immunized with either a recombinant SREHP/maltose-binding protein (MBP) fusion, recombinant MBP alone, or phosphate-buffered saline (PBS), all combined with complete Freund's adjuvant. In the first trial (group 1), gerbils received a primary and two booster immunizations intraperitoneally; in the second trial (group 2), gerbils were immunized by a single intradermal injection. SREHP/MBP-immunized gerbils in both groups produced antibody to native SHEHP and developed delayed-type hypersensitivity responses to recombinant SREHP. All gerbils were challenged by an intrahepatic injection with 5 x 10(4) virulent E. histolytica HM1-IMSS trophozoites. Complete protection from amebic liver abscess was seen in 64% of the SHEHP/MBP-immunized gerbils in group 1 and in 100% of the SREHP/MBP-immunized gerbils in group 2. There was no protection observed in MBP- or PBS-immunized gerbils in either group. Our results indicate that the SREHP molecule has potential as a vaccine to prevent amebic infection and demonstrate that successful vaccination of animals with recombinant E. histolytica antigen vaccines is possible. Images PMID:8132322

  15. The MAK16 Gene of Entamoeba histolytica and Its Identification in Isolates from Patients

    PubMed Central

    Crisóstomo-Vázquez, María del Pilar; Marevelez-Acosta, Víctor Alberto; Flores-Luna, Andrés

    2014-01-01

    To identify sequences of Entamoeba histolytica associated with the development of amebic liver abscess (ALA) in hamsters, subtractive hybridization of cDNA from E. histolytica HM-1:IMSS under 2 growth conditions was performed: 1) cultured in axenic medium and 2) isolated from experimental ALA in hamsters. For this procedure, 6 sequences were obtained. Of these sequences, the mak16 gene was selected for amplification in 29 cultures of E. histolytica isolated from the feces of 10 patients with intestinal symptoms and 19 asymptomatic patients. Only 5 of the 10 isolates obtained from symptomatic patients developed ALA and amplified the mak16 gene, whereas the 19 isolates from asymptomatic patients did not amplify the mak16 gene nor did they develop ALA. Based on the results of Fisher's exact test (P<0.001), an association was inferred between the presence of the mak16 gene of E. histolytica and the ability to develop ALA in hamsters and with the patient's symptoms (P=0.02). The amplification of the mak16 gene suggests that it is an important gene in E. histolytica because it was present in the isolates from hamsters that developed liver damage. PMID:25246723

  16. Preparation of Recombinant Human Monoclonal Antibody Fab Fragments Specific for Entamoeba histolytica

    PubMed Central

    Tachibana, Hiroshi; Cheng, Xun-Jia; Watanabe, Katsuomi; Takekoshi, Masataka; Maeda, Fumiko; Aotsuka, Satoshi; Kaneda, Yoshimasa; Takeuchi, Tsutomu; Ihara, Seiji

    1999-01-01

    Genes coding for human antibody Fab fragments specific for Entamoeba histolytica were cloned and expressed in Escherichia coli. Lymphocytes were separated from the peripheral blood of a patient with an amebic liver abscess. Poly(A)+ RNA was isolated from the lymphocytes, and then genes coding for the light chain and Fd region of the heavy chain were amplified by a reverse transcriptase PCR. The amplified DNA fragments were ligated with a plasmid vector and were introduced into Escherichia coli. Three thousand colonies were screened for the production of antibodies to E. histolytica HM-1:IMSS by an indirect fluorescence-antibody (IFA) test. Lysates from five Escherichia coli clones were positive. Analysis of the DNA sequences of the five clones showed that three of the five heavy-chain sequences and four of the five light-chain sequences differed from each other. When the reactivities of the Escherichia coli lysates to nine reference strains of E. histolytica were examined by the IFA test, three Fab fragments with different DNA sequences were found to react with all nine strains and another Fab fragment was found to react with seven strains. None of the four human monoclonal antibody Fab fragments reacted with Entamoeba dispar reference strains or with other enteric protozoan parasites. These results indicate that the bacterial expression system reported here is effective for the production of human monoclonal antibodies specific for E. histolytica. The recombinant human monoclonal antibody Fab fragments may be applicable for distinguishing E. histolytica from E. dispar and for use in the serodiagnosis of amebiasis. PMID:10225840

  17. The oxygen reduction pathway and heat shock stress response are both required for Entamoeba histolytica pathogenicity.

    PubMed

    Olivos-García, Alfonso; Saavedra, Emma; Nequiz, Mario; Santos, Fabiola; Luis-García, Erika Rubí; Gudiño, Marco; Pérez-Tamayo, Ruy

    2016-05-01

    Several species belonging to the genus Entamoeba can colonize the mouth or the human gut; however, only Entamoeba histolytica is pathogenic to the host, causing the disease amoebiasis. This illness is responsible for one hundred thousand human deaths per year worldwide, affecting mainly underdeveloped countries. Throughout its entire life cycle and invasion of human tissues, the parasite is constantly subjected to stress conditions. Under in vitro culture, this microaerophilic parasite can tolerate up to 5 % oxygen concentrations; however, during tissue invasion the parasite has to cope with the higher oxygen content found in well-perfused tissues (4-14 %) and with reactive oxygen and nitrogen species derived from both host and parasite. In this work, the role of the amoebic oxygen reduction pathway (ORP) and heat shock response (HSP) are analyzed in relation to E. histolytica pathogenicity. The data suggest that in contrast with non-pathogenic E. dispar, the higher level of ORP and HSPs displayed by E. histolytica enables its survival in tissues by diminishing and detoxifying intracellular oxidants and repairing damaged proteins to allow metabolic fluxes, replication and immune evasion. PMID:26589893

  18. Comparison of a Stool Antigen Detection Kit and PCR for Diagnosis of Entamoeba histolytica and Entamoeba dispar Infections in Asymptomatic Cyst Passers in Iran

    PubMed Central

    Solaymani-Mohammadi, Shahram; Rezaian, Mostafa; Babaei, Zahra; Rajabpour, Azam; Meamar, Ahmad R.; Pourbabai, Ahmad A.; Petri, William A.

    2006-01-01

    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. PMID:16757634

  19. The Essentials of Protein Import in the Degenerate Mitochondrion of Entamoeba histolytica

    PubMed Central

    Dolezal, Pavel; Dagley, Michael J.; Kono, Maya; Wolynec, Peter; Likić, Vladimir A.; Foo, Jung Hock; Sedinová, Miroslava; Tachezy, Jan; Bachmann, Anna; Bruchhaus, Iris; Lithgow, Trevor

    2010-01-01

    Several essential biochemical processes are situated in mitochondria. The metabolic transformation of mitochondria in distinct lineages of eukaryotes created proteomes ranging from thousands of proteins to what appear to be a much simpler scenario. In the case of Entamoeba histolytica, tiny mitochondria known as mitosomes have undergone extreme reduction. Only recently a single complete metabolic pathway of sulfate activation has been identified in these organelles. The E. histolytica mitosomes do not produce ATP needed for the sulfate activation pathway and for three molecular chaperones, Cpn60, Cpn10 and mtHsp70. The already characterized ADP/ATP carrier would thus be essential to provide cytosolic ATP for these processes, but how the equilibrium of inorganic phosphate could be maintained was unknown. Finally, how the mitosomal proteins are translocated to the mitosomes had remained unclear. We used a hidden Markov model (HMM) based search of the E. histolytica genome sequence to discover candidate (i) mitosomal phosphate carrier complementing the activity of the ADP/ATP carrier and (ii) membrane-located components of the protein import machinery that includes the outer membrane translocation channel Tom40 and membrane assembly protein Sam50. Using in vitro and in vivo systems we show that E. histolytica contains a minimalist set up of the core import components in order to accommodate a handful of mitosomal proteins. The anaerobic and parasitic lifestyle of E. histolytica has produced one of the simplest known mitochondrial compartments of all eukaryotes. Comparisons with mitochondria of another amoeba, Dictystelium discoideum, emphasize just how dramatic the reduction of the protein import apparatus was after the loss of archetypal mitochondrial functions in the mitosomes of E. histolytica. PMID:20333239

  20. Chew on this: Amoebic trogocytosis and host cell killing by Entamoeba histolytica

    PubMed Central

    Ralston, Katherine S.

    2015-01-01

    Entamoeba histolytica was named “histolytica” (histo-: tissue; lytic-: dissolving) for its ability to destroy host tissues. Direct killing of host cells by the amoebae is likely to be the driving factor that underlies tissue destruction, but the mechanism was unclear. We recently showed that after attaching to host cells, amoebae bite off and ingest distinct host cell fragments, and that this contributes to cell killing. Here we review this process, termed “amoebic trogocytosis” (trogo-: nibble), and how this process interplays with phagocytosis, or whole cell ingestion, in this organism. “Nibbling” processes have been described in other microbes and in multicellular organisms. The discovery of amoebic trogocytosis in E. histolytica may also shed light on an evolutionarily conserved process for intercellular exchange. PMID:26070402

  1. Viruses of Entamoeba histolytica. I. Identification of transmissible virus-like agents.

    PubMed

    Diamond, L S; Mattern, C F; Bartgis, I L

    1972-02-01

    This and a companion report deal with the identification and morphogenesis of viruses in axenized cultures of Entamoeba histolytica. There are probably two different types of virus each producing a different pathological picture in different amoebal strains, or, less likely, there is one type of agent having widely different morphological and morphogenetical pictures in different strains of E. histolytica. Both types of agent produce a lytic response in axenized amoebae and have been serially passaged to an extent assuring their replicating nature. One appears to replicate in the nucleus as multiple clusters of fine filaments which ultimately lyse the nucleus, causing cell death. The second type of agent appears to be a typical polyhedral virus, seen only in the cytoplasm and also resulting in lysis of the cell. A particle morphologically indistinguishable from this second agent is also found in late passages of the agent producing the nuclear pathology. PMID:4335522

  2. The Role of Lipopeptidophosphoglycan in the Immune Response to Entamoeba histolytica

    PubMed Central

    Wong-Baeza, Isabel; Alcántara-Hernández, Marcela; Mancilla-Herrera, Ismael; Ramírez-Saldívar, Itzmel; Arriaga-Pizano, Lourdes; Ferat-Osorio, Eduardo; López-Macías, Constantino; Isibasi, Armando

    2010-01-01

    The sensing of Pathogen Associated Molecular Patterns (PAMPs) by innate immune receptors, such as Toll-like receptors (TLRs), is the first step in the inflammatory response to pathogens. Entamoeba histolytica, the etiological agent of amebiasis, has a surface molecule with the characteristics of a PAMP. This molecule, which was termed lipopeptidophosphoglycan (LPPG), is recognized through TLR2 and TLR4 and leads to the release of cytokines from human monocytes, macrophages, and dendritic cells; LPPG-activated dendritic cells have increased expression of costimulatory molecules. LPPG activates NKT cells in a CD1d-dependent manner, and this interaction limits amebic liver abscess development. LPPG also induces antibody production, and anti-LPPG antibodies prevent disease development in animal models of amebiasis. Because LPPG is recognized by both the innate and the adaptive immune system (it is a “Pamptigen”), it may be a good candidate to develop a vaccine against E. histolytica infection and an effective adjuvant. PMID:20145703

  3. The role of lipopeptidophosphoglycan in the immune response to Entamoeba histolytica.

    PubMed

    Wong-Baeza, Isabel; Alcántara-Hernández, Marcela; Mancilla-Herrera, Ismael; Ramírez-Saldívar, Itzmel; Arriaga-Pizano, Lourdes; Ferat-Osorio, Eduardo; López-Macías, Constantino; Isibasi, Armando

    2010-01-01

    The sensing of Pathogen Associated Molecular Patterns (PAMPs) by innate immune receptors, such as Toll-like receptors (TLRs), is the first step in the inflammatory response to pathogens. Entamoeba histolytica, the etiological agent of amebiasis, has a surface molecule with the characteristics of a PAMP. This molecule, which was termed lipopeptidophosphoglycan (LPPG), is recognized through TLR2 and TLR4 and leads to the release of cytokines from human monocytes, macrophages, and dendritic cells; LPPG-activated dendritic cells have increased expression of costimulatory molecules. LPPG activates NKT cells in a CD1d-dependent manner, and this interaction limits amebic liver abscess development. LPPG also induces antibody production, and anti-LPPG antibodies prevent disease development in animal models of amebiasis. Because LPPG is recognized by both the innate and the adaptive immune system (it is a "Pamptigen"), it may be a good candidate to develop a vaccine against E. histolytica infection and an effective adjuvant. PMID:20145703

  4. Heterotrimeric G-protein signaling is critical to pathogenic processes in Entamoeba histolytica.

    PubMed

    Bosch, Dustin E; Kimple, Adam J; Muller, Robin E; Giguère, Patrick M; Machius, Mischa; Willard, Francis S; Temple, Brenda R S; Siderovski, David P

    2012-01-01

    Heterotrimeric G-protein signaling pathways are vital components of physiology, and many are amenable to pharmacologic manipulation. Here, we identify functional heterotrimeric G-protein subunits in Entamoeba histolytica, the causative agent of amoebic colitis. The E. histolytica Gα subunit EhGα1 exhibits conventional nucleotide cycling properties and is seen to interact with EhGβγ dimers and a candidate effector, EhRGS-RhoGEF, in typical, nucleotide-state-selective fashions. In contrast, a crystal structure of EhGα1 highlights unique features and classification outside of conventional mammalian Gα subfamilies. E. histolytica trophozoites overexpressing wildtype EhGα1 in an inducible manner exhibit an enhanced ability to kill host cells that may be wholly or partially due to enhanced host cell attachment. EhGα1-overexpressing trophozoites also display enhanced transmigration across a Matrigel barrier, an effect that may result from altered baseline migration. Inducible expression of a dominant negative EhGα1 variant engenders the converse phenotypes. Transcriptomic studies reveal that modulation of pathogenesis-related trophozoite behaviors by perturbed heterotrimeric G-protein expression includes transcriptional regulation of virulence factors and altered trafficking of cysteine proteases. Collectively, our studies suggest that E. histolytica possesses a divergent heterotrimeric G-protein signaling axis that modulates key aspects of cellular processes related to the pathogenesis of this infectious organism. PMID:23166501

  5. Heterotrimeric G-protein Signaling Is Critical to Pathogenic Processes in Entamoeba histolytica

    PubMed Central

    Muller, Robin E.; Giguère, Patrick M.; Machius, Mischa; Willard, Francis S.; Temple, Brenda R. S.; Siderovski, David P.

    2012-01-01

    Heterotrimeric G-protein signaling pathways are vital components of physiology, and many are amenable to pharmacologic manipulation. Here, we identify functional heterotrimeric G-protein subunits in Entamoeba histolytica, the causative agent of amoebic colitis. The E. histolytica Gα subunit EhGα1 exhibits conventional nucleotide cycling properties and is seen to interact with EhGβγ dimers and a candidate effector, EhRGS-RhoGEF, in typical, nucleotide-state-selective fashions. In contrast, a crystal structure of EhGα1 highlights unique features and classification outside of conventional mammalian Gα subfamilies. E. histolytica trophozoites overexpressing wildtype EhGα1 in an inducible manner exhibit an enhanced ability to kill host cells that may be wholly or partially due to enhanced host cell attachment. EhGα1-overexpressing trophozoites also display enhanced transmigration across a Matrigel barrier, an effect that may result from altered baseline migration. Inducible expression of a dominant negative EhGα1 variant engenders the converse phenotypes. Transcriptomic studies reveal that modulation of pathogenesis-related trophozoite behaviors by perturbed heterotrimeric G-protein expression includes transcriptional regulation of virulence factors and altered trafficking of cysteine proteases. Collectively, our studies suggest that E. histolytica possesses a divergent heterotrimeric G-protein signaling axis that modulates key aspects of cellular processes related to the pathogenesis of this infectious organism. PMID:23166501

  6. Nitroimidazole Action in Entamoeba histolytica: A Central Role for Thioredoxin Reductase

    PubMed Central

    Leitsch, David; Kolarich, Daniel; Wilson, Iain B. H; Altmann, Friedrich; Duchêne, Michael

    2007-01-01

    Metronidazole, a 5-nitroimidazole drug, has been the gold standard for several decades in the treatment of infections with microaerophilic protist parasites, including Entamoeba histolytica. For activation, the drug must be chemically reduced, but little is known about the targets of the active metabolites. Applying two-dimensional gel electrophoresis and mass spectrometry, we searched for protein targets in E. histolytica. Of all proteins visualized, only five were found to form adducts with metronidazole metabolites: thioredoxin, thioredoxin reductase, superoxide dismutase, purine nucleoside phosphorylase, and a previously unknown protein. Recombinant thioredoxin reductase carrying the modification displayed reduced enzymatic activity. In treated cells, essential non-protein thiols such as free cysteine were also affected by covalent adduct formation, their levels being drastically reduced. Accordingly, addition of cysteine allowed E. histolytica to survive in the presence of otherwise lethal metronidazole concentrations and reduced protein adduct formation. Finally, we discovered that thioredoxin reductase reduces metronidazole and other nitro compounds, suggesting a new model of metronidazole activation in E. histolytica with a central role for thioredoxin reductase. By reducing metronidazole, the enzyme renders itself and associated thiol-containing proteins vulnerable to adduct formation. Because thioredoxin reductase is a ubiquitous enzyme, similar processes could occur in other eukaryotic or prokaryotic organisms. PMID:17676992

  7. Overexpression of specific cysteine peptidases confers pathogenicity to a nonpathogenic Entamoeba histolytica clone.

    PubMed

    Matthiesen, Jenny; Bär, Ann-Katrein; Bartels, Anne-Kathrin; Marien, Dennis; Ofori, Susann; Biller, Laura; Tannich, Egbert; Lotter, Hannelore; Bruchhaus, Iris

    2013-01-01

    Cysteine peptidases (CPs) of Entamoeba histolytica are considered to be important pathogenicity factors. Previous studies have found that under standard axenic culture conditions, only four (ehcp-a1, ehcp-a2, ehcp-a5, and ehcp-a7) out of 35 papain-like ehcp genes present in the E. histolytica genome are expressed at high levels. Little is known about the expression of CPs in E. histolytica during amoebic liver abscess (ALA) formation. In the current study, a quantitative real-time PCR assay was developed to determine the expression of the various ehcp genes during ALA formation in animal models. Increased expression of four ehcp genes (ehcp-a3, -a4, -a10, and -c13) was detected in the gerbil and mouse models. Increased expression of another three ehcp genes (ehcp-a5, -a6, and -a7) was detected in the mouse model only, and two other ehcp genes (ehcp-b8 and -b9) showed increased expression in the gerbil model only. Trophozoites of the nonpathogenic E. histolytica HM-1:IMSS clone A1, which was unable to induce ALAs, were transfected with vectors enabling overexpression of those CPs that are expressed at high levels under culture conditions or during ALA formation. Interestingly, overexpression of ehcp-b8, -b9, and -c13 restored the pathogenic phenotype of the nonpathogenic clone A1 whereas overexpression of various other peptidase genes had no effect on the pathogenicity of this clone. PMID:23532975

  8. New insights into host–pathogen interactions during Entamoeba histolytica liver infection

    PubMed Central

    Faust, D. M.; Marquay Markiewicz, J.; Santi-Rocca, J.

    2011-01-01

    Amoebiasis is the third worldwide disease due to a parasite. The causative agent of this disease, the unicellular eukaryote Entamoeba histolytica, causes dysentery and liver abscesses associated with inflammation and human cell death. During liver invasion, before entering the parenchyma, E. histolytica trophozoites are in contact with liver sinusoidal endothelial cells (LSEC). We present data characterizing human LSEC responses to interaction with E. histolytica and identifying amoebic factors involved in the process of cell death in this cell culture model potentially relevant for early steps of hepatic amoebiasis. E. histolytica interferes with host cell adhesion signalling and leads to diminished adhesion and target cell death. Contact with parasites induces disruption of actin stress fibers and focal adhesion complexes. We conclude that interference with LSEC signalling may result from amoeba-triggered changes in the mechanical forces in the vicinity of cells in contact with parasites, sensed and transmitted by focal adhesion complexes. The study highlights for the first time the potential role in the onset of hepatic amoebiasis of the loss of liver endothelium integrity by disturbance of focal adhesion function and adhesion signalling. Among the amoebic factors required for changed LSEC adherence properties we identified the Gal/GalNAC lectin, cysteine proteases and KERP1. PMID:24466432

  9. ANTIPARASITIC ACTIVITY OF SILVER AND COPPER OXIDE NANOPARTICLES AGAINST ENTAMOEBA HISTOLYTICA AND CRYPTOSPORIDIUM PARVUM CYSTS.

    PubMed

    Saad, Halim A; Soliman, Mohamed I; Azzam, Ahmed M; Mostafa, B

    2015-12-01

    Nanoparticles (NPs) have received more attention as antiparasitic agents. In the present study, silver and copper nanoparticles were synthesized and characterized using scanning electron microscopy (SEM), transmission electron microscope (TEM) and X-ray fluorescence (XRF). The antiparasitic activity of Ag and CuO nanoparticles were tested against two of the most environmentally spread parasites in Egypt (Entamoeba histolytica and Cryptosporidium parvum). The average sizes of synthesized Ag NPs and CuO NPs were 9 & 29 nm respectively and a significant reduction for cysts viability (p > 0.05) was observed for CuO NPs against E. histolytica cysts and Ag NPs against C. parvum oocysts. Moreover, LC50-3h of CuO NPs for E. histolytica and C. parvum were 0.13 and 0.72 mg/l, while Ag NPs recorded 0.34 and 0.54 mg/l respectively. Accordingly, these NPs could be suggested as a new nanoform agent for safe and effective treatment of E. histolytica and C. parvum parasites. PMID:26939237

  10. Entamoeba histolytica antigenic protein detected in pus aspirates from patients with amoebic liver abscess.

    PubMed

    Othman, Nurulhasanah; Mohamed, Zeehaida; Yahya, Maya Mazuwin; Leow, Voon Meng; Lim, Boon Huat; Noordin, Rahmah

    2013-08-01

    Entamoeba histolytica is a causative agent of amoebic liver abscess (ALA) and is endemic in many underdeveloped countries. We investigated antigenic E. histolytica proteins in liver abscess aspirates using proteomics approach. Pus samples were first tested by real-time PCR to confirm the presence of E. histolytica DNA and the corresponding serum samples tested for E. histolytica-specific IgG by a commercial ELISA. Proteins were extracted from three and one pool(s) of pus samples from ALA and PLA (pyogenic liver abscess) patients respectively, followed by analysis using isoelectric focussing, SDS-PAGE and Western blot. Unpurified pooled serum samples from infected hamsters and pooled human amoebic-specific IgG were used as primary antibodies. The antigenic protein band was excised from the gel, digested and analysed by MALDI-TOF/TOF and LC-MS/MS. The results using both primary antibodies showed an antigenic protein band of ∼14kDa. Based on the mass spectrum analysis, putative tyrosine kinase is the most probable identification of the antigenic band. PMID:23680184

  11. Simple standardized enzyme-linked immunosorbent assay for human antibodies to Entamoeba histolytica.

    PubMed Central

    Lin, T M; Halbert, S P; Chiu, C T; Zarco, R

    1981-01-01

    A simple solid-phase enzyme-linked immunosorbent assay procedure for the detection of human antibodies to Entamoeba histolytica was developed which showed a high degree of correlation with the agar gel diffusion, counterelectrophoresis, and indirect hemagglutination methods, as well as with clinical data. The enzyme-linked immunosorbent assay is rapid (1 h 15 min, total incubation time), and the reported values are referenced to a positive control so that they correlate with levels of antibody sufficient to be detected by the gel diffusion methods. The enzyme-linked immunosorbent assay is highly reproducible, specific, and sensitive; it can be used qualitatively or quantitatively. PMID:6262370

  12. [Immunofluorescent diagnosis of Entamoeba histolytica trophocoites in preserved stool specimens of patients (author's transl)].

    PubMed

    Hess, U; Fröhlich, A

    1979-09-01

    A fixative and examination technique is described for identification of Entamoeba histolytica trophocoites in faeces with the aid of the indirect immunofluorescence technique. Magnaforms in bloody-dysenteric specimens and Minutaforms in spontaneous or saline purged specimens give equally good fluorescence. The specifity of the rabbit antiserum against axenically grown E. hist. is good: There is strong positive reaction only with trophocoites of E. hist. Weak crossreactions are encountered with trophocoites of E. coli, E. hartmanni and E. polecki. Negative reactions are encountered with trophocoites of Endolimax nana, Jodamoeba bāutschlii, and Dientamoeba fragilis. Amebic cysts, flagellates, leucocytes, epithelial cells and Blastocytis give negative reactions, too. PMID:94475

  13. An ex-vivo Human Intestinal Model to Study Entamoeba histolytica Pathogenesis

    PubMed Central

    Bansal, Devendra; Ave, Patrick; Kerneis, Sophie; Frileux, Pascal; Boché, Olivier; Baglin, Anne Catherine; Dubost, Geneviève; Leguern, Anne-Sophie; Prevost, Marie-Christine; Bracha, Rivka; Mirelman, David; Guillén, Nancy; Labruyère, Elisabeth

    2009-01-01

    Amoebiasis (a human intestinal infection affecting 50 million people every year) is caused by the protozoan parasite Entamoeba histolytica. To study the molecular mechanisms underlying human colon invasion by E. histolytica, we have set up an ex vivo human colon model to study the early steps in amoebiasis. Using scanning electron microscopy and histological analyses, we have established that E. histolytica caused the removal of the protective mucus coat during the first two hours of incubation, detached the enterocytes, and then penetrated into the lamina propria by following the crypts of Lieberkühn. Significant cell lysis (determined by the release of lactodehydrogenase) and inflammation (marked by the secretion of pro-inflammatory molecules such as interleukin 1 beta, interferon gamma, interleukin 6, interleukin 8 and tumour necrosis factor) were detected after four hours of incubation. Entamoeba dispar (a closely related non-pathogenic amoeba that also colonizes the human colon) was unable to invade colonic mucosa, lyse cells or induce an inflammatory response. We also examined the behaviour of trophozoites in which genes coding for known virulent factors (such as amoebapores, the Gal/GalNAc lectin and the cysteine protease 5 (CP-A5), which have major roles in cell death, adhesion (to target cells or mucus) and mucus degradation, respectively) were silenced, together with the corresponding tissue responses. Our data revealed that the signalling via the heavy chain Hgl2 or via the light chain Lgl1 of the Gal/GalNAc lectin is not essential to penetrate the human colonic mucosa. In addition, our study demonstrates that E. histolytica silenced for CP-A5 does not penetrate the colonic lamina propria and does not induce the host's pro-inflammatory cytokine secretion. PMID:19936071

  14. Evaluation of the Triage Micro Parasite Panel for Detection of Giardia lamblia, Entamoeba histolytica/Entamoeba dispar, and Cryptosporidium parvum in Patient Stool Specimens

    PubMed Central

    Sharp, Susan E.; Suarez, Clarisa A.; Duran, Yolanda; Poppiti, Robert J.

    2001-01-01

    A study comparing the Triage Micro Parasite Panel (Biosite Diagnostics, Inc., San Diego, Calif.) to conventional O&P examination (O&P) was performed using patient fecal specimens. Five hundred twenty-three stool samples were compared. Nineteen specimens were found to be positive by Triage, and 29 were found to be positive by O&P. Seven specimens were positive for Giardia lamblia, four were positive for Entamoeba histolytica/E. dispar, and three were positive for Cryptosporidium parvum as determined by both methods. There was one false positive by Triage (C. parvum) and four false negatives by O&P (two G. lamblia, one E. histolytica/E. dispar, and one C. parvum). The Triage test accurately detected all 18 specimens that contained one of the three organisms that it was designed to detect. The Triage test is a rapid, easy-to-use enzyme immunoassay for the detection of G. lamblia, E. histolytica/E. dispar, and C. parvum in fresh or fresh-frozen fecal specimens. These data suggest that the Triage test can be used as a screen for the immediate testing of stool specimens for these three pathogenic parasites. If Triage test results are negative, O&P can be performed if parasitic infections other than G. lamblia, E. histolytica/E. dispar, or C. parvum are suspected. PMID:11136793

  15. Molecular epidemiology of amoebiasis in Malaysia: highlighting the different risk factors of Entamoeba histolytica and Entamoeba dispar infections among Orang Asli communities.

    PubMed

    Anuar, Tengku Shahrul; Al-Mekhlafi, Hesham M; Abdul Ghani, Mohamed Kamel; Abu Bakar, Edariah; Azreen, Siti Nor; Salleh, Fatmah Md; Ghazali, Nuraffini; Bernadus, Mekadina; Moktar, Norhayati

    2012-12-01

    Currently, species-specific information on Entamoeba infections is unavailable in Malaysia and is restricted worldwide due to the re-description of pathogenic Entamoeba histolytica and non-pathogenic Entamoeba dispar and Entamoeba moshkovskii. Therefore, this cross-sectional study was conducted to provide the first known documented data on the true prevalence of these three species in western Malaysia using a molecular method. Another aim of this study was to determine the association of potential risk factors associated with each Entamoeba sp. A total of 500 stool samples from three Orang Asli tribes were randomly collected. The overall prevalence of E. histolytica, E. dispar and E. moshkovskii determined by microscopy was 18.6% (93/500). Molecular analysis revealed that while most Entamoeba-positive individuals were infected with E. dispar (13.4%), followed by E. histolytica (3.2%) and E. moshkovskii (1.0%), the present findings show low prevalence rates of mixed infections with E. histolytica and E. dispar (2%), E. dispar and E. moshkovskii (1.2%) and association infections of E. histolytica, E. dispar and E. moshkovskii (0.4%). Logistical regression analysis indicates that the dynamics of the transmission of the three Entamoeba spp. was different. Of six statistically significant variables observed in the univariate analysis, three were retained as significant risk factors for E. histolytica infection in the logistical regression model. These factors were (i) not washing hands after playing with soil or gardening (Odds ratio (OR)=4.7; 95% confidence level (CI)=1.38, 16.14; P=0.013), (ii) indiscriminate defecation in the river or bush (OR=5.7; 95% CI=1.46, 21.95; P=0.012) and (iii) close contact with domestic animals (OR=5.4; 95% CI=1.36, 2.51; P=0.017). However, subjects with family members who were infected with E. histolytica/E. dispar/E. moshkovskii (OR=3.8; 95 CI=2.11, 6.86; P<0.001) and those who consumed raw vegetables (OR=1.8; 95% CI=1.01, 3.23; P=0

  16. Protection of gerbils from amebic liver abscess by immunization with a recombinant protein derived from the 170-kilodalton surface adhesin of Entamoeba histolytica.

    PubMed Central

    Zhang, T; Stanley, S L

    1994-01-01

    The protozoan parasite Entamoeba histolytica causes extensive morbidity and mortality worldwide through intestinal infection and amebic liver abscess. Here we show that vaccination of gerbils, a standard model for amebic liver abscess, with recombinant proteins derived from the 170-kDa galactose-binding adhesin of E. histolytica and the serine-rich E. histolytica protein or a combination of the two recombinant antigens provides excellent protection against subsequent hepatic challenge with virulent E. histolytica trophozoites. PMID:8188384

  17. Transfection and continuous expression of heterologous genes in the protozoan parasite Entamoeba histolytica.

    PubMed Central

    Hamann, L; Nickel, R; Tannich, E

    1995-01-01

    To provide tools for functional molecular genetics of the protozoan parasite Entamoeba histolytica, we investigated the use of the prokaryotic neomycin phosphotransferase (NEO) gene as a selectable marker for the transfection of the parasite. An Escherichia coli-derived plasmid vector was constructed (pA5'A3'NEO) containing the NEO coding region flanked by untranslated 5' and 3' sequences of an Ent. histolytica actin gene. Preceding experiments had revealed that amoebae are highly sensitive to the neomycin analogue G418 and do not survive in the presence of as little as 2 micrograms/ml. Transfection of circular pA5'A3'NEO via electroporation resulted in Ent. histolytica trophozoites resistant to G418 up to 100 micrograms/ml. DNA and RNA analyses of resistant cells indicated that (i) the transfected DNA was not integrated into the amoeba genome but was segregated episomally, (ii) in the amoebae, the plasmid replicated autonomously, (iii) the copy number of the plasmid and the expression of NEO-specific RNA were proportional to the amount of G418 used for selection, and (iv) under continuous selection, the plasmid was propagated over an observation period of 6 months. Moreover, the plasmid could be recloned into E. coli and was found to be unrearranged. To investigate the use of pA5'A3'NEO to coexpress other genes in Ent. histolytica, a second marker, the prokaryotic chloramphenicol acetyltransferase (CAT) gene under control of an Ent. histolytica lectin gene promoter was introduced into the plasmid. Transfection of the amoebae with this construct also conferred G418 resistance and, in addition, allowed continuous expression of CAT activity in quantities corresponding to the amount of G418 used for selection. When selection was discontinued, transfected plasmids were lost as indicated by an exponential decline of CAT activity in trophozoite extracts. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7568055

  18. Entamoeba histolytica: a unicellular organism containing two active genes encoding for members of the TBP family.

    PubMed

    Castañon-Sanchez, Carlos Alberto; Luna-Arias, Juan Pedro; de Dios-Bravo, Ma Guadalupe; Herrera-Aguirre, Maria Esther; Olivares-Trejo, Jose J; Orozco, Esther; Hernandez, Jose Manuel

    2010-03-01

    Entamoeba histolytica is the protozoan parasite which causes human amoebiasis. In this parasite, few encoding genes for transcription factors have been cloned and characterized. The E. histolytica TATA-box binding protein (EhTBP) is the first basal transcription factor that has been studied. To continue with the identification of other members of the basal transcription machinery, we performed an in silico analysis of the E. histolytica genome and found three loci encoding for polypeptides with similarity to EhTBP. One locus has a 100% identity to the previously Ehtbp gene reported by our group. The second locus encodes for a 212 aa polypeptide that is 100% identical to residues 23-234 from EhTBP. The third one encodes for a 216 aa polypeptide of 24kDa that showed 42.6% identity and 73.7% similarity to EhTBP. This protein was named E. histolytica TBP-related factor 1 (EhTRF1). Ehtrf1 gene was expressed in bacteria and the purified 28kDa recombinant polypeptide showed the capacity to bind to TATTTAAA-box by electrophoretic mobility shift assays. K(D) values for rEhTBP and rEhTRF1 were (1.71+/-2.90)x10(-12)M and (1.12+/-0.160)x10(-11)M, respectively. Homology modeling of EhTRF1 and EhTBP revealed that, although they were very similar, they showed some differences on their surfaces. Thus, E. histolytica is a unicellular organism having two members of the TBP family. PMID:20026212

  19. Crystal structure and putative substrate identification for the Entamoeba histolytica low molecular weight tyrosine phosphatase.

    PubMed

    Linford, Alicia S; Jiang, Nona M; Edwards, Thomas E; Sherman, Nicholas E; Van Voorhis, Wesley C; Stewart, Lance J; Myler, Peter J; Staker, Bart L; Petri, William A

    2014-01-01

    Entamoeba histolytica is a eukaryotic intestinal parasite of humans, and is endemic in developing countries. We have characterized the E. histolytica putative low molecular weight protein tyrosine phosphatase (LMW-PTP). The structure for this amebic tyrosine phosphatase was solved, showing the ligand-induced conformational changes necessary for binding of substrate. In amebae, it was expressed at low but detectable levels as detected by immunoprecipitation followed by immunoblotting. A mutant LMW-PTP protein in which the catalytic cysteine in the active site was replaced with a serine lacked phosphatase activity, and was used to identify a number of trapped putative substrate proteins via mass spectrometry analysis. Seven of these putative substrate protein genes were cloned with an epitope tag and overexpressed in amebae. Five of these seven putative substrate proteins were demonstrated to interact specifically with the mutant LMW-PTP. This is the first biochemical study of a small tyrosine phosphatase in Entamoeba, and sets the stage for understanding its role in amebic biology and pathogenesis. PMID:24548880

  20. Crystal structure and putative substrate identification for the Entamoeba histolytica low molecular weight tyrosine phosphatase

    PubMed Central

    Linford, Alicia S.; Jiang, Nona M.; Edwards, Thomas E.; Sherman, Nicholas E.; Van Voorhis, Wesley C.; Stewart, Lance J.; Myler, Peter J.; Staker, Bart L.; Petri, William A.

    2014-01-01

    Entamoeba histolytica is a eukaryotic intestinal parasite of humans, and is endemic in developing countries. We have characterized the E. histolytica putative low molecular weight protein tyrosine phosphatase (LMW-PTP). The structure for this amebic tyrosine phosphatase was solved, showing the ligand-induced conformational changes necessary for binding of substrate. In amebae, it was expressed at low but detectable levels as detected by immunoprecipitation followed by immunoblotting. A mutant LMW-PTP protein in which the catalytic cysteine in the active site was replaced with a serine lacked phosphatase activity, and was used to identify a number of trapped putative substrate proteins via mass spectrometry analysis. Seven of these putative substrate protein genes were cloned with an epitope tag and overexpressed in amebae. Five of these seven putative substrate proteins were demonstrated to interact specifically with the mutant LMW-PTP. This is the first biochemical study of a small tyrosine phosphatase in Entamoeba, and sets the stage for understanding its role in amebic biology and pathogenesis. PMID:24548880

  1. Antisense Inhibition of Expression of Cysteine Proteinases Affects Entamoeba histolytica-Induced Formation of Liver Abscess in Hamsters

    PubMed Central

    Ankri, Serge; Stolarsky, Tamara; Bracha, Rivka; Padilla-Vaca, Felipe; Mirelman, David

    1999-01-01

    Trophozoites of virulent Entamoeba histolytica transfected with the antisense gene encoding cysteine proteinase 5 (CP5) have only 10% of the CP activity but retain their cytopathic activity on mammalian monolayers. In the present study we found that the transfected trophozoites with low levels of CP activity were incapable of inducing the formation of liver lesions in hamsters. PMID:9864246

  2. (1)H, (13)C and (15)N NMR assignments of a calcium-binding protein from Entamoeba histolytica.

    PubMed

    Verma, Deepshikha; Bhattacharya, Alok; Chary, Kandala V R

    2016-04-01

    We report almost complete sequence specific (1)H, (13)C and (15)N NMR assignments of a 150-residue long calmodulin-like calcium-binding protein from Entamoeba histolytica (EhCaBP6), as a prelude to its structural and functional characterization. PMID:26377206

  3. Prevalence of Entamoeba histolytica, Giardia lamblia, and Cryptosporidium spp. in Libya: 2000–2015

    PubMed Central

    Ghenghesh, Khalifa Sifaw; Ghanghish, Khaled; BenDarif, Elloulu T.; Shembesh, Khaled; Franka, Ezzadin

    2016-01-01

    Introduction The intestinal protozoa Entamoeba histolytica, Giardia lamblia, and Cryptosporidium spp. are the causative agents of giardiasis, amebiasis, and cryptosporidiosis, respectively. Adequate knowledge of the geographical distribution of parasites and the demographic variables that influence their prevalence is important for effective control of infection in at-risk populations. Methods The data were obtained by an English language literature search of Medline and PubMed for papers using the search terms ‘intestinal parasites and Libya, G. lamblia and Libya, E. histolytica and Libya and Cryptosporidium and Libya’ for the period 2000–2015. Results The data obtained for the period 2000–2015 showed prevalence rates of 0.8–36.6% (mean 19.9%) for E. histolytica/dispar, 1.2–18.2% (mean 4.6%) for G. lamblia and 0.9–13% (mean 3.4%) for Cryptosporidium spp. among individuals in Libya with gastroenteritis (GE). On the other hand, prevalence rates of 0.8–16.3% (mean 8.3%), 1.8–28.8% (mean 4.8%), and 1.0–2.5% (mean=2.4), respectively, were observed for individuals without GE. The mean prevalence rate of E. histolytica/dispar was significantly higher among individuals with GE compared with those without GE (p<0.0000001, OR=2.74). No significant difference in prevalence rate of the three organisms was found according to gender, but most of infections were observed in children aged 10 years or younger. Conclusion The reviewed data suggest that E. histolytica, G. lamblia, and Cryptosporidium spp. may play a minor role in GE in Libya. The observed high prevalence rates of E. histolytica/dispar reported from Libya could be due mainly to the non-pathogenic E. dispar and E. moshkovskii. However, more studies are needed in the future using E. histolytica-specific enzyme immunoassays and/or molecular methods to confirm this observation. PMID:27363524

  4. A mutation in the leptin receptor is associated with Entamoeba histolytica infection in children

    PubMed Central

    Duggal, Priya; Guo, Xiaoti; Haque, Rashidul; Peterson, Kristine M.; Ricklefs, Stacy; Mondal, Dinesh; Alam, Faisal; Noor, Zannatun; Verkerke, Hans P.; Marie, Chelsea; Leduc, Charles A.; Jr., Streamson C. Chua; Jr., Martin G. Myers; Leibel, Rudolph L.; Houpt, Eric; Gilchrist, Carol A.; Sher, Alan; Porcella, Stephen F.; Jr., William A. Petri

    2011-01-01

    Malnutrition substantially increases susceptibility to Entamoeba histolytica in children. Leptin is a hormone produced by adipocytes that inhibits food intake, influences the immune system, and is suppressed in malnourished children. Therefore we hypothesized that diminished leptin function may increase susceptibility to E. histolytica infection. We prospectively observed a cohort of children, beginning at preschool age, for infection by the parasite E. histolytica every other day over 9 years and evaluated them for genetic variants in leptin (LEP) and the leptin receptor (LEPR). We found increased susceptibility to intestinal infection by this parasite associated with an amino acid substitution in the cytokine receptor homology domain 1 of LEPR. Children carrying the allele for arginine (223R) were nearly 4 times more likely to have an infection compared with those homozygous for the ancestral glutamine allele (223Q). An association of this allele with amebic liver abscess was also determined in an independent cohort of adult patients. In addition, mice carrying at least 1 copy of the R allele of Lepr were more susceptible to infection and exhibited greater levels of mucosal destruction and intestinal epithelial apoptosis after amebic infection. These findings suggest that leptin signaling is important in mucosal defense against amebiasis and that polymorphisms in the leptin receptor explain differences in susceptibility of children in the Bangladesh cohort to amebiasis. PMID:21393862

  5. Serum stress responsive gene EhslncRNA of Entamoeba histolytica is a novel long noncoding RNA

    PubMed Central

    Saha, Arpita; Bhattacharya, Sudha; Bhattacharya, Alok

    2016-01-01

    Non coding RNAs are known to play important roles in regulating gene expression at the transcriptional and posttranscriptional levels in metazoans. There is very little information available about non coding RNAs in protists such as Entamoeba histolytica. Antisense and micro RNAs have been reported in E. histolytica, however no long non coding RNAs has been reported yet. Here, we report our findings on an in vitro serum stress-inducible gene EhslncRNA, a member of B1 transmembrane kinase family of E. histolytica. EhslncRNA encodes a transcript of 2.6 kb and sequence analysis revealed that there is no ORF >150 bp within this transcript. The transcript was found to be polyadenylated and mainly associated with monosomes in the cytoplasm under serum starvation. In normal proliferating cells this RNA is mainly present in the nucleus. The promoter element was mapped between 437 to 346 nucleotides upstream of transcriptional start site and has both positive and negative regulatory elements. Deletion of the negative element converted the promoter to serum inducible type. Oxygen and heat stress also increased expression levels of EhslncRNA. These observations suggest that EhslncRNA may be a long non coding RNA and likely to help cells withstand stressful conditions in the host. PMID:27273618

  6. Serum stress responsive gene EhslncRNA of Entamoeba histolytica is a novel long noncoding RNA.

    PubMed

    Saha, Arpita; Bhattacharya, Sudha; Bhattacharya, Alok

    2016-01-01

    Non coding RNAs are known to play important roles in regulating gene expression at the transcriptional and posttranscriptional levels in metazoans. There is very little information available about non coding RNAs in protists such as Entamoeba histolytica. Antisense and micro RNAs have been reported in E. histolytica, however no long non coding RNAs has been reported yet. Here, we report our findings on an in vitro serum stress-inducible gene EhslncRNA, a member of B1 transmembrane kinase family of E. histolytica. EhslncRNA encodes a transcript of 2.6 kb and sequence analysis revealed that there is no ORF >150 bp within this transcript. The transcript was found to be polyadenylated and mainly associated with monosomes in the cytoplasm under serum starvation. In normal proliferating cells this RNA is mainly present in the nucleus. The promoter element was mapped between 437 to 346 nucleotides upstream of transcriptional start site and has both positive and negative regulatory elements. Deletion of the negative element converted the promoter to serum inducible type. Oxygen and heat stress also increased expression levels of EhslncRNA. These observations suggest that EhslncRNA may be a long non coding RNA and likely to help cells withstand stressful conditions in the host. PMID:27273618

  7. Nanoimaging and ultra structure of Entamoeba histolytica and its pseudopods by using atomic force microscope

    NASA Astrophysics Data System (ADS)

    Joshi, Narahari V.; Medina, Honorio; Urdaneta, H.; Barboza, J.

    2000-04-01

    Nan-imaging of Entamoeba histolytica was carried out by using Atomic Force Microscope (AFM). The structure of the nucleus, endoplasm and ectoplasm were studied separately. The diameter of the nucleus in living E. histolytica was found to be of the order of 10 micrometers which is slightly higher than the earlier reported value. The presence of karysome was detected in the nucleus. Well-organized patterns of chromatoid bodies located within the endoplasm, were detected and their repetitive patterns were examined. The organized structure was also extended within the ectoplasm. The dimensions and form of the organization suggest that chromatic bodies are constituted with ribosomes ordered in the form of folded sheet. Such structures were found to be absent in non-living E. histolytica. AFM images were also captured just in the act when ameba was extending its pseudopods. Alteration in the ultrastructure caused during the process of extension was viewed. Well marked canals of width 694.05 nm. And height 211.05 nm are clearly perceptible towards the direction of the pseudopods. 3D images are presented to appreciate the height variation, which can not be achieved by conventional well-established techniques such as electron microscopy.

  8. Entamoeba histolytica TATA-box binding protein binds to different TATA variants in vitro.

    PubMed

    de Dios-Bravo, Guadalupe; Luna-Arias, Juan Pedro; Riverón, Ana María; Olivares-Trejo, José J; López-Camarillo, César; Orozco, Esther

    2005-03-01

    The ability of Entamoeba histolytica TATA binding protein (EhTBP) to interact with different TATA boxes in gene promoters may be one of the key factors to perform an efficient transcription in this human parasite. In this paper we used several TATA variants to study the in vitro EhTBP DNA-binding activity and to determine the TATA-EhTBP dissociation constants. The presence of EhTBP in complexes formed by nuclear extracts (NE) and the TATTTAAA oligonucleotide, which corresponds to the canonical TATA box for E. histolytica, was demonstrated by gel-shift assays. In these experiments a single NE-TATTTAAA oligonucleotide complex was detected. Complex was retarded by anti-EhTBP Igs in supershift experiments and antibodies also recognized the cross-linked complex in Western blot assays. Recombinant EhTBP formed specific complexes with TATA variants found in E. histolytica gene promoters and other TATA variants generated by mutation of TATTTAAA sequence. The dissociation constants of recombinant EhTBP for TATA variants ranged between 1.04 (+/-0.39) x 10(-11) and 1.60 (+/-0.37) x 10(-10) m. TATTTAAA and TAT_ _AAA motifs presented the lowest KD values. Intriguingly, the recombinant EhTBP affinity for TATA variants is stronger than other TBPs reported. In addition, EhTBP is more promiscuous than human and yeast TBPs, probably due to modifications in amino acids involved in TBP-DNA binding. PMID:15752353

  9. Inhibitory Effects of Iranian Thymus vulgaris Extracts on in Vitro Growth of Entamoeba histolytica

    PubMed Central

    Behnia, Maryam; Komeylizadeh, Hossein; Tabaei, Seyyed-Javad Seyyed; Abadi, Alireza

    2008-01-01

    One of the most common drugs used against a wide variety of anaerobic protozoan parasites is metronidazole. However, this drug is mutagenic for bacteria and is a potent carcinogen for rodents. Thymus vulgaris is used for cough suppression and relief of dyspepsia. Also it has antibacterial and antifungal properties. The aim of this study was to investigate antiamebic effect of Thymus vulgaris against Entamoeba histolytica in comparison with metronidazole. One hundred gram air-dried T. vulgaris plant was obtained and macerated at 25℃ for 14 days using n-hexane and a mixture of ethanol and water. For essential oil isolation T. vulgaris was subjected to hydrodistillation using a clevenger-type apparatus for 3 hr. E. histolytica, HM-1: IMSS strain was used in all experiments. It was found that the minimal inhibitory concentration (MIC) for T. vulgaris hydroalcoholic, hexanic extracts, and the essential oil after 24 hr was 4 mg/mL, 4 mg/mL, and 0.7 mg/mL, respectively. After 48 hr the MIC for T. vulgaris hydroalcoholic and hexanic extracts was 3 and 3 mg/mL, respectively. Therefore, it can be concluded that the Iranian T. vulgaris is effective against the trophozoites of E. histolytica. PMID:18830054

  10. Entamoeba histolytica and other parasitic infections in south Kanara district, Karnataka.

    PubMed

    Subbannayya, K; Babu, M H; Kumar, A; Rao, T S; Shivananda, P G

    1989-09-01

    Single stool specimens collected from 1,020 apparently healthy people of the South Kanara District, were processed for intestinal parasites using three parasitological methods viz. (a) Direct smear in physiological saline and D'Antoni's iodine, (b) Zinc sulfate concentration method and (c) by culture in modified Boek and Drbolhav medium. Of these 781 (76.51 per cent) were found to be infected with parasites. The prevalence of various intestinal parasites was: Ascaris lumbricoides (48.33 per cent), Necator americanus (46.86 per cent), Trichuris trichiura (42.75 per cent), Entamoeba coli (23.24 per cent), Entamoeba histolytica (7.94 per cent), Enterobius vermicularis (2.84 per cent), Giardia intestinalis (2.45 per cent), Iodamoeba buitschlii (1.57 per cent), Entamoeba hartmanni (1.37 per cent), Trichomonas hominis (0.88 per cent), Strongyloides stercoralis (0.68 per cent), Hymenolepis nana (0.49 per cent), Chilomastis mesnili (0.10 per cent) and Endolimax nana (0.10 per cent). High incidence of parasitic infections was recorded in females and age group of 6-14 years. PMID:2559118

  11. Cell-matrix interactions of Entamoeba histolytica and E. dispar. A comparative study by electron-, atomic force- and confocal microscopy

    SciTech Connect

    Talamás-Lara, Daniel; Talamás-Rohana, Patricia; Fragoso-Soriano, Rogelio Jaime; Espinosa-Cantellano, Martha; Chávez-Munguía, Bibiana; González-Robles, Arturo; Martínez-Palomo, Adolfo

    2015-10-01

    Invasion of tissues by Entamoeba histolytica is a multistep process that initiates with the adhesion of the parasite to target tissues. The recognition of the non-invasive Entamoeba dispar as a distinct, but closely related protozoan species raised the question as to whether the lack of its pathogenic potential could be related to a weaker adhesion due to limited cytoskeleton restructuring capacity. We here compared the adhesion process of both amebas to fibronectin through scanning, transmission, atomic force, and confocal microscopy. In addition, electrophoretic and western blot assays of actin were also compared. Adhesion of E. histolytica to fibronectin involves a dramatic reorganization of the actin network that results in a tighter contact to and the subsequent focal degradation of the fibronectin matrix. In contrast, E. dispar showed no regions of focal adhesion, the cytoskeleton was poorly reorganized and there was little fibronectin degradation. In addition, atomic force microscopy using topographic, error signal and phase modes revealed clear-cut differences at the site of contact of both amebas with the substrate. In spite of the morphological and genetic similarities between E. histolytica and E. dispar the present results demonstrate striking differences in their respective cell-to-matrix adhesion processes, which may be of relevance for understanding the invasive character of E. histolytica. - Highlights: • Striking differences in adhesion to FN between E. histolytica and E. dispar. • A greater degree of cell stiffness in E. histolytica with respect to E. dispar. • E. histolytica but not E. dispar forms regions of close contact with FN. • The actin cytoskeleton is involved in the pathogenicity of E. histolytica.

  12. Entamoeba histolytica and Trichomonas vaginalis: trophozoite growth inhibition by metronidazole electro-transferred water.

    PubMed

    Heredia-Rojas, J Antonio; Torres-Flores, Antonio Cayetano; Rodríguez-De la Fuente, Abraham O; Mata-Cárdenas, Benito David; Rodríguez-Flores, Laura E; Barrón-González, María Porfiria; Torres-Pantoja, Antonio Cayetano; Alcocer-González, Juan M

    2011-01-01

    The influence of low-frequency electromagnetic (LF-EM) waves on microorganisms has been a subject of experimental investigations for more than two decades and the results are promising. In parallel, an interesting procedure known as biophysical-information-therapy or bioresonance therapy (BRT) which in principle is based on LF-EM stimulation, has emerged. BRT was discovered in the late 1980's but it is still poorly studied. This paper demonstrates that by transferring metronidazole information to water samples by an electronic amplifier (BRT device), the growth of axenically cultured trophozoites of Entamoeba histolytica and Trichomonasvaginalis is significantly inhibited, compared with those cultures treated with non and sham electro-transferred water samples. A positive control of metronidazole, a well-known cytotoxic drug against parasites, was used as a reference. PMID:20603119

  13. Characterization of a cytochalasin D-resistant mutant of Entamoeba histolytica.

    PubMed

    de la Garza, M; Gallegos, B; Meza, I

    1989-01-01

    Characterization of a cytochalasin D-resistant mutant of the human parasite Entamoeba histolytica capable of growing at 10 microM cytochalasin is described. The mutant cells also show resistance to 5 mM colchicine and 100 microM cytochalasin B, drugs proved deleterious for wild type trophozoites. The mutants show increased osmotic fragility and electric mobility but reduced phagocytic activity, and agglutination by Concanavalin A. On the other hand pinocytic activity remains unaltered when compared with the wild type cells. Polymerized actin, seen by staining with phalloidin, often appears polarized to one end of the trophozoites and forms few of the endocytic invaginations found in wild type amebas. An altered distribution of part of the actin could explain the differences in surface properties and motility observed in the mutant amebas. PMID:2557444

  14. Prevalence and Risk Factors Associated with Entamoeba histolytica/dispar/moshkovskii Infection among Three Orang Asli Ethnic Groups in Malaysia

    PubMed Central

    Shahrul Anuar, Tengku; M. Al-Mekhlafi, Hesham; Abdul Ghani, Mohamed Kamel; Osman, Emelia; Mohd Yasin, Azlin; Nordin, Anisah; Nor Azreen, Siti; Md Salleh, Fatmah; Ghazali, Nuraffini; Bernadus, Mekadina; Moktar, Norhayati

    2012-01-01

    Background Entamoeba histolytica/Entamoeba dispar/Entamoeba moshkovskii infection is still prevalent in rural Malaysia especially among Orang Asli communities. Currently, information on prevalence of this infection among different ethnic groups of Orang Asli is unavailable in Malaysia. To contribute to a better comprehension of the epidemiology of this infection, a cross-sectional study aimed at providing the first documented data on the prevalence and risk factors associated with E. histolytica/E. dispar/E. moshkovskii infection was carried out among three Orang Asli ethnic groups (Proto-Malay, Negrito, and Senoi) in selected villages in Negeri Sembilan, Perak, and Pahang states, Malaysia. Methods/Findings Faecal samples were examined by formalin-ether sedimentation and trichrome staining techniques. Of 500 individuals, 8.7% (13/150) of Proto-Malay, 29.5% (41/139) of Negrito, and 18.5% (39/211) of Senoi were positive for E. histolytica/E. dispar/E. moshkovskii, respectively. The prevalence of this infection showed an age-dependency relationship, with higher rates observed among those aged less than 15 years in all ethnic groups studied. Multivariate analysis confirmed that not washing hands after playing with soils or gardening and presence of other family members infected with E. histolytica/E. dispar/E. moshkovskii were significant risk factors of infection among all ethnic groups. However, eating with hands, the consumption of raw vegetables, and close contact with domestic animals were identified as significant risk factors in Senoi. Conclusions Essentially, the findings highlighted that E. histolytica/E. dispar/E. moshkovskii parasites are still prevalent in Malaysia. Further studies using molecular approaches to distinguish the morphologically identical species of pathogenic, E. histolytica from the non-pathogenic, E. dispar and E. moshkovskii are needed. The establishment of such data will be beneficial for the public health authorities in the planning and

  15. Detection of Entamoeba histolytica DNA in the Saliva of Amoebic Liver Abscess Patients Who Received Prior Treatment with Metronidazole

    PubMed Central

    Khairnar, Krishna; Parija, Subhash Chandra

    2008-01-01

    Saliva is an easily-accessible and a non-invasive clinical specimen alternate to blood and liver pus. An attempt was made to detect Entamoeba histolytica DNA released in the saliva of amoebic liver abscess (ALA) patients by applying 16S-like rRNA gene-based nested multiplex polymerase chain reaction (NM-PCR). The NM-PCR detected E. histolytica DNA in the saliva of eight (28.6%) of 28 ALA patients. The NM-PCR result was negative for E. histolytica DNA in the saliva of all the eight ALA patients who were tested prior to treatment with metronidazole but was positive in the saliva of eight (40%) of 20 ALA patient who were tested after therapy with metronidazole. The NM-PCR detected E. histolytica DNA in liver abscess pus of all 28 (100%) patients with ALA. The TechLab E. histolytica II enzyme-linked immunosorbent assay was positive for E. histolytica Gal/GalNAc lectin antigen in the liver abscess pus of 13 (46.4%) of the 28 ALA patients. The indirect haemagglutination (IHA) test was positive for anti-amoebic antibodies in the serum of 22 (78.6%) of the 28 ALA patients and 2 (5.7%) of 35 healthy controls. The present study, for the first time, demonstrates the release of E. histolytica DNA in the saliva of ALA patients by applying NM-PCR. PMID:19069620

  16. Detection of Entamoeba histolytica DNA in the saliva of amoebic liver abscess patients who received prior treatment with metronidazole.

    PubMed

    Khairnar, Krishna; Parija, Subhash Chandra

    2008-12-01

    Saliva is an easily-accessible and a non-invasive clinical specimen alternate to blood and liver pus. An attempt was made to detect Entamoeba histolytica DNA released in the saliva of amoebic liver abscess (ALA) patients by applying 16S-like rRNA gene-based nested multiplex polymerase chain reaction (NM-PCR). The NM-PCR detected E. histolytica DNA in the saliva of eight (28.6%) of 28 ALA patients. The NM-PCR result was negative for E. histolytica DNA in the saliva of all the eight ALA patients who were tested prior to treatment with metronidazole but was positive in the saliva of eight (40%) of 20 ALA patient who were tested after therapy with metronidazole. The NM-PCR detected E. histolytica DNA in liver abscess pus of all 28 (100%) patients with ALA. The TechLab E. histolytica II enzyme-linked immunosorbent assay was positive for E. histolytica Gal/GalNAc lectin antigen in the liver abscess pus of 13 (46.4%) of the 28 ALA patients. The indirect haemagglutination (IHA) test was positive for anti-amoebic antibodies in the serum of 22 (78.6%) of the 28 ALA patients and 2 (5.7%) of 35 healthy controls. The present study, for the first time, demonstrates the release of E. histolytica DNA in the saliva of ALA patients by applying NM-PCR. PMID:19069620

  17. Real-time analysis of gut flora in Entamoeba histolytica infected patients of Northern India

    PubMed Central

    2012-01-01

    Background Amebic dysentery is caused by the protozoan parasite Entamoeba histolytica and the ingestion of quadrinucleate cyst of E. histolytica from fecally contaminated food or water initiates infection. Excystation occurs in the lumen of small intestine, where motile and potentially invasive trophozoites germinate from cysts. The ability of trophozoites to interact and digest gut bacteria is apparently important for multiplication of the parasite and its pathogenicity; however the contribution of resident bacterial flora is not well understood. We quantified the population of Bacteroides, Bifidobacterium, Ruminococcus, Lactobacillus, Clostridium leptum subgroup, Clostridium coccoides subgroup, Eubacterium, Campylobacter, Methanobrevibacter smithii and Sulphur reducing bacteria using genus specific primers in healthy (N = 22) vs amebic patients (E. histolytica positive, N = 17) stool samples by Real-time PCR. Results Absolute quantification of Bacteroides (p = .001), Closrtridium coccoides subgroup (p = 0.002), Clostridium leptum subgroup (p = 0.0001), Lactobacillus (p = 0.037), Campylobacter (p = 0.0014) and Eubacterium (p = 0.038) show significant drop in their population however, significant increase in Bifdobacterium (p = 0.009) was observed where as the population of Ruminococcus (p = 0.33) remained unaltered in healthy vs amebic patients (E. histolytica positive). We also report high prevalence of nimE gene in stool samples of both healthy volunteers and amebic patients. No significant decrease in nimE gene copy number was observed before and after the treatment with antiamebic drug. Conclusions Our results show significant alteration in predominant gut bacteria in E. histolytica infected individuals. The frequent episodes of intestinal amoebic dysentery thus result in depletion of few predominant genera in gut that may lead to poor digestion and absorption of food in intestine. It further disturbs the homeostasis

  18. Viruses of Entamoeba histolytica. 3. Properties of the polyhedral virus of the HB-301 strain.

    PubMed

    Hruska, J F; Mattern, C F; Diamond, L S; Keister, D B

    1973-01-01

    Some biological characteristics and bioassay of a polyhedral virus isolated from normal-appearing strain HB-301 of Entamoeba histolytica are described. The virus produced lysis of susceptible strains of E. histolytica, yet it caused no cytopathological effect in the host strain, HB-301. The virus and host amoeba existed in an equilibrium; approximately 10(4) mean infective dose units of virus were produced per million HB-301 amoebae. Superinfection and antiviral antiserum treatment failed to disturb this equilibrium permanently. The mechanism of viral persistence in strain HB-301 amoebae remains to be determined. Purification of the virus was attempted. Ninety-nine percent of the viral infectivity was associated with a low-speed pellet which consisted of complexes of virus and cellular membranes. Various treatments of this low-speed pellet failed to release virus. Biologically active, membrane-free virus of low titer was prepared by differential centrifugation of supernatant solutions and employed in electron microscopy and other studies. PMID:4346278

  19. Structural determinants of RGS-RhoGEF signaling critical to Entamoeba histolytica pathogenesis.

    PubMed

    Bosch, Dustin E; Kimple, Adam J; Manning, Alyssa J; Muller, Robin E; Willard, Francis S; Machius, Mischa; Rogers, Stephen L; Siderovski, David P

    2013-01-01

    G protein signaling pathways, as key components of physiologic responsiveness and timing, are frequent targets for pharmacologic intervention. Here, we identify an effector for heterotrimeric G protein α subunit (EhGα1) signaling from Entamoeba histolytica, the causative agent of amoebic colitis. EhGα1 interacts with this effector and guanosine triphosphatase-accelerating protein, EhRGS-RhoGEF, in a nucleotide state-selective fashion. Coexpression of EhRGS-RhoGEF with constitutively active EhGα1 and EhRacC leads to Rac-dependent spreading in Drosophila S2 cells. EhRGS-RhoGEF overexpression in E. histolytica trophozoites leads to reduced migration toward serum and lower cysteine protease activity, as well as reduced attachment to, and killing of, host cells. A 2.3 Å crystal structure of the full-length EhRGS-RhoGEF reveals a putative inhibitory helix engaging the Dbl homology domain Rho-binding surface and the pleckstrin homology domain. Mutational analysis of the EhGα1/EhRGS-RhoGEF interface confirms a canonical "regulator of G protein signaling" domain rather than a RhoGEF-RGS ("rgRGS") domain, suggesting a convergent evolution toward heterotrimeric and small G protein cross-talk. PMID:23260656

  20. Structural Determinants of RGS-RhoGEF Signaling Critical to Entamoeba histolytica Pathogenesis

    PubMed Central

    Bosch, Dustin E.; Kimple, Adam J.; Manning, Alyssa J.; Muller, Robin E.; Willard, Francis S.; Machius, Mischa; Rogers, Stephen L.; Siderovski, David P.

    2012-01-01

    Summary G-protein signaling pathways, as key components of physiologic responsiveness and timing, are frequent targets for pharmacologic intervention. Here, we identify an effector for heterotrimeric G-protein α subunit (EhGα1) signaling from Entamoeba histolytica, the causative agent of amoebic colitis. EhGα1 interacts with this effector and GTPase-accelerating protein (GAP), EhRGS-RhoGEF, in a nucleotide state-selective fashion. Co-expression of EhRGS-RhoGEF with constitutively active EhGα1 and EhRacC leads to Rac-dependent spreading in Drosophila S2 cells. EhRGS-RhoGEF overexpression in E. histolytica trophozoites leads to reduced migration toward serum and lower cysteine protease activity, as well as reduced attachment to, and killing of, host cells. A 2.3 Å crystal structure of the full-length EhRGS-RhoGEF reveals a putative inhibitory helix engaging the DH domain Rho-binding surface and the PH domain. Mutational analysis of the EhGα1/EhRGS-RhoGEF interface confirms a canonical RGS domain rather than a RhoGEF-RGS (“rgRGS”) domain, suggesting a convergent evolution toward heterotrimeric and small G-protein cross-talk. PMID:23260656

  1. Archaeological occurrences and historical review of the human amoeba, Entamoeba histolytica, over the past 6000years.

    PubMed

    Le Bailly, Matthieu; Maicher, Céline; Dufour, Benjamin

    2016-08-01

    Understanding parasite history and the evolution of host/parasite relationships is one of the most important aspects of paleoparasitology. Within the framework of this research topic, this paper focuses on the human pathogenic amoeba, Entamoeba histolytica. The compilation of all the available archaeological data concerning this parasite leads to a first glimpse of the history of this parasite of current medical importance. Paleoparasitological investigation into this parasite uses immunological techniques and shows that the modern strain of E. histolytica has been present in Western Europe since at least the Neolithic period (3700yearsBCE), and could have originated in the Old World. The appearance of the modern amoeba strain in the pre-Columbian Americas and the Middle East around the 12th century CE gives rise to hypotheses as to how human migrations (Atlantic or Pacific routes) contributed to the diffusion of this pathogen, resulting in its current distribution. This compilation proves that parasites are valuable proxies for studying past human and animal migrations, and should be given more consideration in the future. PMID:27130884

  2. Trogocytosis by Entamoeba histolytica contributes to cell killing and tissue invasion.

    PubMed

    Ralston, Katherine S; Solga, Michael D; Mackey-Lawrence, Nicole M; Somlata; Bhattacharya, Alok; Petri, William A

    2014-04-24

    Entamoeba histolytica is the causative agent of amoebiasis, a potentially fatal diarrhoeal disease in the developing world. The parasite was named "histolytica" for its ability to destroy host tissues, which is probably driven by direct killing of human cells. The mechanism of human cell killing has been unclear, although the accepted model was that the parasites use secreted toxic effectors to kill cells before ingestion. Here we report the discovery that amoebae kill by ingesting distinct pieces of living human cells, resulting in intracellular calcium elevation and eventual cell death. After cell killing, amoebae detach and cease ingestion. Ingestion of human cell fragments is required for cell killing, and also contributes to invasion of intestinal tissue. The internalization of fragments of living human cells is reminiscent of trogocytosis (from Greek trogo, nibble) observed between immune cells, but amoebic trogocytosis differs because it results in death. The ingestion of live cell material and the rejection of corpses illuminate a stark contrast to the established model of dead cell clearance in multicellular organisms. These findings change the model for tissue destruction in amoebiasis and suggest an ancient origin of trogocytosis as a form of intercellular exchange. PMID:24717428

  3. In vitro induction of Entamoeba histolytica cyst-like structures from trophozoites.

    PubMed

    Aguilar-Díaz, Hugo; Díaz-Gallardo, Martha; Laclette, Juan P; Carrero, Julio C

    2010-01-01

    Inhibition of encystment can be conceived as a potentially useful mechanism to block the transmission of Entamoeba histolytica under natural conditions. Unfortunately, amoeba encystment has not been achieved in vitro and drugs inhibiting the formation of cysts are not available. Luminal conditions inducing encystment in vivo are also unknown, but cellular stress such as exposure to reactive oxygen species from immune cells or intestinal microbiota could be involved. A role for certain divalent cations as cofactors of enzymes involved in excystment has also been described. In this study, we show that trophozoite cultures, treated with hydrogen peroxide in the presence of trace amounts of several cations, transform into small-sized spherical and refringent structures that exhibit resistance to different detergents. Ultrastructural analysis under scanning and transmission electron microscopy revealed multinucleated structures (some with four nuclei) with smooth, thick membranes and multiple vacuoles. Staining with calcofluor white, as well as an ELISA binding assay using wheat germ agglutinin, demonstrated the presence of polymers of N-acetylglucosamine (chitin), which is the primary component of the natural cyst walls. Over-expression of glucosamine 6-phosphate isomerase, likely to be the rate-limiting enzyme in the chitin synthesis pathway, was also confirmed by RT-PCR. These results suggest that E. histolytica trophozoites activated encystment pathways when exposed to our treatment. PMID:20169067

  4. Rat and human colonic mucins bind to and inhibit adherence lectin of Entamoeba histolytica.

    PubMed Central

    Chadee, K; Petri, W A; Innes, D J; Ravdin, J I

    1987-01-01

    Establishment of adherence by Entamoeba histolytica is mediated by a 170-kD Gal/GalNAc inhibitable lectin and is required for cytolysis and phagocytosis of mammalian target cells. We studied the biochemical mechanisms of the in vitro interaction between rat and human colonic mucins and axenic E. histolytica trophozoites. Crude mucus prevented amebic adherence to Chinese hamster ovary (CHO) cells by up to 70%. Purification of the colonic mucins by Sepharose 4B chromatography, nuclease digestion, and cesium chloride gradient centrifugation resulted in a 1,000-fold enrichment of the inhibitory mucins. Purified rat mucin inhibited amebic adherence to and cytolysis of homologous rat colonic epithelial cells. Oxidation and enzymatic cleavage of rat mucin Gal and GalNAc residues completely abrogated mucin inhibition of amebic adherence. The binding of rat 125I-mucin to amebae was galactose specific, saturable, reversible, and pH dependent. A monoclonal antibody specific for the 170-kD amebic Gal/GalNAc lectin completely inhibited the binding of rat 125I-mucin. Rat mucin bound to Affigel affinity purified the amebic lectin from conditioned medium. Colonic mucin glycoproteins act as an important host defense by binding to the parasite's adherence lectin, thus preventing amebic attachment to and cytolysis of host epithelial cells. Images PMID:2890655

  5. Kinetics and strain variation of phagosome proteins of Entamoeba histolytica by proteomic analysis.

    PubMed

    Okada, Mami; Huston, Christopher D; Oue, Miho; Mann, Barbara J; Petri, William A; Kita, Kiyoshi; Nozaki, Tomoyoshi

    2006-02-01

    The protozoan parasite Entamoeba histolytica ingests and feeds on microorganisms and mammalian cells. Phagocytosis is essential for cell growth and implicated in pathogenesis of E. histolytica. We report here the dynamic changes of phagosome proteins during phagosome maturation by proteomic analysis using reversed-phase capillary liquid chromatography and ion trap tandem mass spectrometry. Phagosomes were isolated at various intervals after internalization of latex beads. Immunoblot analysis and electron microscopy verified successful isolation of phagosomes. A total of 159 proteins were identified from the reference strain HM1 at different stages of phagosome maturation. Approximately 70% of them were detected in a time-dependent fashion, suggesting dynamism of phagosome biogenesis. The kinetics of representative proteins were verified by immunoblots and also by video microscopy of live transgenic amebae expressing green fluorescent protein-fused EhRab7A. Furthermore, we observed significant differences in phagosome profiles between HM1 and two recent clinical isolates. Approximately 60% of 229 proteins detected in at least one of these three strains were identified only in one strain, while approximately 20% of these proteins were detected in all three strains. These data should provide significant insights into molecular characterization of phagosome biogenesis, and help to elucidate the pathogenesis of this important infection. PMID:16290089

  6. A Proteomic and Cellular Analysis of Uropods in the Pathogen Entamoeba histolytica

    PubMed Central

    Marquay Markiewicz, Jacques; Syan, Sylvie; Hon, Chung-Chau; Weber, Christian; Faust, Daniela; Guillen, Nancy

    2011-01-01

    Exposure of Entamoeba histolytica to specific ligands induces cell polarization via the activation of signalling pathways and cytoskeletal elements. The process leads to formation of a protruding pseudopod at the front of the cell and a retracting uropod at the rear. In the present study, we show that the uropod forms during the exposure of trophozoites to serum isolated from humans suffering of amoebiasis. To investigate uropod assembly, we used LC-MS/MS technology to identify protein components in isolated uropod fractions. The galactose/N-acetylgalactosamine lectin, the immunodominant antigen M17 (which is specifically recognized by serum from amoeba-infected persons) and a few other cells adhesion-related molecules were primarily involved. Actin-rich cytoskeleton components, GTPases from the Rac and Rab families, filamin, α-actinin and a newly identified ezrin-moesin-radixin protein were the main factors found to potentially interact with capped receptors. A set of specific cysteine proteases and a serine protease were enriched in isolated uropod fractions. However, biological assays indicated that cysteine proteases are not involved in uropod formation in E. histolytica, a fact in contrast to the situation in human motile immune cells. The surface proteins identified here are testable biomarkers which may be either recognized by the immune system and/or released into the circulation during amoebiasis. PMID:21483708

  7. Repurposing the Open Access Malaria Box To Discover Potent Inhibitors of Toxoplasma gondii and Entamoeba histolytica

    PubMed Central

    Fokou, Patrick V. T.; Tchokouaha, Lauve R. Y.; Spangenberg, Thomas; Mfopa, Alvine N.; Kouipou, Ruffin M. T.; Mbouna, Cedric J.; Donfack, Valerie F. Donkeng; Zollo, Paul H. A.

    2014-01-01

    Toxoplasmosis and amebiasis are important public health concerns worldwide. The drugs currently available to control these diseases have proven limitations. Therefore, innovative approaches should be adopted to identify and develop new leads from novel scaffolds exhibiting novel modes of action. In this paper, we describe results from the screening of compounds in the Medicines for Malaria Venture (MMV) open access Malaria Box in a search for new anti-Toxoplasma and anti-Entamoeba agents. Standard in vitro phenotypic screening procedures were adopted to assess their biological activities. Seven anti-Toxoplasma compounds with a 50% inhibitory concentration (IC50) of <5 μM and selectivity indexes (SI) of >6 were identified. The most interesting compound was MMV007791, a piperazine acetamide, which has an IC50 of 0.19 μM and a selectivity index of >157. Also, we identified two compounds, MMV666600 and MMV006861, with modest activities against Entamoeba histolytica, with IC50s of 10.66 μM and 15.58 μM, respectively. The anti-Toxoplasma compounds identified in this study belong to scaffold types different from those of currently used drugs, underscoring their novelty and potential as starting points for the development of new antitoxoplasmosis drugs with novel modes of action. PMID:25049259

  8. Nitroimidazole carboxamides as antiparasitic agents targeting Giardia lamblia, Entamoeba histolytica and Trichomonas vaginalis.

    PubMed

    Jarrad, A M; Debnath, A; Miyamoto, Y; Hansford, K A; Pelingon, R; Butler, M S; Bains, T; Karoli, T; Blaskovich, M A T; Eckmann, L; Cooper, M A

    2016-09-14

    Diarrhoeal diseases caused by the intestinal parasites Giardia lamblia and Entamoeba histolytica constitute a major global health burden. Nitroimidazoles are first-line drugs for the treatment of giardiasis and amebiasis, with metronidazole 1 being the most commonly used drug worldwide. However, treatment failures in giardiasis occur in up to 20% of cases and development of resistance to metronidazole is of concern. We have re-examined 'old' nitroimidazoles as a foundation for the systematic development of next-generation derivatives. Using this approach, derivatisation of the nitroimidazole carboxamide scaffold provided improved antiparasitic agents. Thirty-three novel nitroimidazole carboxamides were synthesised and evaluated for activity against G. lamblia and E. histolytica. Several of the new compounds exhibited potent activity against G. lamblia strains, including metronidazole-resistant strains of G. lamblia (EC50 = 0.1-2.5 μM cf. metronidazole EC50 = 6.1-18 μM). Other compounds showed improved activity against E. histolytica (EC50 = 1.7-5.1 μM cf. metronidazole EC50 = 5.0 μM), potent activity against Trichomonas vaginalis (EC50 = 0.6-1.4 μM cf. metronidazole EC50 = 0.8 μM) and moderate activity against the intestinal bacterial pathogen Clostridium difficile (0.5-2 μg/mL, cf. metronidazole = 0.5 μg/mL). The new compounds had low toxicity against mammalian kidney and liver cells (CC50 > 100 μM), and selected antiparasitic hits were assessed for human plasma protein binding and metabolic stability in liver microsomes to demonstrate their therapeutic potential. PMID:27236016

  9. Production of mouse monoclonal antibodies which inhibit in vitro adherence of Entamoeba histolytica trophozoites.

    PubMed Central

    Ravdin, J I; Petri, W A; Murphy, C F; Smith, R D

    1986-01-01

    Adherence by axenic Entamoeba histolytica trophozoites to mammalian cells is mediated by an N-acetylgalactosamine (GalNAc)-inhibitable adhesin on the surface of the parasite. We isolated 35 hybridoma cell lines producing antibodies to E. histolytica as indicated by ELISA with sonicated amebic protein or by immunofluorescence assay with fixed whole trophozoites. Tissue culture supernatants were further screened for subcloning by the ability to bind to Chinese hamster ovary (CHO) cells which were first exposed to a partially purified soluble preparation of the amebic GalNAc-inhibitable lectin. Eight tissue culture supernatants were positive in this assay. Antibodies from four subcloned cell lines (D3-14, H8-5, I12-2, and I1-21) inhibited amebic adherence to CHO cells (P less than 0.01). Of the original 35 tissue culture supernatants, 3 also inhibited amebic adherence (P less than 0.01; F1, F14, and J10); monoclonal antibodies in these supernatants did not bind to lectin-exposed CHO cells. Three purified monoclonal antibodies (H8-5, I12-2, and I1-21) inhibited amebic adherence at greater than or equal to 2 micrograms/10(4) amebae (P less than 0.05). None of these inhibitory monoclonal antibodies immunoprecipitated with a soluble amebic protein preparation following sodium dodecyl sulfate-polyacrylamide gel electrophoresis under denaturing conditions. Monoclonal antibodies which inhibit in vitro adherence by E. histolytica will be useful in purification of the GalNAc-inhibitable lectin. PMID:2873102

  10. Adherence of Entamoeba histolytica trophozoites to rat and human colonic mucosa.

    PubMed Central

    Ravdin, J I; John, J E; Johnston, L I; Innes, D J; Guerrant, R L

    1985-01-01

    We studied the adherence of [3H]thymidine-labeled axenic Entamoeba histolytica (strain HM1-IMSS) to in vitro preparations of rat and human colonic mucosa. Studies were performed with fixed or unfixed rat colonic mucosa, unfixed rat mucosa exposed to trypsin, unfixed rat submucosa, and fixed human colonic mucosa. Twenty percent of the amebae adhered to fixed rat colonic mucosa; adherence was specifically inhibited by N-acetyl-D-galactosamine (GalNAc), galactose, and asialofetuin. The adherence of amebae to fixed human colonic mucosa was also GalNAc inhibitable. Greater adherence was found with unfixed rat colonic mucosa (40.9%) and was not GalNAc inhibitable unless the tissue was first exposed to trypsin. However, GalNAc did inhibit the adherence of amebae to unfixed rat submucosa. Glutaraldehyde fixation of amebae inactivates known amebic adhesion proteins; there was a markedly decreased adherence of fixed amebae to trypsin-exposed mucosa or fixed rat colonic mucosa. However, fixed or viable amebae had equal levels of adherence to unfixed rat colonic mucosa, suggesting the presence of a host adhesion protein that binds to receptors on amebae. Human (10%) and rabbit (5%) immune sera reduced the adherence of viable amebae to fixed rat colonic mucosa. We concluded that the GalNAc-inhibitable adhesion protein on the surface of E. histolytica trophozoites mediated adherence to fixed rat mucosa, fixed human colonic mucosa, trypsin-exposed unfixed rat mucosa, and unfixed rat submucosa. The surface of unfixed rat colonic mucosa contained a glutaraldehyde- and trypsin-sensitive host adhesion protein, perhaps in the overlying mucus blanket, which bound viable or fixed E. histolytica trophozoites. Images PMID:2580787

  11. Data on docking and dynamics simulation of Entamoeba histolytica EhADH (an ALIX protein) and lysobisphosphatidic acid.

    PubMed

    Castellanos-Castro, Silvia; Montaño, Sarita; Orozco, Esther

    2016-06-01

    Entamoeba histolytica is the protozoan agent responsible for human amoebiasis. Trophozoites are highly phagocytic cells and the lysobisphosphatidic acid (LBPA) is involved in endocytosis. LBPA interacts with EhADH protein (an ALIX family member) also participating in phagocytosis, as it is referred in the research article Identification of the phospholipid lysobisphosphatidic acid in the protozoan Entamoeba histolytica: an active molecule in endocytosis (Castellanos-Castro et al., 2016) [1]. To unveil the interaction site between EhADH and LBPA, here we performed molecular modeling, dynamics simulation and docking. Molecular modeling and docking predictions revealed that EhADH interacts with LBPA through the Bro1 domain, located at the N-terminus of the protein and through the adherence domain at the C-terminus. In silico mutation abolished these interactions, supporting the data obtained in molecular dynamic and docking in silico assays. PMID:27014730

  12. Data on docking and dynamics simulation of Entamoeba histolytica EhADH (an ALIX protein) and lysobisphosphatidic acid

    PubMed Central

    Castellanos-Castro, Silvia; Montaño, Sarita; Orozco, Esther

    2016-01-01

    Entamoeba histolytica is the protozoan agent responsible for human amoebiasis. Trophozoites are highly phagocytic cells and the lysobisphosphatidic acid (LBPA) is involved in endocytosis. LBPA interacts with EhADH protein (an ALIX family member) also participating in phagocytosis, as it is referred in the research article Identification of the phospholipid lysobisphosphatidic acid in the protozoan Entamoeba histolytica: an active molecule in endocytosis (Castellanos-Castro et al., 2016) [1]. To unveil the interaction site between EhADH and LBPA, here we performed molecular modeling, dynamics simulation and docking. Molecular modeling and docking predictions revealed that EhADH interacts with LBPA through the Bro1 domain, located at the N-terminus of the protein and through the adherence domain at the C-terminus. In silico mutation abolished these interactions, supporting the data obtained in molecular dynamic and docking in silico assays. PMID:27014730

  13. Identification of natural inhibitors of Entamoeba histolytica cysteine synthase from microbial secondary metabolites

    PubMed Central

    Mori, Mihoko; Jeelani, Ghulam; Masuda, Yui; Sakai, Kazunari; Tsukui, Kumiko; Waluyo, Danang; Tarwadi; Watanabe, Yoshio; Nonaka, Kenichi; Matsumoto, Atsuko; Ōmura, Satoshi; Nozaki, Tomoyoshi; Shiomi, Kazuro

    2015-01-01

    Amebiasis is a common worldwide diarrheal disease, caused by the protozoan parasite, Entamoeba histolytica. Metronidazole has been a drug of choice against amebiasis for decades despite its known side effects and low efficacy against asymptomatic cyst carriers. E. histolytica is also capable of surviving sub-therapeutic levels of metronidazole in vitro. Novel drugs with different mode of action are therefore urgently needed. The sulfur assimilatory de novo L-cysteine biosynthetic pathway is essential for various cellular activities, including the proliferation and anti-oxidative defense of E. histolytica. Since the pathway, consisting of two reactions catalyzed by serine acetyltransferase (SAT) and cysteine synthase (CS, O-acetylserine sulfhydrylase), does not exist in humans, it is a rational drug target against amebiasis. To discover inhibitors against the CS of E. histolytica (EhCS), the compounds of Kitasato Natural Products Library were screened against two recombinant CS isozymes: EhCS1 and EhCS3. Nine compounds inhibited EhCS1 and EhCS3 with IC50 values of 0.31–490 μM. Of those, seven compounds share a naphthoquinone moiety, indicating the structural importance of the moiety for binding to the active site of EhCS1 and EhCS3. We further screened >9,000 microbial broths for CS inhibition and purified two compounds, xanthofulvin and exophillic acid from fungal broths. Xanthofulvin inhibited EhCS1 and EhCS3. Exophillic acid showed high selectivity against EhCS1, but exhibited no inhibition against EhCS3. In vitro anti-amebic activity of the 11 EhCS inhibitors was also examined. Deacetylkinamycin C and nanaomycin A showed more potent amebicidal activity with IC50 values of 18 and 0.8 μM, respectively, in the cysteine deprived conditions. The differential sensitivity of trophozoites against deacetylkinamycin C in the presence or absence of L-cysteine in the medium and the IC50 values against EhCS suggest the amebicidal effect of deacetylkinamycin C is due to

  14. Technical advances in trigger-induced RNA interference gene silencing in the parasite Entamoeba histolytica.

    PubMed

    Khalil, Mohamed I; Foda, Bardees M; Suresh, Susmitha; Singh, Upinder

    2016-03-01

    Entamoeba histolytica has a robust endogenous RNA interference (RNAi) pathway. There are abundant 27 nucleotide (nt) anti-sense small RNAs (AS sRNAs) that target genes for silencing and the genome encodes many genes involved in the RNAi pathway such as Argonaute proteins. Importantly, an E. histolytica gene with numerous AS sRNAs can function as a "trigger" to induce silencing of a gene that is fused to the trigger. Thus, the amebic RNAi pathway regulates gene expression relevant to amebic biology and has additionally been harnessed as a tool for genetic manipulation. In this study we have further improved the trigger-induced gene silencing method. We demonstrate that rather than using the full-length gene, a short portion of the coding region fused to a trigger is sufficient to induce silencing; the first 537 bp of the E. histolytica rhomboid gene (EhROM1) fused in-frame to the trigger was sufficient to silence EhROM1. We also demonstrated that the trigger method could silence two amebic genes concomitantly; fusion of the coding regions of EhROM1 and transcription factor, EhMyb, in-frame to a trigger gene resulted in both genes being silenced. Alternatively, two genes can be silenced sequentially: EhROM1-silenced parasites with no drug selection plasmid were transfected with trigger-EhMyb, resulting in parasites with both EhROM1 and EhMyb silenced. With all approaches tested, the trigger-mediated silencing was substantive and silencing was maintained despite loss of the G418 selectable marker. All gene silencing was associated with generation of AS sRNAs to the silenced gene. We tested the reversibility of the trigger system using inhibitors of histone modifications but found that the silencing was highly stable. This work represents a technical advance in the trigger gene silencing method in E. histolytica. Approaches that readily silence multiple genes add significantly to the genetic toolkit available to the ameba research community. PMID:26747561

  15. Pathogenic Entamoeba histolytica: cDNA cloning of a histone H3 with a divergent primary structure.

    PubMed

    Födinger, M; Ortner, S; Plaimauer, B; Wiedermann, G; Scheiner, O; Duchêne, M

    1993-06-01

    Entamoeba histolytica has an unusual nuclear structure characterized by a low degree of chromatin condensation and the absence of stainable metaphase chromosomes. Although nucleosome-like particles were observed, no information about histones was available so far. In this paper we describe a cDNA clone with significant homology to H3 histones that was isolated from a library of pathogenic E. histolytica. The complete cDNA encodes a 15-kDa polypeptide, which like the histone sequence from Volvox carteri is shorter by one residue than the human homologue. The amino acid sequence has only 69% identity with human H3.3 histone and 67% identity with the human H3.1 histone. This is the highest degree of sequence divergence observed for any eukaryote H3 histone sequence. Our results indicate that this divergence may contribute to the unusual chromatin structure of E. histolytica. PMID:8341328

  16. A double-antibody sandwich ELISA for the detection of Entamoeba histolytica antigen in stool samples of humans.

    PubMed

    Baumann, D; Gottstein, B

    1987-06-01

    A double-antibody sandwich ELISA was developed to detect detergent-solubilized antigens of Entamoeba histolytica in stool samples of humans. The test system was evaluated for its methodical and diagnostic sensitivity and specificity. In recovery experiments the lower limit of detection was 400 ng E. histolytica (HK9) protein/ml stool, corresponding to approximately 2000 amoebic trophozoites/ml stool. Samples of 97 patients with suspected intestinal amoebiasis were examined. Specific antigens were detected by ELISA (= positive reaction) in 14 (93%) out of 15 stool samples containing trophozoites of E. histolytica. In contrast, 68 (93%) of 73 samples with other protozoa, including Entamoeba coli, E. hartmanni, Endolimax nana, Iodamoeba buetschlii and Giardia lamblia, did not react in the test system (= negative reaction). The test was shown to detect only trophozoites of E. histolytica and not the cyst stage. This fact could facilitate the differentiation between cyst carriers and persons excreting trophozoites. The results of this preliminary study justify a further large scale evaluation of the test system. PMID:2888183

  17. The epidemiology of Entamoeba histolytica in a rural and an urban area of Mexico. A pilot survey II.

    PubMed

    Sargeaunt, P G; Williams, J E; Bhojnani, R; Campos, J E; Gomez, A

    1982-01-01

    Stocks of intestinal amoebae grown in monoxenic culture, were compared against each other and against those previously reported from Mexico City. These were isolated from subjects in San Cristobal de las Casas, Chiapas (rural area) and hospital patients in Merida, Yucatan (urban area). Electrophoretic patterns of the four enzymes: glucose phosphate isomerase (GPI), phosphoglucomutase (PGM), L-malate: NADP+ oxido-reductase (oxalacetate-decarboxylating) (ME) and hexokinase (HK) demonstrated the presence of five groups (zymodemes) of Entamoeba histolytica already described from Mexico City, together with two new zymodemes, one of which gave a recognizable pathogenic pattern, whilst the other gave a contradictory pattern. Zymodemes of Entamoeba coli, Entamoeba hartmanni, Iodamoeba buetschlii and Dientamoeba fragilis, previously described were also isolated. One new zymodeme of D. fragilis was demonstrated. PMID:6285556

  18. Growth of the protozoan parasite Entamoeba histolytica in 5-azacytidine has limited effects on parasite gene expression

    PubMed Central

    Ali, Ibne Karim M; Ehrenkaufer, Gretchen M; Hackney, Jason A; Singh, Upinder

    2007-01-01

    Background In higher eukaryotes DNA methylation regulates important biological functions including silencing of gene expression and protection from adverse effects of retrotransposons. In the protozoan parasite Entamoeba histolytica, a DNA methyltransferase has been identified and treatment with 5-azacytidine (5-AzaC), a potent inhibitor of DNA methyltransferase, has been reported to attenuate parasite virulence. However, the overall extent of DNA methylation and its subsequent effects on global gene expression in this parasite are currently unknown. Results In order to identify the genome-wide effects of DNA methylation in E. histolytica, we used a short oligonucleotide microarray representing 9,435 genes (~95% of all annotated amebic genes) and compared the expression profile of E. histolytica HM-1:IMSS parasites with those treated with 23 μM 5-AzaC for up to one week. Overall, 2.1% of genes tested were transcriptionally modulated under these conditions. 68 genes were upregulated and 131 genes down regulated (2-fold change; p-value < 0.05). Sodium-bisulfite treatment and sequencing of genes indicated that there were at least two subsets of genes with genomic DNA methylation in E. histolytica: (i) genes that were endogenously silenced by genomic DNA methylation and for which 5-AzaC treatment induced transcriptional de-repression, and (ii) genes that have genomic DNA methylation, but which were not endogenously silenced by the methylation. We identified among the genes down regulated by 5-AzaC treatment a cysteine proteinase (2.m00545) and lysozyme (52.m00148) both of which have known roles in amebic pathogenesis. Decreased expression of these genes in the 5-AzaC treated E. histolytica may account in part for the parasites reduced cytolytic abilities. Conclusion This work represents the first genome-wide analysis of DNA-methylation in Entamoeba histolytica and indicates that DNA methylation has relatively limited effects on gene expression in this parasite. PMID

  19. Unique Structural and Nucleotide Exchange Features of the Rho1 GTPase of Entamoeba histolytica

    SciTech Connect

    Bosch, Dustin E.; Wittchen, Erika S.; Qiu, Connie; Burridge, Keith; Siderovski, David P.

    2012-08-10

    The single-celled human parasite Entamoeba histolytica possesses a dynamic actin cytoskeleton vital for its intestinal and systemic pathogenicity. The E. histolytica genome encodes several Rho family GTPases known to regulate cytoskeletal dynamics. EhRho1, the first family member identified, was reported to be insensitive to the Rho GTPase-specific Clostridium botulinum C3 exoenzyme, raising the possibility that it may be a misclassified Ras family member. Here, we report the crystal structures of EhRho1 in both active and inactive states. EhRho1 is activated by a conserved switch mechanism, but diverges from mammalian Rho GTPases in lacking a signature Rho insert helix. EhRho1 engages a homolog of mDia, EhFormin1, suggesting a role in mediating serum-stimulated actin reorganization and microtubule formation during mitosis. EhRho1, but not a constitutively active mutant, interacts with a newly identified EhRhoGDI in a prenylation-dependent manner. Furthermore, constitutively active EhRho1 induces actin stress fiber formation in mammalian fibroblasts, thereby identifying it as a functional Rho family GTPase. EhRho1 exhibits a fast rate of nucleotide exchange relative to mammalian Rho GTPases due to a distinctive switch one isoleucine residue reminiscent of the constitutively active F28L mutation in human Cdc42, which for the latter protein, is sufficient for cellular transformation. Nonconserved, nucleotide-interacting residues within EhRho1, revealed by the crystal structure models, were observed to contribute a moderating influence on fast spontaneous nucleotide exchange. Collectively, these observations indicate that EhRho1 is a bona fide member of the Rho GTPase family, albeit with unique structural and functional aspects compared with mammalian Rho GTPases.

  20. [A Case of Peristomal Cutaneous Ulcer Following Amebic Colitis Caused by Entamoeba histolytica].

    PubMed

    Sasaki, Yu; Yoshida, Tetsuya; Suzuki, Jun; Kobayashi, Seiki; Sato, Tomotaka

    2016-01-01

    A 66-year-old Japanese male with a history of a rectal ulcer and rectovesical fistula following brachytherapy and radiotherapy for prostate cancer, who had undergone colostomy and vesicotomy presented with a painful peristomal ulcer of approximately 5 x 2.5cm adjacent to the direction of 6 o'clock of the stoma in his left lower abdomen. Although he was admitted to be treated with intravenous antibiotics and topical debridement, the ulcer was rapidly increasing. In the laboratory findings, WBC was 12,400/μL, CRP was 16.9 mg/dL, ESR was 105mm in the first hour. Contrast enhanced CT images showed a wide high density area of skin and subcutaneous tissue around the stoma and dillitation of the transverse and descending colon. Colonoscopy showed furred profound ulcers in the rectum. A biopsy from the ulcer floor submitted to histopathology showed necrotic tissue with a mixed inflammatory infiltrates mainly composed of neutrophils and lymphocytes in the dermis. We suspected pyoderma gangrenosum with an inflammatory bowel disease in the beginning. Although he was started on oral prednisolone 60 mg daily, the ulcer did not respond to treatment. Additional methylprednisolone pulse therapy, intravenous cyclosporine and granulocytapheresis were also ineffective. A biopsy specimen from the skin ulcer margin showed erythrophagocytosis by trophozoites of amebae which were identified on PAS stained slides. The PCR method and stool examination showed positive for Entamoeba histolytica (E. histolytica), but serum antibodies were negative. Within two weeks of treatment with oral metronidazole 2,250 mg/day and topical metronidazole ointment, resolution of the ulcer was observed, then the prednisolone dosage was tapered. A split-thickness skin graft was used to cover the ulcer with a successful result. Even though we originally misdiagnosed this case, we finally reached a diagnosis of amebiasis. It is important to take account of amebiasis in the differential diagnosis of intractable

  1. Relationship of free intracellular calcium to the cytolytic activity of Entamoeba histolytica.

    PubMed

    Ravdin, J I; Moreau, F; Sullivan, J A; Petri, W A; Mandell, G L

    1988-06-01

    Entamoeba histolytica adherence and destruction of host cells is required for in vivo pathogenicity; amebic in vitro adherence is mediated by a galactose- or N-acetyl-D-galactosamine-inhibitable surface lectin (Gal/GalNAc adherence lectin). Free intracellular Ca2+ concentration [( Ca2+]i) was measured in living amebae and target cells during amebic cytolysis of Chinese hamster ovary (CHO) cells and human polymorphonuclear neutrophils by utilizing the Ca2+ probe Fura-2 and computer-enhanced digitized microscopy. Motile E. histolytica trophozoites had oscillatory increases in [Ca2+]i in head or tail regions; however, there was no increase in regional or total amebic [Ca2+]i upon contact with a target CHO cell. Target CHO cells and polymorphonuclear neutrophils demonstrated marked irreversible increases in [Ca2+]i within 30 to 300 s following contact by an ameba (P less than 0.01); increased [Ca2+]i preceded the occurrence of nonspecific surface membrane permeability and death of the target cell. Target CHO cells contiguous on a monolayer to a cell contacted by an ameba experienced a rapid but reversible rise in [Ca2+]i (P less than 0.01) and were not killed. Galactose (40 mg/ml) totally abrogated the rise in target CHO cell [Ca2+]i that followed contact by amebae (P less than 0.01); immunoaffinity-purified amebic Gal/GalNAc adherence lectin (0.25 micrograms/ml) induced a rapid and reversible rise in CHO cell [Ca2+]i (P less than 0.01) which was inhibited by galactose. Amebic [Ca2+]i was not elevated following parasite adherence to target cells; a rapid and substantial rise in target cell [Ca2+]i occurred which was mediated, at least in part, by the Gal/GalNAc adherence lectin of the parasite and led to the death of target cells. PMID:2897335

  2. Relationship of free intracellular calcium to the cytolytic activity of Entamoeba histolytica.

    PubMed Central

    Ravdin, J I; Moreau, F; Sullivan, J A; Petri, W A; Mandell, G L

    1988-01-01

    Entamoeba histolytica adherence and destruction of host cells is required for in vivo pathogenicity; amebic in vitro adherence is mediated by a galactose- or N-acetyl-D-galactosamine-inhibitable surface lectin (Gal/GalNAc adherence lectin). Free intracellular Ca2+ concentration [( Ca2+]i) was measured in living amebae and target cells during amebic cytolysis of Chinese hamster ovary (CHO) cells and human polymorphonuclear neutrophils by utilizing the Ca2+ probe Fura-2 and computer-enhanced digitized microscopy. Motile E. histolytica trophozoites had oscillatory increases in [Ca2+]i in head or tail regions; however, there was no increase in regional or total amebic [Ca2+]i upon contact with a target CHO cell. Target CHO cells and polymorphonuclear neutrophils demonstrated marked irreversible increases in [Ca2+]i within 30 to 300 s following contact by an ameba (P less than 0.01); increased [Ca2+]i preceded the occurrence of nonspecific surface membrane permeability and death of the target cell. Target CHO cells contiguous on a monolayer to a cell contacted by an ameba experienced a rapid but reversible rise in [Ca2+]i (P less than 0.01) and were not killed. Galactose (40 mg/ml) totally abrogated the rise in target CHO cell [Ca2+]i that followed contact by amebae (P less than 0.01); immunoaffinity-purified amebic Gal/GalNAc adherence lectin (0.25 micrograms/ml) induced a rapid and reversible rise in CHO cell [Ca2+]i (P less than 0.01) which was inhibited by galactose. Amebic [Ca2+]i was not elevated following parasite adherence to target cells; a rapid and substantial rise in target cell [Ca2+]i occurred which was mediated, at least in part, by the Gal/GalNAc adherence lectin of the parasite and led to the death of target cells. Images PMID:2897335

  3. Detection of Giardia lamblia, Entamoeba histolytica/Entamoeba dispar, and Cryptosporidium parvum Antigens in Human Fecal Specimens Using the Triage Parasite Panel Enzyme Immunoassay

    PubMed Central

    Garcia, Lynne S.; Shimizu, Robyn Y.; Bernard, Caroline N.

    2000-01-01

    The Triage parasite panel (BIOSITE Diagnostics, San Diego, Calif.) is a new qualitative enzyme immunoassay (EIA) panel for the detection of Giardia lamblia, Entamoeba histolytica/E. dispar, and Cryptosporidium parvum in fresh or fresh, frozen, unfixed human fecal specimens. By using specific antibodies, antigens specific for these organisms are captured and immobilized on a membrane. Panel performance was evaluated with known positive and negative stool specimens (a total of 444 specimens) that were tested by the standard ova and parasite (O&P) examination as the “gold standard,” including staining with both trichrome and modified acid-fast stains. Specimens with discrepant results between the reference and Triage methods were retested by a different method, either EIA or immunofluorescence. A number of samples with discrepant results with the Triage device were confirmed to be true positives. After resolution of discrepant results, the number of positive specimens and the sensitivity and specificity results were as follows: for G. lamblia, 170, 95.9%, and 97.4%, respectively; for E. histolytica/E. dispar, 99, 96.0%, and 99.1%, respectively; and for C. parvum, 60, 98.3%, and 99.7%, respectively. There was no cross-reactivity with other parasites found in stool specimens, including eight different protozoa (128 challenges) and three different helminths (83 challenges). The ability to perform the complete O&P examination should remain an option for those patients with negative parasite panel results but who are still symptomatic. PMID:10970380

  4. Evidence for the bacterial origin of genes encoding fermentation enzymes of the amitochondriate protozoan parasite Entamoeba histolytica.

    PubMed Central

    Rosenthal, B; Mai, Z; Caplivski, D; Ghosh, S; de la Vega, H; Graf, T; Samuelson, J

    1997-01-01

    Entamoeba histolytica is an amitochondriate protozoan parasite with numerous bacterium-like fermentation enzymes including the pyruvate:ferredoxin oxidoreductase (POR), ferredoxin (FD), and alcohol dehydrogenase E (ADHE). The goal of this study was to determine whether the genes encoding these cytosolic E. histolytica fermentation enzymes might derive from a bacterium by horizontal transfer, as has previously been suggested for E. histolytica genes encoding heat shock protein 60, nicotinamide nucleotide transhydrogenase, and superoxide dismutase. In this study, the E. histolytica por gene and the adhE gene of a second amitochondriate protozoan parasite, Giardia lamblia, were sequenced, and their phylogenetic positions were estimated in relation to POR, ADHE, and FD cloned from eukaryotic and eubacterial organisms. The E. histolytica por gene encodes a 1,620-amino-acid peptide that contained conserved iron-sulfur- and thiamine pyrophosphate-binding sites. The predicted E. histolytica POR showed fewer positional identities to the POR of G. lamblia (34%) than to the POR of the enterobacterium Klebsiella pneumoniae (49%), the cyanobacterium Anabaena sp. (44%), and the protozoan Trichomonas vaginalis (46%), which targets its POR to anaerobic organelles called hydrogenosomes. Maximum-likelihood, neighbor-joining, and parsimony analyses also suggested as less likely E. histolytica POR sharing more recent common ancestry with G. lamblia POR than with POR of bacteria and the T. vaginalis hydrogenosome. The G. lamblia adhE encodes an 888-amino-acid fusion peptide with an aldehyde dehydrogenase at its amino half and an iron-dependent (class 3) ADH at its carboxy half. The predicted G. lamblia ADHE showed extensive positional identities to ADHE of Escherichia coli (49%), Clostridium acetobutylicum (44%), and E. histolytica (43%) and lesser identities to the class 3 ADH of eubacteria and yeast (19 to 36%). Phylogenetic analyses inferred a closer relationship of the E

  5. Proteomic Study of Entamoeba histolytica Trophozoites, Cysts, and Cyst-Like Structures.

    PubMed

    Luna-Nácar, Milka; Navarrete-Perea, José; Moguel, Bárbara; Bobes, Raúl J; Laclette, Juan P; Carrero, Julio C

    2016-01-01

    The cyst stage of Entamoeba histolytica is a promising therapeutic target against human amoebiasis. Our research team previously reported the production in vitro of Cyst-Like Structures (CLS) sharing structural features with cysts, including rounded shape, size reduction, multinucleation, and the formation of a chitin wall coupled to the overexpression of glucosamine 6-phosphate isomerase, the rate-limiting enzyme of the chitin synthesis pathway. A proteomic study of E. histolytica trophozoites, cysts, and in vitro-produced CLS is reported herein to determine the nature of CLS, widen our knowledge on the cyst stage, and identify possible proteins and pathways involved in the encystment process. Total protein extracts were obtained from E. histolytica trophozoites, CLS, and partially purified cysts recovered from the feces of amoebic human patients; extracts were trypsin-digested and analyzed by LC-MS/MS. In total, 1029 proteins were identified in trophozoites, 550 in CLS, and 411 in cysts, with 539, 299, and 84 proteins unique to each sample, respectively, and only 74 proteins shared by all three stages. About 70% of CLS proteins were shared with trophozoites, even though differences were observed in the relative protein abundance. While trophozoites showed a greater abundance of proteins associated to a metabolically active cell, CLS showed higher expression of proteins related to proteolysis, redox homeostasis, and stress response. In addition, the expression of genes encoding for the cyst wall proteins Jessie and Jacob was detected by RT-PCR and the Jacob protein identified by Western blotting and immunofluorescence in CLS. However, the proteomic profile of cysts as determined by LC-MS/MS was very dissimilar to that of trophozoites and CLS, with almost 40% of hypothetical proteins. Our global results suggest that CLS are more alike to trophozoites than to cysts, and they could be generated as a rapid survival response of trophozoites to a stressful condition

  6. Proteomic Study of Entamoeba histolytica Trophozoites, Cysts, and Cyst-Like Structures

    PubMed Central

    Luna-Nácar, Milka; Navarrete-Perea, José; Moguel, Bárbara; Bobes, Raúl J.; Laclette, Juan P.; Carrero, Julio C.

    2016-01-01

    The cyst stage of Entamoeba histolytica is a promising therapeutic target against human amoebiasis. Our research team previously reported the production in vitro of Cyst-Like Structures (CLS) sharing structural features with cysts, including rounded shape, size reduction, multinucleation, and the formation of a chitin wall coupled to the overexpression of glucosamine 6-phosphate isomerase, the rate-limiting enzyme of the chitin synthesis pathway. A proteomic study of E. histolytica trophozoites, cysts, and in vitro-produced CLS is reported herein to determine the nature of CLS, widen our knowledge on the cyst stage, and identify possible proteins and pathways involved in the encystment process. Total protein extracts were obtained from E. histolytica trophozoites, CLS, and partially purified cysts recovered from the feces of amoebic human patients; extracts were trypsin-digested and analyzed by LC-MS/MS. In total, 1029 proteins were identified in trophozoites, 550 in CLS, and 411 in cysts, with 539, 299, and 84 proteins unique to each sample, respectively, and only 74 proteins shared by all three stages. About 70% of CLS proteins were shared with trophozoites, even though differences were observed in the relative protein abundance. While trophozoites showed a greater abundance of proteins associated to a metabolically active cell, CLS showed higher expression of proteins related to proteolysis, redox homeostasis, and stress response. In addition, the expression of genes encoding for the cyst wall proteins Jessie and Jacob was detected by RT-PCR and the Jacob protein identified by Western blotting and immunofluorescence in CLS. However, the proteomic profile of cysts as determined by LC-MS/MS was very dissimilar to that of trophozoites and CLS, with almost 40% of hypothetical proteins. Our global results suggest that CLS are more alike to trophozoites than to cysts, and they could be generated as a rapid survival response of trophozoites to a stressful condition

  7. cDNA sequence analysis of a 29-kDa cysteine-rich surface antigen of pathogenic Entamoeba histolytica

    SciTech Connect

    Torian, B.E.; Stroeher, V.L.; Stamm, W.E. ); Flores, B.M. ); Hagen, F.S. )

    1990-08-01

    A {lambda}gt11 cDNA library was constructed from poly(U)-Spharose-selected Entamoeba histolytica trophozoite RNA in order to clone and identify surface antigens. The library was screened with rabbit polyclonal anti-E. histolytica serum. A 700-base-pair cDNA insert was isolated and the nucleotide sequence was determined. The deduced amino acid sequence of the cDNA revealed a cysteine-rich protein. DNA hybridizations showed that the gene was specific to E. histolytica since the cDNA probe reacted with DNA from four axenic strains of E. histolytica but did not react with DNA from Entamoeba invadens, Acanthamoeba castellanii, or Trichomonas vaginalis. The insert was subcloned into the expression vector pGEX-1 and the protein was expressed as a fusion with the C terminus of glutathione S-transferase. Purified fusion protein was used to generate 22 monoclonal antibodies (mAbs) and a mouse polyclonal antiserum specific for the E. histolytica portion of the fusion protein. A 29-kDa protein was identified as a surface antigen when mAbs were used to immunoprecipitate the antigen from metabolically {sup 35}S-labeled live trophozoites. The surface location of the antigen was corroborated by mAb immunoprecipitation of a 29-kDa protein from surface-{sup 125}I-labeled whole trophozoites as well as by the reaction of mAbs with live trophozoites in an indirect immunofluorescence assay performed at 4{degree}C. Immunoblotting with mAbs demonstrated that the antigen was present on four axenic isolates tested. mAbs recognized epitopes on the 29-kDa native antigen on some but not all clinical isolates tested.

  8. In vivo identification of the steps that control energy metabolism and survival of Entamoeba histolytica.

    PubMed

    Pineda, Erika; Encalada, Rusely; Vázquez, Citlali; Néquiz, Mario; Olivos-García, Alfonso; Moreno-Sánchez, Rafael; Saavedra, Emma

    2015-01-01

    The steps that control the Entamoeba histolytica glycolytic flux were here identified by elasticity analysis, an experimental approach of metabolic control analysis. The concentrations of glycolytic metabolites were gradually varied in live trophozoites by (a) feeding with different glucose concentrations and (b) inhibiting the final pathway steps; in parallel, the changes in the pathway flux were determined. From the metabolite concentration-flux relationship, the elasticity coefficients of individual or groups of pathway reactions were determined and used to calculate their respective degrees of control on the glycolytic flux (flux control coefficients). The results indicated that the pathway flux was mainly controlled (72-86%) by the glucose transport/hexokinase/glycogen degradation group of reactions and by bifunctional aldehyde-alcohol dehydrogenase (ADHE; 18%). Further, inhibition of the first pathway reactions with 2-deoxyglucose (2DOG) decreased the glycolytic flux and ATP content by 75% and 50%, respectively. Cell viability was also decreased by 2DOG (25%) and more potently (50%) by 2DOG plus the ADHE inhibitor tetraethylthiuram disulfide (disulfiram). Biosate as an alternative carbon (amino acid) source was unable to replace glucose for ATP supply, which indicated that glucose was the main nutrient for amoebal ATP synthesis and survival. These results indicated that glycolysis in the parasite is mainly controlled by the initial pathway reactions and that their inhibition can decrease the parasite energy load and survival. PMID:25350227

  9. Recombinant expression and biochemical characterization of an NADPH:flavin oxidoreductase from Entamoeba histolytica.

    PubMed Central

    Bruchhaus, I; Richter, S; Tannich, E

    1998-01-01

    The gene encoding a putative NADPH:flavin oxidoreductase of the protozoan parasite Entamoeba histolytica (Eh34) was recombinantly expressed in Escherichia coli. The purified recombinant protein (recEh34) has a molecular mass of about 35 kDa upon SDS/PAGE analysis, exhibits a flavoprotein-like absorption spectrum and contains 1 mol of non-covalently bound FMN per mol of protein. RecEh34 reveals two different enzymic activities. It catalyses the NADPH-dependent reduction of oxygen to hydrogen peroxide (H2O2), as well as of disulphides such as 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and cystine. The disulphide reductase but not the H2O2-forming NADPH oxidase activity is inhibitable by sulphydryl-active compounds, indicating that a thiol component is part of the active site for the disulphide reductase activity, whereas for the H2O2-forming NADPH oxidase activity only the flavin is required. Compared with the recombinant protein, similar activities are present in amoebic extracts. Native Eh34 is active in a monomeric as well as in a dimeric state. In contrast to recEh34, no flavin was associated with the native protein. However, both NADPH oxidase as well as DTNB reductase activity were found to be dependent on the addition of FAD or FMN. PMID:9494088

  10. Entamoeba histolytica: cloning and expression of the poly(A) polymerase EhPAP.

    PubMed

    García-Vivas, Jessica; López-Camarillo, César; Azuara-Liceaga, Elisa; Orozco, Esther; Marchat, Laurence A

    2005-07-01

    In eukaryotes, polyadenylation of pre-mRNA 3' end is essential for mRNA export, stability, and translation. Here we identified and cloned a gene codifying for a putative nuclear poly(A) polymerase (EhPAP) in Entamoeba histolytica. Protein sequence alignments with eukaryotic PAPs showed that EhPAP has the RNA-binding region and the PAP central domain with the catalytic nucleotidyl transferase domain described for other nuclear PAPs. Recombinant EhPAP expressed in bacteria was used to generate specific antibodies, which recognized two EhPAP isoforms of 60 and 63kDa in nuclear and cytoplasmic extracts by Western blot assays. RT-PCR assays showed that EhPap mRNA expression varies in multidrug-resistant trophozoites growing in different emetine concentrations. Moreover, EhPap mRNA expression is about 10- and 7-fold increased in G1 and S phase, respectively, through cell cycle progression. These results suggest the existence of a link between EhPAP expression and MDR and cell cycle regulation, respectively. PMID:15955317

  11. Calpain-like: A Ca(2+) dependent cystein protease in Entamoeba histolytica cell death.

    PubMed

    Monroy, Virginia Sánchez; Flores, Olivia Medel; García, Consuelo Gómez; Maya, Yesenia Chávez; Fernández, Tania Domínguez; Pérez Ishiwara, D Guillermo

    2015-12-01

    Entamoeba histolytica programmed cell death (PCD) induced by G418 is characterized by the release of important amounts of intracellular calcium from reservoirs. Nevertheless, no typical caspases have been detected in the parasite, the PCD phenotype is inhibited by the cysteine protease inhibitor E-64. These results strongly suggest that Ca(2+)-dependent proteases could be involved in PCD. In this study, we evaluate the expression and activity of a specific dependent Ca(2+) protease, the calpain-like protease, by real-time quantitative PCR (RTq-PCR), Western blot assays and a enzymatic method during the induction of PCD by G418. Alternatively, using cell viability and TUNEL assays, we also demonstrated that the Z-Leu-Leu-Leu-al calpain inhibitor reduced the rate of cell death. The results demonstrated 4.9-fold overexpression of calpain-like gene 1.5 h after G418 PCD induction, while calpain-like protein increased almost two-fold with respect to basal calpain-like expression after 3 h of induction, and calpain activity was found to be approximately three-fold higher 6 h after treatment compared with untreated trophozoites. Taken together, these results suggest that this Ca(2+)-dependent protease could be involved in the executory phase of PCD. PMID:26496790

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

    PubMed

    Sanuki, J; Asai, T; Okuzawa, E; Kobayashi, S; Takeuchi, T

    1997-01-01

    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. PMID:9000244

  13. Activity, stability and folding analysis of the chitinase from Entamoeba histolytica.

    PubMed

    Muñoz, Patricia L A; Minchaca, Alexis Z; Mares, Rosa E; Ramos, Marco A

    2016-02-01

    Human amebiasis, caused by the parasitic protozoan Entamoeba histolytica, remains as a significant public health issue in developing countries. The life cycle of the parasite compromises two main stages, trophozoite and cyst, linked by two major events: encystation and excystation. Interestingly, the cyst stage has a chitin wall that helps the parasite to withstand harsh environmental conditions. Since the amebic chitinase, EhCHT1, has been recognized as a key player in both encystation and excystation, it is plausible to consider that specific inhibition could arrest the life cycle of the parasite and, thus, stop the infection. However, to selectively target EhCHT1 it is important to recognize its unique biochemical features to have the ability to control its cellular function. Hence, to gain further insights into the structure-function relationship, we conducted an experimental approach to examine the effects of pH, temperature, and denaturant concentration on the enzymatic activity and protein stability. Additionally, dependence on in vivo oxidative folding was further studied using a bacterial model. Our results attest the potential of EhCHT1 as a target for the design and development of new or improved anti-amebic therapeutics. Likewise, the potential of the oxidoreductase EhPDI, involved in oxidative folding of amebic proteins, was also confirmed. PMID:26526675

  14. Binding and Endocytosis of Bovine Hololactoferrin by the Parasite Entamoeba histolytica

    PubMed Central

    Ortíz-Estrada, Guillermo; Calderón-Salinas, Víctor; Shibayama-Salas, Mineko; León-Sicairos, Nidia; de la Garza, Mireya

    2015-01-01

    Entamoeba histolytica is a human parasite that requires iron (Fe) for its metabolic function and virulence. Bovine lactoferrin (B-Lf) and its peptides can be found in the digestive tract after dairy products are ingested. The aim of this study was to compare virulent trophozoites recently isolated from hamster liver abscesses with nonvirulent trophozoites maintained for more than 30 years in cultures in vitro regarding their interaction with iron-charged B-Lf (B-holo-Lf). We performed growth kinetics analyses of trophozoites in B-holo-Lf and throughout several consecutive transfers. The virulent parasites showed higher growth and tolerance to iron than nonvirulent parasites. Both amoeba variants specifically bound B-holo-Lf with a similar Kd. However, averages of 9.45 × 105 and 6.65 × 106 binding sites/cell were found for B-holo-Lf in nonvirulent and virulent amoebae, respectively. Virulent amoebae bound more efficiently to human and bovine holo-Lf, human holo-transferrin, and human and bovine hemoglobin than nonvirulent amoebae. Virulent amoebae showed two types of B-holo-Lf binding proteins. Although both amoebae endocytosed this glycoprotein through clathrin-coated vesicles, the virulent amoebae also endocytosed B-holo-Lf through a cholesterol-dependent mechanism. Both amoeba variants secreted cysteine proteases cleaving B-holo-Lf. These data demonstrate that the B-Lf endocytosis is more efficient in virulent amoebae. PMID:26090404

  15. Flavodiiron oxygen reductase from Entamoeba histolytica: modulation of substrate preference by tyrosine 271 and lysine 53.

    PubMed

    Gonçalves, Vera L; Vicente, João B; Pinto, Liliana; Romão, Célia V; Frazão, Carlos; Sarti, Paolo; Giuffrè, Alessandro; Teixeira, Miguel

    2014-10-10

    Flavodiiron proteins (FDPs) are a family of enzymes endowed with bona fide oxygen- and/or nitric-oxide reductase activity, although their substrate specificity determinants remain elusive. After a comprehensive comparison of available three-dimensional structures, particularly of FDPs with a clear preference toward either O2 or NO, two main differences were identified near the diiron active site, which led to the construction of site-directed mutants of Tyr(271) and Lys(53) in the oxygen reducing Entamoeba histolytica EhFdp1. The biochemical and biophysical properties of these mutants were studied by UV-visible and electron paramagnetic resonance (EPR) spectroscopies coupled to potentiometry. Their reactivity with O2 and NO was analyzed by stopped-flow absorption spectroscopy and amperometric methods. These mutations, whereas keeping the overall properties of the redox cofactors, resulted in increased NO reductase activity and faster inactivation of the enzyme in the reaction with O2, pointing to a role of the mutated residues in substrate selectivity. PMID:25151360

  16. Identification of a pathogenic isolate-specific 30,000-Mr antigen of Entamoeba histolytica by using a monoclonal antibody.

    PubMed

    Tachibana, H; Kobayashi, S; Kato, Y; Nagakura, K; Kaneda, Y; Takeuchi, T

    1990-04-01

    A monoclonal antibody (MAb) produced against trophozoites of Entamoeba histolytica strain HM-1:IMSS, reacted with all of 42 isolates and 4 clones showing pathogenic zymodeme (Z) patterns, i.e., Z-II, Z-II alpha-, Z-II (glucose phosphate isomerase: gamma +), Z-VII, Z-VII (glucose phosphate isomerase: alpha lack, gamma +), Z-XI, Z-XIV, and Z-XIX, regardless of culture conditions, geographical origins, or host symptoms in an indirect fluorescence antibody test. In contrast, the MAb failed to react with 14 isolates possessing nonpathogenic zymodemes Z-I and Z-VIII and did not react with other enteric protozoan parasites, such as E. histolytica-like Laredo, Entamoeba hartmanni, Entamoeba coli, Endolimax nana, Dientamoeba fragilis, Trichomonas hominis, and Giardia lamblia. Western immunoblotting analysis showed that the molecular weight of the antigenic component recognized by the MAb was exclusively 30,000 in pathogenic isolates of different zymodemes. These results suggest that the 30,000-molecular-weight antigen is a marker of pathogenic isolates and that the indirect fluorescent-antibody test with the MAb is useful for the accurate discrimination of pathogenic amebae. PMID:2180826

  17. TNFα-Mediated Liver Destruction by Kupffer Cells and Ly6Chi Monocytes during Entamoeba histolytica Infection

    PubMed Central

    Ernst, Thomas; Ittrich, Harald; Jacobs, Thomas; Heeren, Joerg; Tacke, Frank; Tannich, Egbert; Lotter, Hannelore

    2013-01-01

    Amebic liver abscess (ALA) is a focal destruction of liver tissue due to infection by the protozoan parasite Entamoeba histolytica (E. histolytica). Host tissue damage is attributed mainly to parasite pathogenicity factors, but massive early accumulation of mononuclear cells, including neutrophils, inflammatory monocytes and macrophages, at the site of infection raises the question of whether these cells also contribute to tissue damage. Using highly selective depletion strategies and cell-specific knockout mice, the relative contribution of innate immune cell populations to liver destruction during amebic infection was investigated. Neutrophils were not required for amebic infection nor did they appear to be substantially involved in tissue damage. In contrast, Kupffer cells and inflammatory monocytes contributed substantially to liver destruction during ALA, and tissue damage was mediated primarily by TNFα. These data indicate that besides direct antiparasitic drugs, modulating innate immune responses may potentially be beneficial in limiting ALA pathogenesis. PMID:23300453

  18. Small GTPase Rab21 Mediates Fibronectin Induced Actin Reorganization in Entamoeba histolytica: Implications in Pathogen Invasion

    PubMed Central

    Emmanuel, Merlyn; Nakano, Yumiko Saito; Nozaki, Tomoyoshi; Datta, Sunando

    2015-01-01

    The protozoan parasite Entamoeba histolytica causes a wide spectrum of intestinal infections. In severe cases, the trophozoites can breach the mucosal barrier, invade the intestinal epithelium and travel via the portal circulation to the liver, where they cause hepatic abscesses, which can prove fatal if left untreated. The host Extra Cellular Matrix (ECM) plays a crucial role in amoebic invasion by triggering an array of cellular responses in the parasite, including induction of actin rich adhesion structures. Similar actin rich protrusive structures, known as ‘invadosomes’, promote chemotactic migration of the metastatic cancer cells and non-transformed cells by remodeling the ECM. Recent studies showed a central role for Rab GTPases, the master regulators of vesicular trafficking, in biogenesis of invadosomes. Here, we showed that fibronectin, a major host ECM component induced actin remodeling in the parasite in a Rab21 dependent manner. The focalized actin structures formed were reminiscent of the mammalian invadosomes. By using various approaches, such as immunofluorescence confocal microscopy and scanning electron microscopy, along with in vitro invasion assay and matrix degradation assay, we show that the fibronectin induced formation of amoebic actin dots depend on the nucleotide status of the GTPase. The ECM components, fibronectin and collagen type I, displayed differential control over the formation of actin dots, with fibronectin positively and collagen type I negatively modulating it. The cell surface adhesion molecule Gal/GalNAc complex was also found to impose additional regulation on this process, which might have implication in collagen type I mediated suppression of actin dots. PMID:25730114

  19. Functional manipulation of a calcium-binding protein from Entamoeba histolytica guided by paramagnetic NMR.

    PubMed

    Rout, Ashok K; Patel, Sunita; Somlata; Shukla, Manish; Saraswathi, Deepa; Bhattacharya, Alok; Chary, Kandala V R

    2013-08-01

    EhCaBP1, one of the calcium-binding proteins from Entamoeba histolytica, is a two-domain EF-hand protein. The two domains of EhCaBP1 are structurally and functionally different from each other. However, both domains are required for structural stability and a full range of functional diversity. Analysis of sequence and structure of EhCaBP1 and other CaBPs indicates that the C-terminal domain of EhCaBP1 possesses a unique structure compared with other family members. This had been attributed to the absence of a Phe-Phe interaction between highly conserved Phe residues at the -4 position in EF-hand III (F[-4]; Tyr(81)) and at the 13th position in EF-hand IV (F[+13]; Phe(129)) of the C-terminal domain. Against this backdrop, we mutated the Tyr residue at the -4th position of EF III to the Phe residue (Y81F), to bring in the Phe-Phe interaction and understand the nature of structural and functional changes in the protein by NMR spectroscopy, molecular dynamics (MD) simulation, isothermal titration calorimetry (ITC), and biological assays, such as imaging and actin binding. The Y81F mutation in EhCaBP1 resulted in a more compact structure for the C-terminal domain of the mutant as in the case of calmodulin and troponin C. The compact structure is favored by the presence of a π-π interaction between Phe(81) and Phe(129) along with several hydrophobic interactions of Phe(81), which are not seen in the wild-type protein. Furthermore, the biological assays reveal preferential membrane localization of the mutant, loss of its colocalization with actin in the phagocytic cups, whereas retaining its ability to bind G- and F-actin. PMID:23782698

  20. A novel nested multiplex polymerase chain reaction (PCR) assay for differential detection of Entamoeba histolytica, E. moshkovskii and E. dispar DNA in stool samples

    PubMed Central

    Khairnar, Krishna; Parija, Subhash C

    2007-01-01

    Background E. histolytica, a pathogenic amoeba, is indistinguishable in its cyst and trophozoite stages from those of non-pathogenic E. moshkovskii and E. dispar by light microscopy. We have developed a nested multiplex PCR targeting a 16S-like rRNA gene for differential detection of all the three morphologically similar forms of E. histolytica, E. moshkovskii and E. dispar simultaneously in stool samples. Results The species specific product size for E. histolytica, E. moshkovskii and E. dispar was 439, 553 and 174 bp respectively, which was clearly different for all the three Entamoeba species. The nested multiplex PCR showed a sensitivity of 94% and specificity of 100% for the demonstration of E. histolytica, E. moshkovskii and E. dispar DNA in stool samples. The PCR was positive for E. histolytica, E. moshkovskii and E. dispar in a total of 190 out of 202 stool specimens (94% sensitive) that were positive for E. histolytica/E. dispar/E. moshkovskii by examination of stool by microscopy and/or culture. All the 35 negative control stool samples that were negative for E. histolytica/E. dispar/E. moshkovskii by microscopy and culture were also found negative by the nested multiplex PCR (100% specific). The result from the study shows that only 34.6% of the patient stool samples that were positive for E. histolytica/E. dispar/E. moshkovskii by examination of stool by microscopy and/or culture, were actually positive for pathogenic E. histolytica and the remaining majority of the stool samples were positive for non-pathogenic E. dispar or E. moshkovskii as demonstrated by the use of nested multiplex PCR. Conclusion The present study reports a new nested multiplex PCR strategy for species specific detection and differentiation of E. histolytica, E. dispar and E. moshkovskii DNA in stool specimens. The test is highly specific, sensitive and also rapid, providing the results within 12 hours of receiving stool specimens. PMID:17524135

  1. A Genomewide Overexpression Screen Identifies Genes Involved in the Phosphatidylinositol 3-Kinase Pathway in the Human Protozoan Parasite Entamoeba histolytica

    PubMed Central

    Koushik, Amrita B.; Welter, Brenda H.; Rock, Michelle L.

    2014-01-01

    Entamoeba histolytica is a protozoan parasite that causes amoebic dysentery and liver abscess. E. histolytica relies on motility, phagocytosis, host cell adhesion, and proteolysis of extracellular matrix for virulence. In eukaryotic cells, these processes are mediated in part by phosphatidylinositol 3-kinase (PI3K) signaling. Thus, PI3K may be critical for virulence. We utilized a functional genomics approach to identify genes whose products may operate in the PI3K pathway in E. histolytica. We treated a population of trophozoites that were overexpressing genes from a cDNA library with a near-lethal dose of the PI3K inhibitor wortmannin. This screen was based on the rationale that survivors would be overexpressing gene products that directly or indirectly function in the PI3K pathway. We sequenced the overexpressed genes in survivors and identified a cDNA encoding a Rap GTPase, a protein previously shown to participate in the PI3K pathway. This supports the validity of our approach. Genes encoding a coactosin-like protein, EhCoactosin, and a serine-rich E. histolytica protein (SREHP) were also identified. Cells overexpressing EhCoactosin or SREHP were also less sensitive to a second PI3K inhibitor, LY294002. This corroborates the link between these proteins and PI3K. Finally, a mutant cell line with an increased level of phosphatidylinositol (3,4,5)-triphosphate, the product of PI3K activity, exhibited increased expression of SREHP and EhCoactosin. This further supports the functional connection between these proteins and PI3K in E. histolytica. To our knowledge, this is the first forward-genetics screen adapted to reveal genes participating in a signal transduction pathway in this pathogen. PMID:24442890

  2. Antiamoebic Activity of Adenophyllum aurantium (L.) Strother and Its Effect on the Actin Cytoskeleton of Entamoeba histolytica.

    PubMed

    Herrera-Martínez, Mayra; Hernández-Ramírez, Verónica I; Hernández-Carlos, Beatriz; Chávez-Munguía, Bibiana; Calderón-Oropeza, Mónica A; Talamás-Rohana, Patricia

    2016-01-01

    In Mexico, the Adenophyllum aurantium (L.) Strother plant is consumed as an infusion to treat intestinal diseases such as amoebiasis, which is an endemic health problem in Mexico and other countries. However, the effect of A. aurantium on Entamoeba histolytica, the causative agent of amoebiasis, is unknown. An aerial part methanolic extract (AaMeA), a root methanolic extract (AaMeR) and a root ethyl acetate extract (AaEaR) were tested on E. histolytica trophozoites. AaMeA and AaMeR did not show antiproliferative activity; however, AaEaR exhibited an in vitro GI50 of 230 μg/ml, and it was able to inhibit the differentiation of Entamoeba invadens trophozoites into cysts. The intraperitoneal administration of AaEaR (2.5 or 5 mg) to hamsters that were infected with E. histolytica inhibited the development of amoebic liver abscesses in 48.5 or 89.0% of the animals, respectively. Adhesion to fibronectin and erythrophagocytosis were 28.7 and 37.5% inhibited by AaEaR, respectively. An ultrastructure analysis of AaEaR-treated trophozoites shows a decrease in the number of vacuoles but no apparent cell damage. Moreover, this extract affected the actin cytoskeleton structuration, and it prevented the formation of contractile rings by mechanism(s) that were independent of reactive oxygen species and RhoA activation pathways. (13)C NMR data showed that the major compounds in the AaEaR extract are thiophenes. Our results suggest that AaEaR may be effective in treatments against amoebiasis, nevertheless, detailed toxicity studies on thiophenes, contained in AaEaR, are required to avoid misuse of this vegetal species. PMID:27445810

  3. Antiamoebic Activity of Adenophyllum aurantium (L.) Strother and Its Effect on the Actin Cytoskeleton of Entamoeba histolytica

    PubMed Central

    Herrera-Martínez, Mayra; Hernández-Ramírez, Verónica I.; Hernández-Carlos, Beatriz; Chávez-Munguía, Bibiana; Calderón-Oropeza, Mónica A.; Talamás-Rohana, Patricia

    2016-01-01

    In Mexico, the Adenophyllum aurantium (L.) Strother plant is consumed as an infusion to treat intestinal diseases such as amoebiasis, which is an endemic health problem in Mexico and other countries. However, the effect of A. aurantium on Entamoeba histolytica, the causative agent of amoebiasis, is unknown. An aerial part methanolic extract (AaMeA), a root methanolic extract (AaMeR) and a root ethyl acetate extract (AaEaR) were tested on E. histolytica trophozoites. AaMeA and AaMeR did not show antiproliferative activity; however, AaEaR exhibited an in vitro GI50 of 230 μg/ml, and it was able to inhibit the differentiation of Entamoeba invadens trophozoites into cysts. The intraperitoneal administration of AaEaR (2.5 or 5 mg) to hamsters that were infected with E. histolytica inhibited the development of amoebic liver abscesses in 48.5 or 89.0% of the animals, respectively. Adhesion to fibronectin and erythrophagocytosis were 28.7 and 37.5% inhibited by AaEaR, respectively. An ultrastructure analysis of AaEaR-treated trophozoites shows a decrease in the number of vacuoles but no apparent cell damage. Moreover, this extract affected the actin cytoskeleton structuration, and it prevented the formation of contractile rings by mechanism(s) that were independent of reactive oxygen species and RhoA activation pathways. 13C NMR data showed that the major compounds in the AaEaR extract are thiophenes. Our results suggest that AaEaR may be effective in treatments against amoebiasis, nevertheless, detailed toxicity studies on thiophenes, contained in AaEaR, are required to avoid misuse of this vegetal species. PMID:27445810

  4. Comparison of the Triage Micro Parasite Panel and Microscopy for the Detection of Entamoeba histolytica/Entamoeba dispar, Giardia lamblia, and Cryptosporidium parvum in Stool Samples Collected in Kenya

    PubMed Central

    Swierczewski, Brett; Odundo, Elizabeth; Ndonye, Janet; Kirera, Ronald; Odhiambo, Cliff; Oaks, Edwin

    2012-01-01

    Entamoeba histolytica, Giardia lamblia, and Cryptosporidium parvum are three of the most important parasitic causes of acute diarrhea worldwide. Laboratory diagnosis of these parasites is usually done by ova and parasite examination (O&P examination) via microscopy. The sensitivity and specificity of O&P examination varies among laboratories and can be labor intensive and time consuming. The Triage Micro Parasite Panel (BioSite, San Diego, California) is an enzyme immunoassay kit that can detect E. histolytica/E. dispar, G. lamblia, and C. parvum simultaneously using fresh or frozen stool. The present study evaluated the Triage Micro Parasite Panel in detecting E. histolytica/E. dispar, G. lamblia, and C. parvum compared to O&P examination in 266 stool samples collected at medical facilities in Kenya. The sensitivity and specificity results for the Triage Micro Parasite Panel were: for E. histolytica/E. dispar: 100%, 100%, G. lamblia: 100%, 100% and C. parvum: 73%, 100%. There was no evidence of cross reactivity using the kit with other parasites identified in the stool specimens. These results indicate that the Triage Micro Parasite Panel is a highly sensitive kit that can be used for screening purposes in large scale studies or outbreak investigations or as a possible alternative to O&P examination. PMID:22848229

  5. Crystallization and preliminary X-ray analysis of RabX3, a tandem GTPase from Entamoeba histolytica.

    PubMed

    Kumar Srivastava, Vijay; Chandra, Mintu; Datta, Sunando

    2014-07-01

    Ras superfamily GTPases regulate signalling pathways that control multiple biological processes by modulating the GTP/GDP cycle. Various Rab GTPases, which are the key regulators of vesicular trafficking pathways, play a vital role in the survival and virulence of the enteric parasite Entamoeba histolytica. The Rab GTPases act as binary molecular switches that utilize the conformational changes associated with the GTP/GDP cycle to elicit responses from target proteins and thereby regulate a broad spectrum of cellular processes including cell proliferation, cytoskeletal assembly, nuclear transport and intracellular membrane trafficking in eukaryotes. Entamoeba histolytica RabX3 (EhRabX3) is a unique GTPase in the amoebic genome, the only member in the eukaryotic Ras superfamily that harbours tandem G-domains and shares only 8-16% sequence identity with other GTPases. Recent studies suggested that EhRabX3 binds to a single guanine nucleotide through its N-terminal G-domain (NTD), while the C-terminal G-domain (CTD) plays a potential role in binding of the nucleotide to the NTD. Thus, understanding the intermolecular regulation between the two GTPase domains is expected to reveal valuable information on the overall action of EhRabX3. To provide structural insights into the inclusive action of this unique GTPase, EhRabX3 was crystallized by successive micro-seeding using the vapour-diffusion method. A complete data set was collected to 3.3 Å resolution using a single native EhRabX3 crystal at 100 K on BM14 at the ESRF, Grenoble, France. The crystal belonged to monoclinic space group C2, with unit-cell parameters a=198.6, b=119.3, c=89.2 Å, β=103.1°. Preliminary analysis of the data using the Matthews Probability Calculator suggested the presence of four to six molecules in the asymmetric unit. PMID:25005092

  6. New Assembly, Reannotation and Analysis of the Entamoeba histolytica Genome Reveal New Genomic Features and Protein Content Information

    PubMed Central

    Lorenzi, Hernan A.; Puiu, Daniela; Miller, Jason R.; Brinkac, Lauren M.; Amedeo, Paolo; Hall, Neil; Caler, Elisabet V.

    2010-01-01

    Background In order to maintain genome information accurately and relevantly, original genome annotations need to be updated and evaluated regularly. Manual reannotation of genomes is important as it can significantly reduce the propagation of errors and consequently diminishes the time spent on mistaken research. For this reason, after five years from the initial submission of the Entamoeba histolytica draft genome publication, we have re-examined the original 23 Mb assembly and the annotation of the predicted genes. Principal Findings The evaluation of the genomic sequence led to the identification of more than one hundred artifactual tandem duplications that were eliminated by re-assembling the genome. The reannotation was done using a combination of manual and automated genome analysis. The new 20 Mb assembly contains 1,496 scaffolds and 8,201 predicted genes, of which 60% are identical to the initial annotation and the remaining 40% underwent structural changes. Functional classification of 60% of the genes was modified based on recent sequence comparisons and new experimental data. We have assigned putative function to 3,788 proteins (46% of the predicted proteome) based on the annotation of predicted gene families, and have identified 58 protein families of five or more members that share no homology with known proteins and thus could be entamoeba specific. Genome analysis also revealed new features such as the presence of segmental duplications of up to 16 kb flanked by inverted repeats, and the tight association of some gene families with transposable elements. Significance This new genome annotation and analysis represents a more refined and accurate blueprint of the pathogen genome, and provides an upgraded tool as reference for the study of many important aspects of E. histolytica biology, such as genome evolution and pathogenesis. PMID:20559563

  7. EhADH112 Is a Bro1 Domain-Containing Protein Involved in the Entamoeba histolytica Multivesicular Bodies Pathway

    PubMed Central

    Bañuelos, Cecilia; García-Rivera, Guillermina; López-Reyes, Israel; Mendoza, Leobardo; González-Robles, Arturo; Herranz, Silvia; Vincent, Olivier; Orozco, Esther

    2012-01-01

    EhADH112 is an Entamoeba histolytica Bro1 domain-containing protein, structurally related to mammalian ALIX and yeast BRO1, both involved in the Endosomal Sorting Complexes Required for Transport (ESCRT)-mediated multivesicular bodies (MVB) biogenesis. Here, we investigated an alternative role for EhADH112 in the MVB protein trafficking pathway by overexpressing 166 amino acids of its N-terminal Bro1 domain in trophozoites. Trophozoites displayed diminished phagocytosis rates and accumulated exogenous Bro1 at cytoplasmic vesicles which aggregated into aberrant complexes at late stages of phagocytosis, probably preventing EhADH112 function. Additionally, the existence of a putative E. histolytica ESCRT-III subunit (EhVps32) presumably interacting with EhADH112, led us to perform pull-down experiments with GST-EhVps32 and [35S]-labeled EhADH112 or EhADH112 derivatives, confirming EhVps32 binding to EhADH112 through its Bro1 domain. Our overall results define EhADH112 as a novel member of ESCRT-accessory proteins transiently present at cellular surface and endosomal compartments, probably contributing to MVB formation during phagocytosis. PMID:22500103

  8. Virtual screening, identification and in vitro testing of novel inhibitors of O-acetyl-L-serine sulfhydrylase of Entamoeba histolytica.

    PubMed

    Nagpal, Isha; Raj, Isha; Subbarao, Naidu; Gourinath, Samudrala

    2012-01-01

    The explosive epidemicity of amoebiasis caused by the facultative gastrointestinal protozoan parasite Entamoeba histolytica is a major public health problem in developing countries. Multidrug resistance and side effects of various available antiamoebic drugs necessitate the design of novel antiamobeic agents. The cysteine biosynthetic pathway is the critical target for drug design due to its significance in the growth, survival and other cellular activities of E. histolytica. Here, we have screened 0.15 million natural compounds from the ZINC database against the active site of the EhOASS enzyme (PDB ID. 3BM5, 2PQM), whose structure we previously determined to 2.4 Å and 1.86 Å resolution. For this purpose, the incremental construction algorithm of GLIDE and the genetic algorithm of GOLD were used. We analyzed docking results for top ranking compounds using a consensus scoring function of X-Score to calculate the binding affinity and using ligplot to measure protein-ligand interactions. Fifteen compounds that possess good inhibitory activity against EhOASS active site were identified that may act as potential high affinity inhibitors. In vitro screening of a few commercially available compounds established their biological activity. The first ranked compound ZINC08931589 had a binding affinity of ∼8.05 µM and inhibited about 73% activity at 0.1 mM concentration, indicating good correlation between in silico prediction and in vitro inhibition studies. This compound is thus a good starting point for further development of strong inhibitors. PMID:22355310

  9. Resveratrol Induces Apoptosis-Like Death and Prevents In Vitro and In Vivo Virulence of Entamoeba histolytica

    PubMed Central

    Pais-Morales, Jonnatan; Betanzos, Abigail; García-Rivera, Guillermina; Chávez-Munguía, Bibiana; Shibayama, Mineko; Orozco, Esther

    2016-01-01

    Entamoeba histolytica causes amoebiasis, an infection that kills 100,000 individuals each year. Metronidazole and its derivatives are currently used against this protozoan, but these drugs present adverse effects on human health. Here, we investigated the effect of resveratrol (a natural compound) on E. histolytica trophozoites viability, as well as its influence on the parasite virulence. Trophozoites growth was arrested by 72 μM resveratrol and the IC50 was determined as 220 μM at 48 h. Cells appeared smaller, rounded and in clusters, with debris-containing vacuoles and with abnormally condensed chromatin. Resveratrol triggered reactive oxygen species production. It caused lipid peroxidation and produced phosphatidylserine externalization and DNA fragmentation this latter evidenced by TUNEL assays. It also provoked an increase of intracellular Ca2+ concentration, activated calpain and decreased superoxide dismutase activity, indicating that an apoptosis-like event occurred; however, autophagy was not detected. Cytopathic activity, phagocytosis, encystment and in vivo virulence were diminished dramatically by pre-incubation of trophozoites with resveratrol, evidencing that resveratrol attenuated the trophozoite virulence in vitro. Interestingly, after the inoculation of virulent trophozoites, animals treated with the drug did not develop or developed very small abscesses. Our findings propose that resveratrol could be an alternative to contend amoebiasis. PMID:26731663

  10. Multinucleation and Polykaryon Formation is Promoted by the EhPC4 Transcription Factor in Entamoeba histolytica

    PubMed Central

    Cruz, Olga Hernández de la; Marchat, Laurence A.; Guillén, Nancy; Weber, Christian; Rosas, Itzel López; Díaz-Chávez, José; Herrera, Luis; Rojo-Domínguez, Arturo; Orozco, Esther; López-Camarillo, César

    2016-01-01

    Entamoeba histolytica is the intestinal parasite responsible for human amoebiasis that is a leading cause of death in developing countries. In this protozoan, heterogeneity in DNA content, polyploidy and genome plasticity have been associated to alterations in mechanisms controlling DNA replication and cell division. Studying the function of the transcription factor EhPC4, we unexpectedly found that it is functionally related to DNA replication, and multinucleation. Site-directed mutagenesis on the FRFPKG motif revealed that the K127 residue is required for efficient EhPC4 DNA-binding activity. Remarkably, overexpression of EhPC4 significantly increased cell proliferation, DNA replication and DNA content of trophozoites. A dramatically increase in cell size resulting in the formation of giant multinucleated trophozoites (polykaryon) was also found. Multinucleation event was associated to cytokinesis failure leading to abortion of ongoing cell division. Consistently, genome-wide profiling of EhPC4 overexpressing trophozoites revealed the up-regulation of genes involved in carbohydrates and nucleic acids metabolism, chromosome segregation and cytokinesis. Forced overexpression of one of these genes, EhNUDC (nuclear movement protein), led to alterations in cytokinesis and partially recapitulated the multinucleation phenotype. These data indicate for the first time that EhPC4 is associated with events related to polyploidy and genome stability in E. histolytica. PMID:26792358

  11. Resveratrol Induces Apoptosis-Like Death and Prevents In Vitro and In Vivo Virulence of Entamoeba histolytica.

    PubMed

    Pais-Morales, Jonnatan; Betanzos, Abigail; García-Rivera, Guillermina; Chávez-Munguía, Bibiana; Shibayama, Mineko; Orozco, Esther

    2016-01-01

    Entamoeba histolytica causes amoebiasis, an infection that kills 100,000 individuals each year. Metronidazole and its derivatives are currently used against this protozoan, but these drugs present adverse effects on human health. Here, we investigated the effect of resveratrol (a natural compound) on E. histolytica trophozoites viability, as well as its influence on the parasite virulence. Trophozoites growth was arrested by 72 μM resveratrol and the IC50 was determined as 220 μM at 48 h. Cells appeared smaller, rounded and in clusters, with debris-containing vacuoles and with abnormally condensed chromatin. Resveratrol triggered reactive oxygen species production. It caused lipid peroxidation and produced phosphatidylserine externalization and DNA fragmentation this latter evidenced by TUNEL assays. It also provoked an increase of intracellular Ca2+ concentration, activated calpain and decreased superoxide dismutase activity, indicating that an apoptosis-like event occurred; however, autophagy was not detected. Cytopathic activity, phagocytosis, encystment and in vivo virulence were diminished dramatically by pre-incubation of trophozoites with resveratrol, evidencing that resveratrol attenuated the trophozoite virulence in vitro. Interestingly, after the inoculation of virulent trophozoites, animals treated with the drug did not develop or developed very small abscesses. Our findings propose that resveratrol could be an alternative to contend amoebiasis. PMID:26731663

  12. Intracellular traffic of the lysine and glutamic acid rich protein KERP1 reveals features of endomembrane organization in Entamoeba histolytica.

    PubMed

    Perdomo, Doranda; Manich, Maria; Syan, Sylvie; Olivo-Marin, Jean-Christophe; Dufour, Alexandre C; Guillén, Nancy

    2016-08-01

    The development of amoebiasis is influenced by the expression of the lysine and glutamic acid rich protein 1 (KERP1), a virulence factor involved in Entamoeba histolytica adherence to human cells. Up to date, it is unknown how the protein transits the parasite cytoplasm towards the plasma membrane, specially because this organism lacks a well-defined endoplasmic reticulum (ER) and Golgi apparatus. In this work we demonstrate that KERP1 is present at the cell surface and in intracellular vesicles which traffic in a pathway that is independent of the ER-Golgi anterograde transport. The intracellular displacement of vesicles enriched in KERP1 relies on the actin-rich cytoskeleton activities. KERP1 is also present in externalized vesicles deposited on the surface of human cells. We further report the interactome of KERP1 with its association to endomembrane components and lipids. The model for KERP1 traffic here proposed hints for the first time elements of the endocytic and exocytic paths of E. histolytica. PMID:26857352

  13. Multinucleation and Polykaryon Formation is Promoted by the EhPC4 Transcription Factor in Entamoeba histolytica.

    PubMed

    Hernández de la Cruz, Olga; Marchat, Laurence A; Guillén, Nancy; Weber, Christian; López Rosas, Itzel; Díaz-Chávez, José; Herrera, Luis; Rojo-Domínguez, Arturo; Orozco, Esther; López-Camarillo, César

    2016-01-01

    Entamoeba histolytica is the intestinal parasite responsible for human amoebiasis that is a leading cause of death in developing countries. In this protozoan, heterogeneity in DNA content, polyploidy and genome plasticity have been associated to alterations in mechanisms controlling DNA replication and cell division. Studying the function of the transcription factor EhPC4, we unexpectedly found that it is functionally related to DNA replication, and multinucleation. Site-directed mutagenesis on the FRFPKG motif revealed that the K127 residue is required for efficient EhPC4 DNA-binding activity. Remarkably, overexpression of EhPC4 significantly increased cell proliferation, DNA replication and DNA content of trophozoites. A dramatically increase in cell size resulting in the formation of giant multinucleated trophozoites (polykaryon) was also found. Multinucleation event was associated to cytokinesis failure leading to abortion of ongoing cell division. Consistently, genome-wide profiling of EhPC4 overexpressing trophozoites revealed the up-regulation of genes involved in carbohydrates and nucleic acids metabolism, chromosome segregation and cytokinesis. Forced overexpression of one of these genes, EhNUDC (nuclear movement protein), led to alterations in cytokinesis and partially recapitulated the multinucleation phenotype. These data indicate for the first time that EhPC4 is associated with events related to polyploidy and genome stability in E. histolytica. PMID:26792358

  14. Crystallization and preliminary X-ray analysis of l-methionine γ-lyase 1 from Entamoeba histolytica

    SciTech Connect

    Sato, Dan; Karaki, Tsuyoshi; Shimizu, Akira; Kamei, Kaeko; Harada, Shigeharu; Nozaki, Tomoyoshi

    2008-08-01

    l-Methionine γ-lyase 1, a key enzyme in sulfur-containing amino-acid degradation, from the protozoan parasite E. histolytica was crystallized in a form suitable for X-ray structure analysis. l-Methionine γ-lyase (MGL) is a pyridoxal phosphate-dependent enzyme that is involved in the degradation of sulfur-containing amino acids. MGL is an attractive drug target against amoebiasis because the mammalian host of its causative agent Entamoeba histolytica lacks MGL. For the development of anti-amoebic agents based on the structure of MGL, one of two MGL isoenzymes (EhMGL1) was crystallized in the monoclinic space group P2{sub 1}, with unit-cell parameters a = 99.12, b = 85.38, c = 115.37 Å, β = 101.82°. The crystals diffract to beyond 2.0 Å resolution. The presence of a tetramer in the asymmetric unit (4 × 42.4 kDa) gives a Matthews coefficient of 2.8 Å{sup 3} Da{sup −1} and a solvent content of 56%. The structure was solved by the molecular-replacement method and structure refinement is now in progress.

  15. The EhADH112 recombinant polypeptide inhibits cell destruction and liver abscess formation by Entamoeba histolytica trophozoites.

    PubMed

    Martínez-López, Carolina; Orozco, Esther; Sánchez, Tomás; García-Pérez, Rosa María; Hernández-Hernández, Fidel; Rodríguez, Mario A

    2004-04-01

    The Entamoeba histolytica EhCPADH complex, formed by a cysteine proteinase (EhCP112) and an adhesin (EhADH112), is involved in adherence, phagocytosis and cytolysis. This makes this complex an attractive candidate as a vaccine against amoebiasis. Here, we produced the recombinant polypeptide EhADH243, which includes the adherence epitope detected by a monoclonal antibody against the EhCPADH complex. EhADH243 was purified, and the effect of the polypeptide on in vitro and in vivo virulence was studied. Antibodies against EhADH243 reacted with the EhCPADH complex and with the recombinant polypeptide. EhADH243 and antibodies against this polypeptide inhibited adherence, phagocytosis and destruction of cell monolayers by live trophozoites, but had little effect on cell monolayer destruction by trophozoite extracts. EhADH243 recognized a 97 kDa protein in the MDCK membrane fraction that could be a putative receptor for E. histolytica trophozoites. Hamsters immunized with EhADH243 developed humoral response against EhCPADH, and animals were partially protected from amoebic liver abscess. PMID:15009028

  16. Expression in fibroblasts and in live animals of Entamoeba histolytica polypeptides EhCP112 and EhADH112.

    PubMed

    Madriz, Xochil; Martínez, Máximo B; Rodríguez, Mario A; Sierra, Gustavo; Martínez-López, Carolina; Riverón, Ana M; Flores, Leopoldo; Orozco, Esther

    2004-05-01

    EhCPADH is an immunogenic, heterodimeric protein that is formed by EhCP112 (cysteine protease) and EhADH112 (adhesin), polypeptides involved in Entamoeba histolytica's cytopathic effect, target-cell adherence and phagocytosis. The EhCPADH complex is located in the plasma membrane and cytoplasmic vacuoles. Here, the independent expression of EhCP112 and EhADH112 in fibroblasts and hamsters was analysed. Also investigated was the immunological response in animals independently inoculated with plasmid pcDNA-Ehcp112, which carries the complete cysteine protease-encoding gene, or with plasmid pcDNA-Ehadh112, which carries the C terminus of the adhesin-encoding gene, or with a mixture of both. Both proteins were expressed in the plasma membranes of the transfected fibroblasts. EhCP112 was toxic for the mammalian cells. Proteins were also independently expressed in hamsters after inoculation with the plasmids. Their expression was indirectly evaluated by the presence of antibodies in the inoculated animals. Remarkably, co-immunization of the animals with the two DNA plasmids resulted in an earlier and higher anti-E. histolytica IgG induction than immunization with separate plasmids. In contrast, the cellular immune response was not noticeably improved by the plasmid mixture. Interestingly, protection against liver abscesses was detected only in animals that received the plasmid mixture and no protection was observed in hamsters independently inoculated with plasmid pcDNA-Ehcp112 or pcDNA-Ehadh112. PMID:15133088

  17. Tyrosine phosphorylation/dephosphorylation of myosin II essential light chains of Entamoeba histolytica trophozoites regulates their motility.

    PubMed

    Bonilla-Moreno, Raúl; Pérez-Yépez, Eloy-Andrés; Villegas-Sepúlveda, Nicolás; Morales, Fernando O; Meza, Isaura

    2016-08-01

    Entamoeba histolytica trophozoites dwell in the human intestine as comensals although under still unclear circumstances become invasive and destroy the host tissues. For these activities, trophozoites relay on remarkable motility provided by the cytoskeleton organization. Amebic actin and some of its actin-associated proteins are well known, while components of the myosin II molecule, although predicted from the E. histolytica genome, need biochemical and functional characterization. Recently, an amebic essential light myosin II chain, named EhMLCI, was identified and reported to be phosphorylated in tyrosines. The phosphorylated form of the protein was associated with the soluble assembly incompetent conformation of the heavy myosin chains, while the non-phosphorylated protein was identified with filamentous heavy chains, organized in an assembly competent conformation. It was postulated that EhMLCI tyrosine phosphorylation could act as a negative regulator of myosin II activity by its phosphorylation/dephosphorylation cycles. To test this hypothesis, we constructed an expression vector containing an EhMLCI DNA sequence where two tyrosine residues, with strong probability of phosphorylation and fall within the single EF-hand domain that interacts with the N-terminus of myosin II heavy chains, were replaced by phenylalanines. Transfected trophozoites, expressing the mutant MutEhMLCI protein cannot process it, thereby not incorporated into the phosphorylation/dephosphorylation cycles required for myosin II activity, results in motility defective trophozoites. PMID:27318258

  18. Free fatty acids released from phospholipids are the major heat-stable hemolytic factor of Entamoeba histolytica trophozoites.

    PubMed Central

    Said-Fernández, S; López-Revilla, R

    1988-01-01

    The major hemolytic activity of Entamoeba histolytica trophozoites is located in a vesicular fraction called P30 and known to be due to heat-labile and heat-stable hemolytic components whose effect increases up to 100 times during preincubation at 36 degrees C. The heat-stable hemolytic activity (HSHA) was found in the chloroform-methanol extract of preincubated P30, whose partition with 2 M KCl yielded a lipid fraction, an interphase, and an aqueous phase. HSHA was detected only in the lipid fraction, where it amounted to 59% of the chloroform-methanol extract activity and increased 50% when supplemented with the interphase material; it was accounted for by the free fatty acids, whose potency increased 33% with the interphase material, and was blocked by delipidated bovine serum albumin. A parallel increase in free fatty acids and lysophospholipids and a corresponding decrease in phospholipids were observed during P30 preincubation. Most of the phospholipase activity of trophozoite homogenates was also found in P30. Therefore, most of the HSHA generated during preincubation was due to free fatty acids released from phospholipids by a P30 phospholipase that may contribute significantly to E. histolytica cytopathogenicity and virulence. Images PMID:2894362

  19. Natural Killer T Cells Activated by a Lipopeptidophosphoglycan from Entamoeba histolytica Are Critically Important To Control Amebic Liver Abscess

    PubMed Central

    Lindner, Buko; Winau, Florian; Isibasi, Armando; Moreno-Lafont, Martha; Ulmer, Artur J.; Holst, Otto; Tannich, Egbert; Jacobs, Thomas

    2009-01-01

    The innate immune response is supposed to play an essential role in the control of amebic liver abscess (ALA), a severe form of invasive amoebiasis due to infection with the protozoan parasite Entamoeba histolytica. In a mouse model for the disease, we previously demonstrated that Jα18-/- mice, lacking invariant natural killer T (iNKT) cells, suffer from more severe abscess development. Here we show that the specific activation of iNKT cells using α-galactosylceramide (α-GalCer) induces a significant reduction in the sizes of ALA lesions, whereas CD1d−/− mice develop more severe abscesses. We identified a lipopeptidophosphoglycan from E. histolytica membranes (EhLPPG) as a possible natural NKT cell ligand and show that the purified phosphoinositol (PI) moiety of this molecule induces protective IFN-γ but not IL-4 production in NKT cells. The main component of EhLPPG responsible for NKT cell activation is a diacylated PI, (1-O-[(28∶0)-lyso-glycero-3-phosphatidyl-]2-O-(C16:0)-Ins). IFN-γ production by NKT cells requires the presence of CD1d and simultaneously TLR receptor signalling through MyD88 and secretion of IL-12. Similar to α-GalCer application, EhLPPG treatment significantly reduces the severity of ALA in ameba-infected mice. Our results suggest that EhLPPG is an amebic molecule that is important for the limitation of ALA development and may explain why the majority of E. histolytica-infected individuals do not develop amebic liver abscess. PMID:19436711

  20. Natural killer T cells activated by a lipopeptidophosphoglycan from Entamoeba histolytica are critically important to control amebic liver abscess.

    PubMed

    Lotter, Hannelore; González-Roldán, Nestor; Lindner, Buko; Winau, Florian; Isibasi, Armando; Moreno-Lafont, Martha; Ulmer, Artur J; Holst, Otto; Tannich, Egbert; Jacobs, Thomas

    2009-05-01

    The innate immune response is supposed to play an essential role in the control of amebic liver abscess (ALA), a severe form of invasive amoebiasis due to infection with the protozoan parasite Entamoeba histolytica. In a mouse model for the disease, we previously demonstrated that Jalpha18(-/-) mice, lacking invariant natural killer T (iNKT) cells, suffer from more severe abscess development. Here we show that the specific activation of iNKT cells using alpha-galactosylceramide (alpha-GalCer) induces a significant reduction in the sizes of ALA lesions, whereas CD1d(-/-) mice develop more severe abscesses. We identified a lipopeptidophosphoglycan from E. histolytica membranes (EhLPPG) as a possible natural NKT cell ligand and show that the purified phosphoinositol (PI) moiety of this molecule induces protective IFN-gamma but not IL-4 production in NKT cells. The main component of EhLPPG responsible for NKT cell activation is a diacylated PI, (1-O-[(28:0)-lyso-glycero-3-phosphatidyl-]2-O-(C16:0)-Ins). IFN-gamma production by NKT cells requires the presence of CD1d and simultaneously TLR receptor signalling through MyD88 and secretion of IL-12. Similar to alpha-GalCer application, EhLPPG treatment significantly reduces the severity of ALA in ameba-infected mice. Our results suggest that EhLPPG is an amebic molecule that is important for the limitation of ALA development and may explain why the majority of E. histolytica-infected individuals do not develop amebic liver abscess. PMID:19436711

  1. Crystal structure of the cysteine protease inhibitor 2 from Entamoeba histolytica: Functional convergence of a common protein fold

    SciTech Connect

    Casados-Vázquez, Luz E.; Lara-González, Samuel; Brieb, Luis G.

    2012-04-18

    Cysteine proteases (CP) are key pathogenesis and virulence determinants of protozoan parasites. Entamoeba histolytica contains at least 50 cysteine proteases; however, only three (EhCP1, EhCP2 and EhCP5) are responsible for approximately 90% of the cysteine protease activity in this parasite. CPs are expressed as inactive zymogens. Because the processed proteases are potentially cytotoxic, protozoan parasites have developed mechanisms to regulate their activity. Inhibitors of cysteine proteases (ICP) of the chagasin-like inhibitor family (MEROPS family I42) were recently identified in bacteria and protozoan parasites. E. histolytica contains two ICP-encoding genes of the chagasin-like inhibitor family. EhICP1 localizes to the cytosol, whereas EhICP2 is targeted to phagosomes. Herein, we report two crystal structures of EhICP2. The overall structure of EhICP2 consists of eight {beta}-strands and closely resembles the immunoglobulin fold. A comparison between the two crystal forms of EhICP2 indicates that the conserved BC, DE and FG loops form a flexible wedge that may block the active site of CPs. The positively charged surface of the wedge-forming loops in EhICP2 contrasts with the neutral surface of the wedge-forming loops in chagasin. We postulate that the flexibility and positive charge observed in the DE and FG loops of EhICP2 may be important to facilitate the initial binding of this inhibitor to the battery of CPs present in E. histolytica.

  2. Cloning and expression of a putative alcohol dehydrogenase gene of Entamoeba histolytica and its application to immunological examination.

    PubMed Central

    Kimura, A; Hara, Y; Kimoto, T; Okuno, Y; Minekawa, Y; Nakabayashi, T

    1996-01-01

    To clone and express the genes encoding major antigens of Entamoeba histolytica, we constructed a lambda gt11 cDNA library for E. histolytica HM1:IMSS and screened it with pooled sera from patients with amoebiasis. A 1,223-bp cDNA was cloned (clone 1223), and its nucleotide sequence was determined. The amino acid sequence predicted to be encoded by the open reading frame of clone 1223 consisted of 396 residues and showed 32.5 and 32.3% homology to the NADH-dependent butanol dehydrogenases I and II (bdhA and bdhB) of Clostridium acetobutylicum, respectively. In addition, 29 of the 34 consensus positions of bdhA and bdhB were also well conserved in clone 1223. The recombinant protein expressed from clone 1223 had an estimated molecular mass of 43.5 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The antigenicity and specificity of the recombinant protein were evaluated by an enzyme-linked immunosorbent assay using sera obtained from two clinical groups of patients with amoebiasis and a group of healthy controls. The recombinant protein had potent and specific antigenicity. In all, 53 serum samples (88.3%) from 60 patients with amoebiasis were positive for immunoglobulin G antibody against the recombinant protein, with a mean optical density value of 0.42. In contrast, 53 of 54 healthy control serum samples were negative, with only 1 positive serum sample showing the lower optical density value. These results suggested that clone 1223 is promising in terms of providing a useful antigen for the accurate serodiagnosis of amoebiasis and that the gene encodes a putative alcohol dehydrogenase of E. histolytica. PMID:8705667

  3. Entamoeba histolytica: differential gene expression during programmed cell death and identification of early pro- and anti-apoptotic signals.

    PubMed

    Monroy, Virginia Sánchez; Flores, Ma Olivia Medel; Villalba-Magdaleno, José D'Artagnan; Garcia, Consuelo Gómez; Ishiwara, David Guillermo Pérez

    2010-12-01

    We have demonstrated that programmed cell death (PCD) in Entamoeba histolytica is induced in vitro by G418 aminoglycoside antibiotic. To ascertain if biochemical and morphological changes previously observed are paired to molecular changes that reflect a genetic program, we looked here for early differential gene expression during the induction of PCD. Using cDNA-amplified fragment length polymorphisms (AFLPs) and in silico derived analysis we showed in E. histolytica a differential gene expression during PCD induced by G418. The genes identified encoded for proteins homologous to Glutaminyl-tRNA synthase, Ribosomal Subunit Proteins 40S and 18S, Saposin-like, Silent Information Regulator-2 (Sir-2), and Grainins 1 and 2. Using real-time quantitative PCR (RT Q-PCR), we found that glutaminyl-tRNA synthetase, sir-2, grainins and saposin-like genes were strongly overexpressed after 30min of PCD induction, while its expression dramatically decreased up to 60min. On the other hand, overexpression of ribosomal genes increased only 7-fold of basal expression, showing a progressive down-regulation up to 90min. glutaminyl-tRNA synthetase, sir-2 and grainins could act as negative regulators of PCD, trying to control the biochemical changes related to PCD activation. Overexpression of saposin-like gene could act as up-regulator of some cell death pathways. Our results give evidence of the first genes identified during the early stage of PCD in E. histolytica that could be implicated in regulation of apoptotic pathways. PMID:20515683

  4. A transposon-derived DNA polymerase from Entamoeba histolytica displays intrinsic strand displacement, processivity and lesion bypass.

    PubMed

    Pastor-Palacios, Guillermo; López-Ramírez, Varinia; Cardona-Felix, Cesar S; Brieba, Luis G

    2012-01-01

    Entamoeba histolytica encodes four family B2 DNA polymerases that vary in amino acid length from 813 to 1279. These DNA polymerases contain a N-terminal domain with no homology to other proteins and a C-terminal domain with high amino acid identity to archetypical family B2 DNA polymerases. A phylogenetic analysis indicates that these family B2 DNA polymerases are grouped with DNA polymerases from transposable elements dubbed Polintons or Mavericks. In this work, we report the cloning and biochemical characterization of the smallest family B2 DNA polymerase from E. histolytica. To facilitate its characterization we subcloned its 660 amino acids C-terminal region that comprises the complete exonuclease and DNA polymerization domains, dubbed throughout this work as EhDNApolB2. We found that EhDNApolB2 displays remarkable strand displacement, processivity and efficiently bypasses the DNA lesions: 8-oxo guanosine and abasic site.Family B2 DNA polymerases from T. vaginalis, G. lambia and E. histolytica contain a Terminal Region Protein 2 (TPR2) motif twice the length of the TPR2 from φ29 DNA polymerase. Deletion studies demonstrate that as in φ29 DNA polymerase, the TPR2 motif of EhDNApolB2 is solely responsible of strand displacement and processivity. Interestingly the TPR2 of EhDNApolB2 is also responsible for efficient abasic site bypass. These data suggests that the 21 extra amino acids of the TPR2 motif may shape the active site of EhDNApolB2 to efficiently incorporate and extended opposite an abasic site. Herein we demonstrate that an open reading frame derived from Politons-Mavericks in parasitic protozoa encode a functional enzyme and our findings support the notion that the introduction of novel motifs in DNA polymerases can confer specialized properties to a conserved scaffold. PMID:23226232

  5. Prevalence of Dientamoeba fragilis, Giardia duodenalis, Entamoeba histolytica/dispar, and Cryptosporidium spp in Da Nang, Vietnam, detected by a multiplex real-time PCR.

    PubMed

    Ögren, Jessica; Van Nguyen, Song; Nguyen, Minh Khac; Dimberg, Jan; Matussek, Andreas

    2016-06-01

    We surveyed the prevalence of Dientamoeba fragilis, Giardia duodenalis, Entamoeba histolytica, Entamoeba dispar, and Cryptosporidium spp in individuals with and without gastrointestinal (GI) symptoms residing in and around Da Nang city, Vietnam. Fecal samples were collected from children (n = 100) and adults (n = 80) with GI symptoms and from healthy individuals (n = 88) reporting no GI symptoms. Parasite detection was performed by multiplex real-time PCR. Overall, except for G. duodenalis, we found a low prevalence (<5%) of D. fragilis and E. dispar and no detection of E. histolytica and C. spp in all participants with GI symptoms. Specifically for D. fragilis this contrasts with findings in European populations of children with GI symptoms showing prevalence up to 73%. Moreover, our results indicate that the prevalence of G. duodenalis is higher in patients with GI symptoms compared to asymptomatic individuals and this difference is most obvious in young patients. PMID:27102222

  6. First report of Entamoeba histolytica infection from Timor-Leste--acute amoebic colitis and concurrent late development of amoebic liver abscess in returned travellers to Australia.

    PubMed

    Nourse, Clare B; Robson, Jennifer M; Whitby, Michael R; Francis, Josh R

    2016-02-01

    This communication reports invasive amoebic colitis and late onset amoebic liver abscess in three members of a group of 12 Australian travellers to Timor-Leste (TL). This is the first report of Entamoeba histolytica infection from TL. Clinicians in Australia need to consider amoebiasis in the differential diagnosis in travellers returning with colitis, abdominal pain and fever. Presentation with amoebic liver abscess months after exposure is rare but should be suspected in symptomatic individuals with a relevant history of travel. PMID:26858275

  7. A new in vitro model of Entamoeba histolytica adhesion, using the human colon carcinoma cell line Caco-2: scanning electron microscopic study.

    PubMed Central

    Rigothier, M C; Coconnier, M H; Servin, A L; Gayral, P

    1991-01-01

    The human colon carcinoma cell line Caco-2, which is widely used to study the adhesion and cytotoxicity of enterobacteria, was used to investigate the adhesion of the trophozoites of Entamoeba histolytica. We observed a high percentage of adhesion of amoebae to Caco-2 cells. Scanning electron microscopy showed that amoebial membrane structures were involved in adhesion and the cytolytic action. These differentiated cells should prove to be a useful model system for investigation of the pathogenic action of amoebae. Images PMID:1937772

  8. Proteomic Identification of Oxidized Proteins in Entamoeba histolytica by Resin-Assisted Capture: Insights into the Role of Arginase in Resistance to Oxidative Stress.

    PubMed

    Shahi, Preeti; Trebicz-Geffen, Meirav; Nagaraja, Shruti; Alterzon-Baumel, Sharon; Hertz, Rivka; Methling, Karen; Lalk, Michael; Ankri, Serge

    2016-01-01

    Entamoeba histolytica is an obligate protozoan parasite of humans, and amebiasis, an infectious disease which targets the intestine and/or liver, is the second most common cause of human death due to a protozoan after malaria. Although amebiasis is usually asymptomatic, E. histolytica has potent pathogenic potential. During host infection, the parasite is exposed to reactive oxygen species that are produced and released by cells of the innate immune system at the site of infection. The ability of the parasite to survive oxidative stress (OS) is essential for a successful invasion of the host. Although the effects of OS on the regulation of gene expression in E. histolytica and the characterization of some proteins whose function in the parasite's defense against OS have been previously studied, our knowledge of oxidized proteins in E. histolytica is lacking. In order to fill this knowledge gap, we performed a large-scale identification and quantification of the oxidized proteins in oxidatively stressed E. histolytica trophozoites using resin-assisted capture coupled to mass spectrometry. We detected 154 oxidized proteins (OXs) and the functions of some of these proteins were associated with antioxidant activity, maintaining the parasite's cytoskeleton, translation, catalysis, and transport. We also found that oxidation of the Gal/GalNAc impairs its function and contributes to the inhibition of E. histolytica adherence to host cells. We also provide evidence that arginase, an enzyme which converts L-arginine into L-ornithine and urea, is involved in the protection of the parasite against OS. Collectively, these results emphasize the importance of OS as a critical regulator of E. histolytica's functions and indicate a new role for arginase in E. histolytica's resistance to OS. PMID:26735309

  9. Proteomic Identification of Oxidized Proteins in Entamoeba histolytica by Resin-Assisted Capture: Insights into the Role of Arginase in Resistance to Oxidative Stress

    PubMed Central

    Shahi, Preeti; Trebicz-Geffen, Meirav; Nagaraja, Shruti; Alterzon-Baumel, Sharon; Hertz, Rivka; Methling, Karen; Lalk, Michael; Ankri, Serge

    2016-01-01

    Entamoeba histolytica is an obligate protozoan parasite of humans, and amebiasis, an infectious disease which targets the intestine and/or liver, is the second most common cause of human death due to a protozoan after malaria. Although amebiasis is usually asymptomatic, E. histolytica has potent pathogenic potential. During host infection, the parasite is exposed to reactive oxygen species that are produced and released by cells of the innate immune system at the site of infection. The ability of the parasite to survive oxidative stress (OS) is essential for a successful invasion of the host. Although the effects of OS on the regulation of gene expression in E. histolytica and the characterization of some proteins whose function in the parasite's defense against OS have been previously studied, our knowledge of oxidized proteins in E. histolytica is lacking. In order to fill this knowledge gap, we performed a large-scale identification and quantification of the oxidized proteins in oxidatively stressed E. histolytica trophozoites using resin-assisted capture coupled to mass spectrometry. We detected 154 oxidized proteins (OXs) and the functions of some of these proteins were associated with antioxidant activity, maintaining the parasite's cytoskeleton, translation, catalysis, and transport. We also found that oxidation of the Gal/GalNAc impairs its function and contributes to the inhibition of E. histolytica adherence to host cells. We also provide evidence that arginase, an enzyme which converts L-arginine into L-ornithine and urea, is involved in the protection of the parasite against OS. Collectively, these results emphasize the importance of OS as a critical regulator of E. histolytica's functions and indicate a new role for arginase in E. histolytica's resistance to OS. PMID:26735309

  10. [Electron-microscopic studies on fine structure and enzyme activity in the axenic and conventional strains of Entamoeba histolytica

    PubMed

    Yong, Tai Soon; Chung, Pyung Rim; Lee, Keun Tae

    1985-12-01

    The metabolism of Entamoeba histolytica would be affected by various environmental factors, and alteration of the environment was known to affect the fine structure of E. histolytica. The present study was designed electronmicroscopically to investigate the ultrastructure and enzyme activities in the axenic and conventional strains of E. histolytica. The trophozoites of axenically cultivated HK-9 strain and conventional YS-27 and YS-49 strains of E. histolytica were collected and fixed with 4 percent paraformaldehyde/0.1 M cacodylate buffer (pH 7.4). After washing them by centrifugation, 1 percent warm agar was added in the sediment. Solidified agar with the trophozoites was cut into 1 mm(3) cubes, and incubated in the various substrates to observe enzyme activities. Then, the specimen was post-fixed with 3 percent glutaraldehyde/0.1 M cacodylate buffer (pH 7.4) and 1 percent osmium tetroxide/0.1 M cacodylate buffer (pH 7.4), dehydrated in ascending ethanol series and embedded in epoxy resin. These were sectioned on an ultramicrotome and observed with a transmission electron microscope. The procedures for the observation of the fine structure were same as the above, except for the incubation in the substrate. The sections were stained with uranyl scetate and lead citrate. For the observation of the surface of the amoebae, scanning electron microscopy was carried out. The results obtained in the present study are summarized as follows: 1. The fuzzy coat around double-layered plasma membrane of E. histolytica was more irregularly and densely distributed in the conventional strains (YS-27, YS-49 strains) than in the axenic strain (HK-9 strain). 2. The endosomes, button bodies and chromatin material were surrounded by a double-layered nuclear membrane having scattered nuclear pores. The paranuclear body, mono- or double-layered vacuoles, vacuolar membrane whorls, rosette-like cylindrical bodies, aggregation of cylindrical bodies and helical bodies were found in the

  11. EhPgp5 mRNA stability is a regulatory event in the Entamoeba histolytica multidrug resistance phenotype.

    PubMed

    López-Camarillo, César; Luna-Arias, Juan Pedro; Marchat, Laurence A; Orozco, Esther

    2003-03-28

    The multidrug resistance (MDR) phenotype in Entamoeba histolytica is characterized by the overexpression of the EhPgp5 gene in trophozoites grown in high drug concentrations. Here we evaluated the role of EhPgp5 mRNA stability on MDR using actinomycin D. EhPgp5 mRNA from trophozoites growing without emetine had a half-life of 2.1 h, which augmented to 3.1 h in cells cultured with 90 microM and to 7.8 h with 225 microM emetine. Polyadenylation sites were detected at 118-, 156-, and 189-nucleotide (nt) positions of the EhPgp5 mRNA 3'-untranslated region. Interestingly, trophozoites grown with 225 microM emetine exhibited an extra polyadenylation site at 19 nt. The 3'-untranslated region sequence is AU-rich and has putative consensus sequences for RNA-binding proteins. We detected a RNA-protein complex in a region that contains a polypyrimidine tract (142-159 nt) and a cytoplasmic polyadenylation element (146-154 nt). A longer poly(A) tail in the EhPgp5 mRNA was seen in trophozoites grown with 225 microM emetine. Emetine stress may affect factors involved in mRNA turnover, including polyadenylation/deadenylation proteins, which could induce changes in the EhPgp5 mRNA half-life and poly(A) tail length. Novel evidence on mechanisms participating in E. histolytica MDR phenotype is provided. PMID:12556531

  12. Susceptibility to Entamoeba histolytica intestinal infection is related to reduction in natural killer T-lymphocytes in C57BL/6 mice.

    PubMed

    Oliveira, Fabrício M S; Horta, Bernardo C; Prata, Luana O; Santiago, Andrezza F; Alves, Andréa C; Faria, Ana M C; Gomes, Maria A; Caliari, Marcelo V

    2012-04-27

    Entamoeba histolytica is a protozoan that causes amoebiasis. Recent studies demonstrated that natural killer T lymphocytes (NKT) are critical for preventing the development of amoebic liver abscess. In spite of that, there are only a handful of studies in the area. Herein, we explored the role of NKT cells in E. histolytica infection using C57BL/6 wild-type and CD1(-/-) mice. Animals were inoculated with E. histolytica and sacrificed 48 hours later to collect caecum samples that were used for quantitative analyses of lesions, trophozoites, NK1.1(+) T lymphocytes and expression of the mucus protein MUC-2 by immunohistochemistry technique. Quantitative analyses confirmed that the frequency of NK1.1(+) T cells was significantly lower in samples from C57BL/6 CD1(-/-) mice as compared to their wild type (WT) counterparts. The extension of necrotic mucosa was larger and the number of trophozoites higher in Entamoeba (Eh)-infected CD1(-/-) mice when compared with Eh-infected WT mice. In mice from both groups, non-infected (CTRL) and Eh-infected CD1(-/-), there was a reduction in the thickness of the caecal mucosa and in the MUC-2-stained area in comparison with CTRL- and Eh-WT mice. Our results showed that NKT lymphocytes contribute to resistance against Entamoeba histolytica infection and to the control of inflammation in the colitis induced by infection. The presence of a normal epithelial layer containing appropriate levels of mucus had also a protective role against infection. PMID:24470941

  13. Production and characterization of monoclonal antibodies against a highly immunogenic fraction of Entamoeba histolytica (NIH:200) and their application in the detection of current amoebic infection.

    PubMed

    Sengupta, K; Das, P; Johnson, T M; Chaudhuri, P P; Das, D; Nair, G B

    1993-01-01

    Six monoclonal antibodies (MAbs) were produced against a highly immunogenic fraction derived by the chromatographic separation of the soluble preparation of axenic Entamoeba histolytica (strain NIH:200) trophozoites. Isotype characterization of the six MAbs revealed that four belonged to the IgM class and one each to the IgG1 and the IgG2a subclasses. The immunoreactivity patterns and the specificity of the MAbs with homologous and heterologous antigens were analyzed by the enzyme-linked immunotransfer blot technique and by the enzyme-linked immunosorbent assay. The MAbs reacted intensely with isolates of E. histolytica (strain NIH:200 as well as a local isolate MX1) but showed no reactivity with Entamoeba coli, Iodamoeba butschlii, Endolimax nana, Entamoeba hartmanni, free-living amoeba (Acanthamoeba harticolus) and other enteric parasites. Using the IgG1 MAb as a detecting antibody, a polyclonal-monoclonal antibody-based enzyme-linked immunosorbent assay was developed for the detection of E. histolytica antigens in stool samples of infected patients. The detection limit of the assay was 8 ng of amoebic antigen. This test was found to be specific and sensitive and yielded 100% positive results in cases with amoebiasis but did not react with controls included in the evaluation. The MAb-based enzyme-linked immunosorbent assay developed in this study will be an important test for the diagnosis of E. histolytica in the feces of infected humans; however, the limitation of the test is the inability to discriminate the pathogenic status of the amoeba detected in the stool. PMID:8292992

  14. Susceptibility to Entamoeba histolytica intestinal infection is related to reduction in natural killer T-lymphocytes in C57BL/6 mice

    PubMed Central

    Oliveira, Fabrício M.S.; Horta, Bernardo C.; Prata, Luana O.; Santiago, Andrezza F.; Alves, Andréa C.; Faria, Ana M.C.; Gomes, Maria A.; Caliari, Marcelo V.

    2012-01-01

    Entamoeba histolytica is a protozoan that causes amoebiasis. Recent studies demonstrated that natural killer T lymphocytes (NKT) are critical for preventing the development of amoebic liver abscess. In spite of that, there are only a handful of studies in the area. Herein, we explored the role of NKT cells in E. histolytica infection using C57BL/6 wild-type and CD1−/− mice. Animals were inoculated with E. histolytica and sacrificed 48 hours later to collect caecum samples that were used for quantitative analyses of lesions, trophozoites, NK1.1+ T lymphocytes and expression of the mucus protein MUC-2 by immunohistochemistry technique. Quantitative analyses confirmed that the frequency of NK1.1+ T cells was significantly lower in samples from C57BL/6 CD1−/− mice as compared to their wild type (WT) counterparts. The extension of necrotic mucosa was larger and the number of trophozoites higher in Entamoeba (Eh)-infected CD1−/− mice when compared with Eh-infected WT mice. In mice from both groups, non-infected (CTRL) and Eh-infected CD1−/−, there was a reduction in the thickness of the caecal mucosa and in the MUC-2-stained area in comparison with CTRL- and Eh-WT mice. Our results showed that NKT lymphocytes contribute to resistance against Entamoeba histolytica infection and to the control of inflammation in the colitis induced by infection. The presence of a normal epithelial layer containing appropriate levels of mucus had also a protective role against infection. PMID:24470941

  15. The antiamoebic effect of a crude drug formulation of herbal extracts against Entamoeba histolytica in vitro and in vivo.

    PubMed

    Sohni, Y R; Kaimal, P; Bhatt, R M

    1995-01-01

    The antiamoebic effect of a crude drug formulation against Entamoeba histolytica was studied. In the traditional system of medicine in India, the formulation has been prescribed for intestinal disorders. It comprises of five medicinal herbs, namely, Boerhavia diffusa, Berberis aristata, Tinospora cordifolia, Terminalia chebula and Zingiber officinale. The dried and pulverized plants were extracted in ethanol together and individually. In vitro amoebicidal activity was studied to determine the minimal inhibitory concentration (MIC) values of all the constituent extracts as well as the whole formulation. The formulation had a MIC of 1000 micrograms/ml as compared with 10 micrograms/ml for metronidazole. In experimental caecal amoebiasis in rats the formulation had a curative rate of 89% with the average degree of infection (ADI) reduced to 0.4 in a group dosed with 500 mg/kg per day as compared with ADI of 3.8 for the sham-treated control group of rats. Metronidazole had a cure rate of 89% (ADI = 0.4) at a dose of 100 mg/kg per day and cured the infection completely (ADI = 0) when the dosage was doubled to 200 mg/kg per day. There were varying degrees of inhibition of the following enzyme activities of crude extracts of axenically cultured amoebae: DNase, RNase, aldolase, alkaline phosphatase, acid phosphatase, alpha-amylase and protease. PMID:7739226

  16. An energy-conserving pyruvate-to-acetate pathway in Entamoeba histolytica. Pyruvate synthase and a new acetate thiokinase.

    PubMed

    Reeves, R E; Warren, L G; Susskind, B; Lo, H S

    1977-01-25

    Under anaerobic conditions, cells of Entamoeba histolytica grown with bacteria produce H2 and acetate while cells grown axenically produce neither. Aerobically, acetate is produced and O2 is consumed by amebae from either type of cells. Centrifuged extracts, 2.4 x 106 x g x min, from both types of cells contain pyruvate synthase (EC 1.2.7.1) and an acetate thiokinase which, together, form a system capable of converting pyruvate to acetate. Pyruvate synthase catalyzes the reaction: pyruvate + CoA leads to CO2 + acetyl-CoA + 2E. Electron acceptors which function with this enzyme are FAD, FMN, riboflavin, ferredoxin, and methyl viologen, but not NAD or NADP. The amebal acetate thiokinase catalyzes the reaction acetyl-CoA + ADP + Pi leads to acetate + ATP + CoA. For this apparently new enzyme we suggest the trivial name acetyl-CoA-synthetase (ADP-forming). Extracts from axenic amebae do not contain hydrogenase, but extracts from cells grown with bacteria do. It is postulated that in bacteria-grown amebae electrons generated at the pyruvate synthase step are utilized anaerobically to produce H2 via the hydrogenase and that the acetyl-CoA is converted to acetate in an energy-conserving step catalyzed by amebal acetyl-CoA synthetase. Aerobically, cells grown under either regimen may utilize the energy-conserving pyruvate-to-acetate pathway since O2 then serves as the ultimate electron acceptor. PMID:13076

  17. Phenotypic and transcriptional profiling in Entamoeba histolytica reveal costs to fitness and adaptive responses associated with metronidazole resistance

    PubMed Central

    Penuliar, Gil M.; Nakada-Tsukui, Kumiko; Nozaki, Tomoyoshi

    2015-01-01

    Antimicrobial chemotherapy is critical in the fight against infectious diseases caused by Entamoeba histolytica. Among the drugs available for the treatment of amebiasis, metronidazole (MTZ) is considered the drug of choice. Recently, in vitro studies have described MTZ resistance and the potential mechanisms involved. Costs to fitness and adaptive responses associated with resistance, however, have not been investigated. In this study we generated an HM-1 derived strain resistant to 12 μM MTZ (MTZR). We examined its phenotypic and transcriptional profile to determine the consequences and mRNA level changes associated with MTZ resistance. Our results indicated increased cell size and granularity, and decreased rates in cell division, adhesion, phagocytosis, cytopathogenicity, and glucose consumption. Transcriptome analysis revealed 142 differentially expressed genes in MTZR. In contrast to other MTZ resistant parasites, MTZR did not down-regulate pyruvate:ferredoxin oxidoreductase, but showed increased expression of genes for a hypothetical protein (HP1) and several iron-sulfur flavoproteins, and downregulation of genes for leucine-rich proteins. Fisher's exact test showed 24 significantly enriched GO terms in MTZR, and a 3-way comparison of modulated genes in MTZR against those of MTZR cultured without MTZ and HM-1 cultured with MTZ, showed that 88 genes were specific to MTZR. Overall, our findings suggested that MTZ resistance is associated with specific transcriptional changes and decreased parasite virulence. PMID:25999919

  18. Entamoeba histolytica Cysteine Proteinase 5 Evokes Mucin Exocytosis from Colonic Goblet Cells via αvβ3 Integrin

    PubMed Central

    Cornick, Steve; Moreau, France; Chadee, Kris

    2016-01-01

    Critical to the pathogenesis of intestinal amebiasis, Entamoeba histolytica (Eh) induces mucus hypersecretion and degrades the colonic mucus layer at the site of invasion. The parasite component(s) responsible for hypersecretion are poorly defined, as are regulators of mucin secretion within the host. In this study, we have identified the key virulence factor in live Eh that elicits the fast release of mucin by goblets cells as cysteine protease 5 (EhCP5) whereas, modest mucus secretion occurred with secreted soluble EhCP5 and recombinant CP5. Coupling of EhCP5-αvβ3 integrin on goblet cells facilitated outside-in signaling by activating SRC family kinases (SFK) and focal adhesion kinase that resulted in the activation/phosphorlyation of PI3K at the site of Eh contact and production of PIP3. PKCδ was activated at the EhCP5-αvβ3 integrin contact site that specifically regulated mucin secretion though the trafficking vesicle marker myristoylated alanine-rich C-kinase substrate (MARCKS). This study has identified that EhCP5 coupling with goblet cell αvβ3 receptors can initiate a signal cascade involving PI3K, PKCδ and MARCKS to drive mucin secretion from goblet cells critical in disease pathogenesis. PMID:27073869

  19. Entamoeba histolytica Cysteine Proteinase 5 Evokes Mucin Exocytosis from Colonic Goblet Cells via αvβ3 Integrin.

    PubMed

    Cornick, Steve; Moreau, France; Chadee, Kris

    2016-04-01

    Critical to the pathogenesis of intestinal amebiasis, Entamoeba histolytica (Eh) induces mucus hypersecretion and degrades the colonic mucus layer at the site of invasion. The parasite component(s) responsible for hypersecretion are poorly defined, as are regulators of mucin secretion within the host. In this study, we have identified the key virulence factor in live Eh that elicits the fast release of mucin by goblets cells as cysteine protease 5 (EhCP5) whereas, modest mucus secretion occurred with secreted soluble EhCP5 and recombinant CP5. Coupling of EhCP5-αvβ3 integrin on goblet cells facilitated outside-in signaling by activating SRC family kinases (SFK) and focal adhesion kinase that resulted in the activation/phosphorlyation of PI3K at the site of Eh contact and production of PIP3. PKCδ was activated at the EhCP5-αvβ3 integrin contact site that specifically regulated mucin secretion though the trafficking vesicle marker myristoylated alanine-rich C-kinase substrate (MARCKS). This study has identified that EhCP5 coupling with goblet cell αvβ3 receptors can initiate a signal cascade involving PI3K, PKCδ and MARCKS to drive mucin secretion from goblet cells critical in disease pathogenesis. PMID:27073869

  20. EhCP112 is an Entamoeba histolytica secreted cysteine protease that may be involved in the parasite-virulence.

    PubMed

    Ocádiz, Ramón; Orozco, Esther; Carrillo, Eduardo; Quintas, Laura Itzel; Ortega-López, Jaime; García-Pérez, Rosa María; Sánchez, Tomas; Castillo-Juárez, Beatriz A; García-Rivera, Guillermina; Rodríguez, Mario A

    2005-02-01

    EhCP112 is an Entamoeba histolytica protease that together with the EhADH112 protein forms the EhCPADH complex involved in trophozoite virulence. Here, we produced the recombinant EhCP112 and studied its relationships with extracellular matrix components and with target cells. A DNA fragment containing the pro-peptide and the mature enzyme was expressed in bacteria as an active enzyme (rEhCP112), whereas the full gene containing the signal peptide, the pro-peptide and the mature enzyme expressed a non-active protein. The fragment only with the mature enzyme was not expressed. rEhCP112 purified by affinity columns digested azocasein and had a strong autoproteolytic activity. Four hours after purification the protein appeared degraded. Anti-tag antibodies, monoclonal antibodies against the EhCP112 and sera from human patients with amoebiasis recognized rEhCP112. rEhCP112 digested gelatin, collagen type I, fibronectin and haemoglobin; it destroyed MDCK cell monolayers and bound to red blood cells. The native EhCP112 was poorly expressed in a virulence-deficient mutant, and in the wild-type clone it was located in secreted vesicles, forming the EhCPADH complex. Altogether these results show that EhCP112 is a molecule able to disrupt cell monolayers and digest proteins of the extracellular matrix and haemoglobin, and it is secreted by the trophozoites. PMID:15659066

  1. Molecular Detection of the Carriage Rate of Four Intestinal Protozoa with Real-Time Polymerase Chain Reaction: Possible Overdiagnosis of Entamoeba histolytica in Nigeria.

    PubMed

    Efunshile, Michael A; Ngwu, Bethrand A F; Kurtzhals, Jørgen A L; Sahar, Sumrin; König, Brigitte; Stensvold, Christen R

    2015-08-01

    Diarrhea remains the second largest killer of children worldwide, and Nigeria ranks number two on the list of global deaths attributable to diarrhea. Meanwhile, prevalence studies on potentially diarrheagenic protozoa in asymptomatic carriers using molecular detection methods remain scarce in sub-Saharan countries. To overcome sensitivity issues related to microscopic detection and identification of cysts in stool concentrates, real-time polymerase chain reaction (PCR) was used to analyze genomic DNAs extracted from stool samples from 199 healthy school children for Entamoeba histolytica, E. dispar, Giardia intestinalis, and Cryptosporidium. Questionnaires were administered for epidemiological data collection. E. histolytica was not detected in any of the samples, whereas Giardia (37.2%), E. dispar (18.6%), and Cryptosporidium (1%) were found. Most of the children sourced their drinking water from community wells (91%), while the majority disposed of feces in the bush (81.9%). Our study is the first to use real-time PCR to evaluate the epidemiology of E. histolytica, Giardia, and Cryptosporidium in Nigeria where previous studies using traditional diagnostic techniques have suggested higher and lower carriage rates of E. histolytica and Giardia, respectively. It is also the first study to accurately identify the prevalence of common potentially diarrheagenic protozoa in asymptomatic carriers in sub-Saharan Africa. PMID:26101274

  2. Prevalence of Entamoeba histolytica -like cysts compared to E. histolytica antigens detected by ELISA in the stools of 600 patients from three socioeconomic communities in the Metropolitan City of Lahore, Pakistan.

    PubMed

    Alam, Muhammad Azhar; Maqbool, Azhar; Nazir, Muhammad Mudasser; Lateef, Muhammad; Khan, Muhammad Sarwar; Ahmed, Atif Nisar; Ziaullah, M; Lindsay, David S

    2015-04-01

    Amoebiasis, caused by Entamoeba histolytica , has a worldwide distribution and is of public health significance in many developing countries. It has a fecal-oral transmission cycle and is most prevalent in developing countries in regions where substandard sanitary conditions exist due to poverty. Little is known about the epidemiology of E. histolytica infection and its presence in different socioeconomic communities in developing countries. We undertook the present study in the city of Lahore, Pakistan, and our prediction was that the prevalence of E. histolytica -like cysts and E. histolytica stool antigen would be lower in patients from upper socioeconomic levels than in individuals from middle or lower socioeconomic levels. We investigated the prevalence of E. histolytica in humans from 3 socioeconomic communities in territories of Lahore, Pakistan. Six hundred fecal samples were collected and examined using both microscopy (triple fecal test) to detect cysts of E. histolytica -like amoeba and ELISA (stool antigen ELISA) to demonstrate diagnostic stool antigens of E. histolytica . Samples were from individuals living under conditions deemed to be upper socioeconomic class (n = 287), middle socioeconomic class (n = 172), and lower socioeconomic class (n = 141). The total prevalence of positive samples was 22.5% (135/600) by triple test and 16.8% (101/600) by stool antigen ELISA in the 600 fecal samples. Statistically, significant (P < 0.05) differences in prevalence were seen between the 3 socioeconomic class groups. Forty-four (15.3%) and 32 (11.1%) of 287 in the fecal samples from the upper socioeconomic class were positive by triple test and by antigen ELISA, respectively. Thirty-nine (22.6%) and 29 (16.8%) of 172 in the fecal samples from the middle socioeconomic class were positive by the triple test and by antigen ELISA, respectively. Fifty-two (36.8%) and 40 (28.3%) of 141 in the fecal samples from the lower socioeconomic class were positive by the triple

  3. An Entamoeba histolytica ADP-ribosyl transferase from the diphtheria toxin family modifies the bacterial elongation factor Tu.

    PubMed

    Avila, Eva E; Rodriguez, Orlando I; Marquez, Jaqueline A; Berghuis, Albert M

    2016-06-01

    ADP-ribosyl transferases are enzymes involved in the post-translational modification of proteins; they participate in multiple physiological processes, pathogenesis and host-pathogen interactions. Several reports have characterized the functions of these enzymes in viruses, prokaryotes and higher eukaryotes, but few studies have reported ADP-ribosyl transferases in lower eukaryotes, such as parasites. The locus EHI_155600 from Entamoeba histolytica encodes a hypothetical protein that possesses a domain from the ADP-ribosylation superfamily; this protein belongs to the diphtheria toxin family according to a homology model using poly-ADP-ribosyl polymerase 12 (PARP12 or ARTD12) as a template. The recombinant protein expressed in Escherichia coli exhibited in vitro ADP-ribosylation activity that was dependent on the time and temperature. Unlabeled βNAD(+), but not ADP-ribose, competed in the enzymatic reaction using biotin-βNAD(+) as the ADP-ribose donor. The recombinant enzyme, denominated EhToxin-like, auto-ADP-ribosylated and modified an acceptor from E. coli that was identified by MS/MS as the elongation factor Tu (EF-Tu). To the best of our knowledge, this is the first report to identify an ADP-ribosyl transferase from the diphtheria toxin family in a protozoan parasite. The known toxins from this family (i.e., the diphtheria toxin, the Pseudomonas aeruginosa toxin Exo-A, and Cholix from Vibrio cholerae) modify eukaryotic elongation factor two (eEF-2), whereas the amoeba EhToxin-like modified EF-Tu, which is another elongation factor involved in protein synthesis in bacteria and mitochondria. PMID:27234208

  4. Rab5-associated vacuoles play a unique role in phagocytosis of the enteric protozoan parasite Entamoeba histolytica.

    PubMed

    Saito-Nakano, Yumiko; Yasuda, Tomoyoshi; Nakada-Tsukui, Kumiko; Leippe, Matthias; Nozaki, Tomoyoshi

    2004-11-19

    In mammals, Rab5 and Rab7 play a specific and coordinated role in a sequential process during phagosome maturation. Here, we report that Rab5 and Rab7 in the enteric protozoan parasite Entamoeba histolytica, EhRab5 and EhRab7A, are involved in steps that are distinct from those known for mammals. EhRab5 and EhRab7A were localized to independent small vesicular structures at steady state. Priming with red blood cells induced the formation of large vacuoles associated with both EhRab5 and EhRab7A ("prephagosomal vacuoles (PPV)") in the amoeba within an incubation period of 5-10 min. PPV emerged de novo physically and distinct from phagosomes. PPV were gradually acidified and matured by fusion with lysosomes containing a digestive hydrolase, cysteine proteinase, and a membrane-permeabilizing peptide amoebapore. After EhRab5 dissociated from PPV, 5-10 min later, the EhRab7A-PPV fused with phagosomes, and EhRab7A finally dissociated from the phagosomes. Immunoelectron and light micrographs showed that PPV contained small vesicle-like structures containing fluid-phase markers and amoebapores, which were not evenly distributed within PPV, suggesting that the mechanism was similar to multivesicular body formation in PPV generation. In contrast to Rab5 from other organisms, EhRab5 was involved exclusively in phagocytosis, but not in endocytosis. Overexpression of wild-type EhRab5 enhanced phagocytosis and the transport of amoebapore to phagosomes. Conversely, expression of an EhRab5Q67L GTP form mutant impaired the formation of PPV and phagocytosis. Altogether, we propose that the amoebic Rab5 plays an important role in the formation of unique vacuoles, which is essential for engulfment of erythrocytes and important for packaging of lysosomal hydrolases, prior to the targeting to phagosomes. PMID:15347665

  5. Expression, purification and crystallization of l-methionine γ-lyase 2 from Entamoeba histolytica

    SciTech Connect

    Sato, Dan; Yamagata, Wataru; Kamei, Kaeko; Nozaki, Tomoyoshi; Harada, Shigeharu

    2006-10-01

    l-Methionine γ-lyase 2 from E. histolytica, a key enzyme in sulfur-containing amino-acid degradation in this protozoan parasite, has been crystallized in a form suitable for X-ray structure analysis. l-Methionine γ-lyase (MGL) is considered to be an attractive target for rational drug development because the enzyme is absent in mammalian hosts. To enable structure-based design of drugs targeting MGL, one of the two MGL isoenzymes (EhMGL2) was crystallized in the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 88.89, b = 102.68, c = 169.87 Å. The crystal diffracted to a resolution of 2.0 Å. The presence of a tetramer in the asymmetric unit (4 × 43.1 kDa) gives a Matthews coefficient of 2.2 Å{sup 3} Da{sup −1}. The structure was solved by the molecular-replacement method and structure refinement is now in progress.

  6. Variation of proteins and proteinases in Entamoeba histolytica lysates containing a protease inhibitor.

    PubMed

    López-Revilla, R; Jiménez-Delgadillo, B; Canto-Ortiz, L; Chávez-Dueñas, L

    1992-01-01

    Sodium dodecyl sulfate (SDS)-lysates of E. histolytica trophozoites were analyzed by electrophoresis in simple and gelatin-containing ("substrate") SDS-polyacrylamide gels. In simple gels, boiled lysates with para hydroxymercuribenzoate (pHMB) had a complex pattern of apparently undegraded proteins; boiled lysates without pHMB showed a major 30 kDa and four minor (43, 46, 63 and 117 kDa) proteins, whereas unheated lysates displayed only the 117 kDa protein. Using substrate gels no gelatinases were detected in heated lysates; unheated lysates without pHMB showed a major 30 kDa and three minor (33, 46 and 68 kDa) gelatinases, whereas those with pHMB presented a major 56 kDa and two minor (70 and 105 kDa) gelatinases. Three caseinase peaks were separated by Sephadex G-75 chromatography from unheated lysates: peak I contained 46, 56 and 117 kDa pHMB-sensitive gelatinases and peaks II and III contained smaller pHMB-resistant caseinases. We conclude that proteins remaining in lysates after SDS-induced proteolysis appear to be mainly proteases relatively resistant to self-digestion whose type and amount changes with the conditions of lysis and the presence of inhibitors; this is exemplified by the finding of the major gelatinase of lysates with pHMB being larger (56 kDa) than in lysates lacking the inhibitor (30 kDa). PMID:1340329

  7. A New Human 3D-Liver Model Unravels the Role of Galectins in Liver Infection by the Parasite Entamoeba histolytica

    PubMed Central

    Petropolis, Debora B.; Faust, Daniela M.; Deep Jhingan, Gagan; Guillen, Nancy

    2014-01-01

    Investigations of human parasitic diseases depend on the availability of appropriate in vivo animal models and ex vivo experimental systems, and are particularly difficult for pathogens whose exclusive natural hosts are humans, such as Entamoeba histolytica, the protozoan parasite responsible for amoebiasis. This common infectious human disease affects the intestine and liver. In the liver sinusoids E. histolytica crosses the endothelium and penetrates into the parenchyma, with the concomitant initiation of inflammatory foci and subsequent abscess formation. Studying factors responsible for human liver infection is hampered by the complexity of the hepatic environment and by the restrictions inherent to the use of human samples. Therefore, we built a human 3D-liver in vitro model composed of cultured liver sinusoidal endothelial cells and hepatocytes in a 3D collagen-I matrix sandwich. We determined the presence of important hepatic markers and demonstrated that the cell layers function as a biological barrier. E. histolytica invasion was assessed using wild-type strains and amoebae with altered virulence or different adhesive properties. We showed for the first time the dependence of endothelium crossing upon amoebic Gal/GalNAc lectin. The 3D-liver model enabled the molecular analysis of human cell responses, suggesting for the first time a crucial role of human galectins in parasite adhesion to the endothelial cells, which was confirmed by siRNA knockdown of galectin-1. Levels of several pro-inflammatory cytokines, including galectin-1 and -3, were highly increased upon contact of E. histolytica with the 3D-liver model. The presence of galectin-1 and -3 in the extracellular medium stimulated pro-inflammatory cytokine release, suggesting a further role for human galectins in the onset of the hepatic inflammatory response. These new findings are relevant for a better understanding of human liver infection by E. histolytica. PMID:25211477

  8. Entamoeba histolytica and E. dispar Calreticulin: Inhibition of Classical Complement Pathway and Differences in the Level of Expression in Amoebic Liver Abscess

    PubMed Central

    Ximénez, Cecilia; González, Enrique; Nieves, Miriam E.; Silva-Olivares, Angélica; Shibayama, Mineko; Galindo-Gómez, Silvia; Escobar-Herrera, Jaime; García de León, Ma del Carmen; Morán, Patricia; Valadez, Alicia; Rojas, Liliana; Hernández, Eric G.; Partida, Oswaldo; Cerritos, René

    2014-01-01

    The role of calreticulin (CRT) in host-parasite interactions has recently become an important area of research. Information about the functions of calreticulin and its relevance to the physiology of Entamoeba parasites is limited. The present work demonstrates that CRT of both pathogenic E. histolytica and nonpathogenic E. dispar species specifically interacted with human C1q inhibiting the activation of the classical complement pathway. Using recombinant EhCRT protein, we demonstrate that CRT interaction site and human C1q is located at the N-terminal region of EhCRT. The immunofluorescence and confocal microscopy experiments show that CRT and human C1q colocalize in the cytoplasmic vesicles and near to the surface membrane of previously permeabilized trophozoites or are incubated with normal human serum which is known to destroy trophozoites. In the presence of peripheral mononuclear blood cells, the distribution of EhCRT and C1q is clearly over the surface membrane of trophozoites. Nevertheless, the level of expression of CRT in situ in lesions of amoebic liver abscess (ALA) in the hamster model is different in both Entamoeba species; this molecule is expressed in higher levels in E. histolytica than in E. dispar. This result suggests that EhCRT may modulate some functions during the early moments of the host-parasite relationship. PMID:24860808

  9. Protection against Invasive Amebiasis by a Single Monoclonal Antibody Directed against a Lipophosphoglycan Antigen Localized on the Surface of Entamoeba histolytica

    PubMed Central

    Marinets, Alexandra; Zhang, Tonghai; Guillén, Nancy; Gounon, Pierre; Bohle, Barbara; Vollmann, Ute; Scheiner, Otto; Wiedermann, Gerhard; Stanley, Samuel L.; Duchêne, Michael

    1997-01-01

    A panel of monoclonal antibodies was raised from mice immunized with a membrane preparation from Entamoeba histolytica, the pathogenic species causing invasive amebiasis in humans. Antibody EH5 gave a polydisperse band in immunoblots from membrane preparations from different E. histolytica strains, and a much weaker signal from two strains of the nonpathogenic species Entamoeba dispar. Although the exact chemical structure of the EH5 antigen is not yet known, the ability of the antigen to be metabolically radiolabeled with [32P]phosphate or [3H]glucose, its sensitivity to digestion by mild acid and phosphatidylinositol-specific phospholipase C, and its specific extraction from E. histolytica trophozoites by a method used to prepare lipophosphoglycans from Leishmania showed that it could be classified as an amebal lipophosphoglycan. Confocal immunofluorescence and immunogold labeling of trophozoites localized the antigen on the outer face of the plasma membrane and on the inner face of internal vesicle membranes. Antibody EH5 strongly agglutinated amebas in a similar way to concanavalin A (Con A), and Con A bound to immunoaffinity-purified EH5 antigen. Therefore, surface lipophosphoglycans may play an important role in the preferential agglutination of pathogenic amebas by Con A. The protective ability of antibody EH5 was tested in a passive immunization experiment in a severe combined immunodeficient (SCID) mouse model. Intrahepatic challenge of animals after administration of an isotype-matched control antibody or without treatment led to the development of a liver abscess in all cases, whereas 11 out of 12 animals immunized with the EH5 antibody developed no liver abscess. Our results demonstrate the importance and, for the first time, the protective capacity of glycan antigens on the surface of the amebas. PMID:9348313

  10. Bioassay-Guided Fractionation of Extracts from Codiaeum variegatum against Entamoeba histolytica Discovers Compounds That Modify Expression of Ceramide Biosynthesis Related Genes

    PubMed Central

    Mfotie Njoya, Emmanuel; Weber, Christian; Hernandez-Cuevas, Nora Adriana; Hon, Chung-Chau; Janin, Yves; Kamini, Melanie F. G.; Moundipa, Paul F.; Guillén, Nancy

    2014-01-01

    Leaves of Codiaeum variegatum (“garden croton”) are used against bloody diarrhoea by local populations in Cameroon. This study aims to search for the active components from C. variegatum against Entamoeba histolytica, and thereby initiate the study of their mechanism of action. A bioassay-guided screening of the aqueous extracts from C. variegatum leaves and various fractions was carried out against trophozoites of E. histolytica axenic culture. We found that the anti-amoebic activity of extracts changed with respect to the collection criteria of leaves. Thereby, optimal conditions were defined for leaves' collection to maximise the anti-amoebic activity of the extracts. A fractionation process was performed, and we identified several sub-fractions (or isolated compounds) with significantly higher anti-amoebic activity compared to the unfractionated aqueous extract. Anti-amoebic activity of the most potent fraction was confirmed with the morphological characteristics of induced death in trophozoites, including cell rounding and lysis. Differential gene expression analysis using high-throughput RNA sequencing implies the potential mechanism of its anti-amoebic activity by targeting ceramide, a bioactive lipid involved in disturbance of biochemical processes within the cell membrane including differentiation, proliferation, cell growth arrest and apoptosis. Regulation of ceramide biosynthesis pathway as a target for anti-amoebic compounds is a novel finding which could be an alternative for drug development against E. histolytica. PMID:24416462

  11. Bioassay-guided fractionation of extracts from Codiaeum variegatum against Entamoeba histolytica discovers compounds that modify expression of ceramide biosynthesis related genes.

    PubMed

    Mfotie Njoya, Emmanuel; Weber, Christian; Hernandez-Cuevas, Nora Adriana; Hon, Chung-Chau; Janin, Yves; Kamini, Melanie F G; Moundipa, Paul F; Guillén, Nancy

    2014-01-01

    Leaves of Codiaeum variegatum ("garden croton") are used against bloody diarrhoea by local populations in Cameroon. This study aims to search for the active components from C. variegatum against Entamoeba histolytica, and thereby initiate the study of their mechanism of action. A bioassay-guided screening of the aqueous extracts from C. variegatum leaves and various fractions was carried out against trophozoites of E. histolytica axenic culture. We found that the anti-amoebic activity of extracts changed with respect to the collection criteria of leaves. Thereby, optimal conditions were defined for leaves' collection to maximise the anti-amoebic activity of the extracts. A fractionation process was performed, and we identified several sub-fractions (or isolated compounds) with significantly higher anti-amoebic activity compared to the unfractionated aqueous extract. Anti-amoebic activity of the most potent fraction was confirmed with the morphological characteristics of induced death in trophozoites, including cell rounding and lysis. Differential gene expression analysis using high-throughput RNA sequencing implies the potential mechanism of its anti-amoebic activity by targeting ceramide, a bioactive lipid involved in disturbance of biochemical processes within the cell membrane including differentiation, proliferation, cell growth arrest and apoptosis. Regulation of ceramide biosynthesis pathway as a target for anti-amoebic compounds is a novel finding which could be an alternative for drug development against E. histolytica. PMID:24416462

  12. A whole-genome RNAi screen uncovers a novel role for human potassium channels in cell killing by the parasite Entamoeba histolytica

    PubMed Central

    Marie, Chelsea; Verkerke, Hans P.; Theodorescu, Dan; Petri, William A.

    2015-01-01

    The parasite Entamoeba histolytica kills human cells resulting in ulceration, inflammation and invasion of the colonic epithelium. We used the cytotoxic properties of ameba to select a genome-wide RNAi library to reveal novel host factors that control susceptibility to amebic killing. We identified 281 candidate susceptibility genes and bioinformatics analyses revealed that ion transporters were significantly enriched among susceptibility genes. Potassium (K+) channels were the most common transporter identified. Their importance was further supported by colon biopsy of humans with amebiasis that demonstrated suppressed K+ channel expression. Inhibition of human K+ channels by genetic silencing, pharmacologic inhibitors and with excess K+ protected diverse cell types from E. histolytica-induced death. Contact with E. histolytica parasites triggered K+ channel activation and K+ efflux by intestinal epithelial cells, which preceded cell killing. Specific inhibition of Ca2+-dependent K+ channels was highly effective in preventing amebic cytotoxicity in intestinal epithelial cells and macrophages. Blockade of K+ efflux also inhibited caspase-1 activation, IL-1β secretion and pyroptotic death in THP-1 macrophages. We concluded that K+ channels are host mediators of amebic cytotoxicity in multiple cells types and of inflammasome activation in macrophages. PMID:26346926

  13. Simultaneous detection of Entamoeba histolytica/dispar, Giardia duodenalis and cryptosporidia by immunochromatographic assay in stool samples from patients living in the Greater Cairo Region, Egypt.

    PubMed

    Banisch, Dagmar M; El-Badry, Ayman; Klinnert, Jorge V; Ignatius, Ralf; El-Dib, Nadia

    2015-08-01

    Gastrointestinal infection due to intestinal parasites is an enormous health problem in developing countries and its reliable diagnosis is demanding. Therefore, this study aimed at evaluating a commercially available immunochromatographic assay (ICA) for the detection of cryptosporidia, Giardia duodenalis, and Entamoeba histolytica/dispar for its usefulness in the Greater Cairo Region, Egypt. Stool samples of 104 patients who presented between October 2012 and March 2013 with gastrointestinal symptoms or for the exclusion of parasites at Kasr-Al-Ainy University Medical School were examined by light microscopy of wet mounts and the triple ICA. Microscopy revealed in 20% of the patients [95% confidence interval (CI), 13.5-29.0%] parasites with Hymenolepis nana, E. histolytica/dispar and Blastocystis hominis being the most frequent ones, but was not able to detect G. duodenalis and cryptosporidia, whereas ICA was positive in 21% (95% CI, 14.3-30.0%) and detected E. histolytica/dispar in 12.5% (95% CI, 7.3-20.4%), cryptosporidia in 6.7% (95% CI, 3.1-13.5%) and G. duodenalis in 15.4% (95% CI, 9.6-23.6%) of the patients. Detection of one or more pathogens was associated with access to water retrieved from a well or pump (p = 0.01). Patients between 20 and 29 years of age (p = 0.08) and patients with symptoms of 5 days or longer (p = 0.07) tended to have a higher risk to be infected than patients of other age groups or with shorter-lasting symptoms. In conclusion, the ICA was easy to perform and timesaving. Importantly, it enabled the detection of cryptosporidia, which cannot be found microscopically in unstained smears, demonstrated a higher sensitivity for the detection of G. duodenalis than microscopy, and was more specific for distinguishing E. histolytica/dispar from apathogenic amoeba. PMID:26018117

  14. Evaluation of the C-Terminal Fragment of Entamoeba histolytica Gal/GalNAc Lectin Intermediate Subunit as a Vaccine Candidate against Amebic Liver Abscess

    PubMed Central

    Guan, Yue; Man, Suqin; Fu, Yongfeng; Cheng, Xunjia; Tachibana, Hiroshi

    2016-01-01

    Background Entamoeba histolytica is an intestinal protozoan parasite that causes amoebiasis, including amebic dysentery and liver abscesses. E. histolytica invades host tissues by adhering onto cells and phagocytosing them depending on the adaptation and expression of pathogenic factors, including Gal/GalNAc lectin. We have previously reported that E. histolytica possesses multiple CXXC sequence motifs, with the intermediate subunit of Gal/GalNAc lectin (i.e., Igl) as a key factor affecting the amoeba's pathogenicity. The present work showed the effect of immunization with recombinant Igl on amebic liver abscess formation and the corresponding immunological properties. Methodology/Principal Findings A prokaryotic expression system was used to prepare the full-length Igl and the N-terminal, middle, and C-terminal fragments (C-Igl) of Igl. Vaccine efficacy was assessed by challenging hamsters with an intrahepatic injection of E. histolytica trophozoites. Hamsters intramuscularly immunized with full-length Igl and C-Igl were found to be 92% and 96% immune to liver abscess formation, respectively. Immune-response evaluation revealed that C-Igl can generate significant humoral immune responses, with high levels of antibodies in sera from immunized hamsters inhibiting 80% of trophozoites adherence to mammalian cells and inducing 80% more complement-mediated lysis of trophozoites compared with the control. C-Igl was further assessed for its cellular response by cytokine-gene qPCR analysis. The productions of IL-4 (8.4-fold) and IL-10 (2-fold) in the spleen cells of immunized hamsters were enhanced after in vitro stimulation. IL-4 expression was also supported by increased programmed cell death 1 ligand 1 gene. Conclusions/Significance Immunobiochemical characterization strongly suggests the potential of recombinant Igl, especially the C-terminal fragment, as a vaccine candidate against amoebiasis. Moreover, protection through Th2-cell participation enabled effective

  15. Identification of Four Entamoeba histolytica Organellar DNA Polymerases of the Family B and Cellular Localization of the Ehodp1 Gene and EhODP1 Protein

    PubMed Central

    Herrera-Aguirre, María Esther; Luna-Arias, Juan Pedro; Labra-Barrios, María Luisa; Orozco, Esther

    2010-01-01

    We report the identification of a family of four active genes (Ehodp1, Ehodp2, Ehodp3, and Ehodp4) encoding putative DNA polymerases in Entamoeba histolytica, the protozoan parasite responsible of human amoebiasis. The four Ehodp genes show similarity to DNA polymerases encoded in fungi and plant mitochondrial plasmids. EhODP polypeptides conserve the 3′-5′ exonuclease II and 5′-3′ polymerization domains, and they have the I, II, and III conserved boxes that characterize them as DNA polymerases of family B. Furthermore, we found in EhODP polymerases two novel A and B boxes, present also in DNA polymerases encoded in fungi mitochondrial plasmids. By in situ PCR, Ehodp1 gene was located in nuclei and in DNA-containing cytoplasmic structures. Additionally, using polyclonal antibodies against a recombinant rEhODP1-168 polypeptide, and confocal microscopy, EhODP1 was located in cytoplasmic DNA-containing structures. PMID:20300437

  16. Expression of EhRAD54, EhRAD51, and EhBLM proteins during DNA repair by homologous recombination in Entamoeba histolytica.

    PubMed

    Charcas-Lopez, Ma del Socorro; Garcia-Morales, Lorena; Pezet-Valdez, Marisol; Lopez-Camarillo, Cesar; Zamorano-Carrillo, Absalom; Marchat, Laurence A

    2014-01-01

    Entamoeba histolytica, the protozoan responsible for human amoebiasis, exhibits a great genome plasticity that is probably related to homologous recombination events. It contains the RAD52 epistasis group genes, including Ehrad51 and Ehrad54, and the Ehblm gene, which are key homologous recombination factors in other organisms. Ehrad51 and Ehrad54 genes are differentially transcribed in trophozoites when DNA double-strand breaks are induced by ultraviolet-C irradiation. Moreover, the EhRAD51 recombinase is overexpressed at 30 min in the nucleus. Here, we extend our analysis of the homologous recombination mechanism in E. histolytica by studying EhRAD51, EhRAD54, and EhBLM expression in response to DNA damage. Bioinformatic analyses show that EhRAD54 has the molecular features of homologous proteins, indicating that it may have similar functions. Western blot assays evidence the differential expression of EhRAD51, EhRAD54, and EhBLM at different times after DNA damage, suggesting their potential roles in the different steps of homologous recombination in this protozoan. PMID:24534563

  17. Detection of the Endosomal Sorting Complex Required for Transport in Entamoeba histolytica and Characterization of the EhVps4 Protein

    PubMed Central

    López-Reyes, Israel; García-Rivera, Guillermina; Bañuelos, Cecilia; Herranz, Silvia; Vincent, Olivier; López-Camarillo, César; Marchat, Laurence A.; Orozco, Esther

    2010-01-01

    Eukaryotic endocytosis involves multivesicular bodies formation, which is driven by endosomal sorting complexes required for transport (ESCRT). Here, we showed the presence and expression of homologous ESCRT genes in Entamoeba histolytica. We cloned and expressed the Ehvps4 gene, an ESCRT member, to obtain the recombinant EhVps4 and generate specific antibodies, which immunodetected EhVps4 in cytoplasm of trophozoites. Bioinformatics and biochemical studies evidenced that rEhVps4 is an ATPase, whose activity depends on the conserved E211 residue. Next, we generated trophozoites overexpressing EhVps4 and mutant EhVps4-E211Q FLAG-tagged proteins. The EhVps4-FLAG was located in cytosol and at plasma membrane, whereas the EhVps4-E211Q-FLAG was detected as abundant cytoplasmic dots in trophozoites. Erythrophagocytosis, cytopathic activity, and hepatic damage in hamsters were not improved in trophozoites overexpressing EhVps4-FLAG. In contrast, EhVps4-E211Q-FLAG protein overexpression impaired these properties. The localization of EhVps4-FLAG around ingested erythrocytes, together with our previous results, strengthens the role for EhVps4 in E. histolytica phagocytosis and virulence. PMID:20508821

  18. Expression of EhRAD54, EhRAD51, and EhBLM proteins during DNA repair by homologous recombination in Entamoeba histolytica

    PubMed Central

    del Socorro Charcas-Lopez, Ma.; Garcia-Morales, Lorena; Pezet-Valdez, Marisol; Lopez-Camarillo, Cesar; Zamorano-Carrillo, Absalom; Marchat, Laurence A.

    2014-01-01

    Entamoeba histolytica, the protozoan responsible for human amoebiasis, exhibits a great genome plasticity that is probably related to homologous recombination events. It contains the RAD52 epistasis group genes, including Ehrad51 and Ehrad54, and the Ehblm gene, which are key homologous recombination factors in other organisms. Ehrad51 and Ehrad54 genes are differentially transcribed in trophozoites when DNA double-strand breaks are induced by ultraviolet-C irradiation. Moreover, the EhRAD51 recombinase is overexpressed at 30 min in the nucleus. Here, we extend our analysis of the homologous recombination mechanism in E. histolytica by studying EhRAD51, EhRAD54, and EhBLM expression in response to DNA damage. Bioinformatic analyses show that EhRAD54 has the molecular features of homologous proteins, indicating that it may have similar functions. Western blot assays evidence the differential expression of EhRAD51, EhRAD54, and EhBLM at different times after DNA damage, suggesting their potential roles in the different steps of homologous recombination in this protozoan. PMID:24534563

  19. The Entamoeba histolytica, Arp2/3 Complex Is Recruited to Phagocytic Cups through an Atypical Kinase EhAK1

    PubMed Central

    Babuta, Mrigya; Mansuri, M Shahid; Bhattacharya, Sudha; Bhattacharya, Alok

    2015-01-01

    The parasite Entamoeba histolytica is the etiological agent of amoebiasis and phagocytosis plays a key role in virulence of this organism. Signaling pathways involved in activation of cytoskeletal dynamics required for phagocytosis remain to be elucidated. Phagocytosis is initiated with sequential recruitment of EhC2PK, EhCaBP1, EhCaBP3 and an atypical kinase EhAK1 after particle attachment. Here we show that EhARPC1, an essential subunit of the actin branching complex Arp 2/3 is recruited to the phagocytic initiation sites by EhAK1. Imaging, expression knockdown of different molecules and pull down experiments suggest that EhARPC1 interacts with EhAK1 and that it is required during initiation of phagocytosis and phagosome formation. Moreover, recruitment of EhARPC2 at the phagocytosis initiation by EhAK1 is also observed, indicating that the Arp 2/3 complex is recruited. In conclusion, these results suggests a novel mechanism of recruitment of Arp 2/3 complex during phagocytosis in E. histolytica. PMID:26646565

  20. A Single RNaseIII Domain Protein from Entamoeba histolytica Has dsRNA Cleavage Activity and Can Help Mediate RNAi Gene Silencing in a Heterologous System.

    PubMed

    Pompey, Justine M; Foda, Bardees; Singh, Upinder

    2015-01-01

    Dicer enzymes process double-stranded RNA (dsRNA) into small RNAs that target gene silencing through the RNA interference (RNAi) pathway. Dicer enzymes are complex, multi-domain RNaseIII proteins, however structural minimalism of this protein has recently emerged in parasitic and fungal systems. The most minimal Dicer, Saccharomyces castellii Dicer1, has a single RNaseIII domain and two double stranded RNA binding domains. In the protozoan parasite Entamoeba histolytica 27nt small RNAs are abundant and mediate silencing, yet no canonical Dicer enzyme has been identified. Although EhRNaseIII does not exhibit robust dsRNA cleavage in vitro, it can process dsRNA in the RNAi-negative background of Saccharomyces cerevisiae, and in conjunction with S. castellii Argonaute1 can partially reconstitute the RNAi pathway. Thus, although EhRNaseIII lacks the domain architecture of canonical or minimal Dicer enzymes, it has dsRNA processing activity that contributes to gene silencing via RNAi. Our data advance the understanding of small RNA biogenesis in Entamoeba as well as broaden the spectrum of non-canonical Dicer enzymes that contribute to the RNAi pathway. PMID:26230096

  1. A Single RNaseIII Domain Protein from Entamoeba histolytica Has dsRNA Cleavage Activity and Can Help Mediate RNAi Gene Silencing in a Heterologous System

    PubMed Central

    Singh, Upinder

    2015-01-01

    Dicer enzymes process double-stranded RNA (dsRNA) into small RNAs that target gene silencing through the RNA interference (RNAi) pathway. Dicer enzymes are complex, multi-domain RNaseIII proteins, however structural minimalism of this protein has recently emerged in parasitic and fungal systems. The most minimal Dicer, Saccharomyces castellii Dicer1, has a single RNaseIII domain and two double stranded RNA binding domains. In the protozoan parasite Entamoeba histolytica 27nt small RNAs are abundant and mediate silencing, yet no canonical Dicer enzyme has been identified. Although EhRNaseIII does not exhibit robust dsRNA cleavage in vitro, it can process dsRNA in the RNAi-negative background of Saccharomyces cerevisiae, and in conjunction with S. castellii Argonaute1 can partially reconstitute the RNAi pathway. Thus, although EhRNaseIII lacks the domain architecture of canonical or minimal Dicer enzymes, it has dsRNA processing activity that contributes to gene silencing via RNAi. Our data advance the understanding of small RNA biogenesis in Entamoeba as well as broaden the spectrum of non-canonical Dicer enzymes that contribute to the RNAi pathway. PMID:26230096

  2. Computational prediction and validation of C/D, H/ACA and Eh_U3 snoRNAs of Entamoeba histolytica

    PubMed Central

    2012-01-01

    Background Small nucleolar RNAs are a highly conserved group of small RNAs found in eukaryotic cells. Genes encoding these RNAs are diversely located throughout the genome. They are functionally conserved, performing post transcriptional modification (methylation and pseudouridylation) of rRNA and other nuclear RNAs. They belong to two major categories: the C/D box and H/ACA box containing snoRNAs. U3 snoRNA is an exceptional member of C/D box snoRNAs and is involved in early processing of pre-rRNA. An antisense sequence is present in each snoRNA which guides the modification or processing of target RNA. However, some snoRNAs lack this sequence and often they are called orphan snoRNAs. Results We have searched snoRNAs of Entamoeba histolytica from the genome sequence using computational programmes (snoscan and snoSeeker) and we obtained 99 snoRNAs (C/D and H/ACA box snoRNAs) along with 5 copies of Eh_U3 snoRNAs. These are located diversely in the genome, mostly in intergenic regions, while some are found in ORFs of protein coding genes, intron and UTRs. The computationally predicted snoRNAs were validated by RT-PCR and northern blotting. The expected sizes were in agreement with the observed sizes for all C/D box snoRNAs tested, while for some of the H/ACA box there was indication of processing to generate shorter products. Conclusion Our results showed the presence of snoRNAs in E. histolytica, an early branching eukaryote, and the structural features of E. histolytica snoRNAs were well conserved when compared with yeast and human snoRNAs. This study will help in understanding the evolution of these conserved RNAs in diverse phylogenetic groups. PMID:22892049

  3. Evaluation of Multiplex Tandem Real-Time PCR for Detection of Cryptosporidium spp., Dientamoeba fragilis, Entamoeba histolytica, and Giardia intestinalis in Clinical Stool Samples ▿

    PubMed Central

    Stark, D.; Al-Qassab, S. E.; Barratt, J. L. N.; Stanley, K.; Roberts, T.; Marriott, D.; Harkness, J.; Ellis, J. T.

    2011-01-01

    The aim of this study was to describe the first development and evaluation of a multiplex tandem PCR (MT-PCR) assay for the detection and identification of 4 common pathogenic protozoan parasites, Cryptosporidium spp., Dientamoeba fragilis, Entamoeba histolytica, and Giardia intestinalis, from human clinical samples. A total of 472 fecal samples submitted to the Department of Microbiology at St. Vincent's Hospital were included in the study. The MT-PCR assay was compared to four real-time PCR (RT-PCR) assays and microscopy by a traditional modified iron hematoxylin stain. The MT-PCR detected 28 G. intestinalis, 26 D. fragilis, 11 E. histolytica, and 9 Cryptosporidium sp. isolates. Detection and identification of the fecal protozoa by MT-PCR demonstrated 100% correlation with the RT-PCR results, and compared to RT-PCR, MT-PCR exhibited 100% sensitivity and specificity, while traditional microscopy of stained fixed fecal smears exhibited sensitivities and specificities of 56% and 100% for Cryptosporidium spp., 38% and 99% for D. fragilis, 47% and 97% for E. histolytica, and 50% and 100% for G. intestinalis. No cross-reactivity was detected in 100 stool samples containing various other bacterial, viral, and protozoan species. The MT-PCR assay was able to provide rapid, sensitive, and specific simultaneous detection and identification of the four most important diarrhea-causing protozoan parasites that infect humans. This study also highlights the lack of sensitivity demonstrated by microscopy, and thus, molecular methods such as MT-PCR must be considered the diagnostic methods of choice for enteric protozoan parasites. PMID:21048004

  4. Prevalence and Spatial Distribution of Entamoeba histolytica/dispar and Giardia lamblia among Schoolchildren in Agboville Area (Côte d'Ivoire)

    PubMed Central

    Ouattara, Mamadou; N'Guéssan, Nicaise A.; Yapi, Ahoua; N'Goran, Eliézer K.

    2010-01-01

    Background New efforts are being made to improve understanding of the epidemiology of the helminths and intensifying the control efforts against these parasites. In contrast, relatively few studies are being carried out in this direction for the intestinal protozoa. To contribute to a better comprehension of the epidemiology of the intestinal protozoa, prevalence, and spatial distribution of Entamoeba histolytica/dispar and Giardia lamblia, and their association with drinking water supplies, were determined in the Agboville department in southeast Côte d'Ivoire. Methods/Findings Stool samples were taken from more than 1,300 schoolchildren in the third year of primary education (CE1) from 30 primary schools and preserved in SAF (sodium acetate-acetic acid-formalin). The samples were analyzed by formalin-ether concentration. Then, a survey questionnaire addressed to schoolchildren and school directors was used to collect data on water supplies. Prevalence of E. histolytica/dispar and G. lamblia were, respectively, 18.8% and 13.9%. No particular focus zone was observed in the spatial distribution of the two species. Significant negative association was observed between use of tap water and high prevalence of E. histolytica/dispar infection (OR = 0.83, p = 0.01). High prevalence of G. lamblia infection was positively associated with use of ponds as the source of drinking water (OR = 1.28, p = 0.009). Conclusion These two species of pathogenic protozoa are present with substantial prevalence in this area of Côte d'Ivoire. Although their spatial distribution is not focused in any one place, determination of the population segments with the highest levels of infection will help to target the chemotherapeutic fight. To reinforce treatment with chemotherapeutic agents, tap water should be made available in all the localities of this area. PMID:20087416

  5. Dimethylated H3K27 Is a Repressive Epigenetic Histone Mark in the Protist Entamoeba histolytica and Is Significantly Enriched in Genes Silenced via the RNAi Pathway.

    PubMed

    Foda, Bardees M; Singh, Upinder

    2015-08-21

    RNA interference (RNAi) is a fundamental biological process that plays a crucial role in regulation of gene expression in many organisms. Transcriptional gene silencing (TGS) is one of the important nuclear roles of RNAi. Our previous data show that Entamoeba histolytica has a robust RNAi pathway that links to TGS via Argonaute 2-2 (Ago2-2) associated 27-nucleotide small RNAs with 5'-polyphosphate termini. Here, we report the first repressive histone mark to be identified in E. histolytica, dimethylation of H3K27 (H3K27Me2), and demonstrate that it is enriched at genes that are silenced by RNAi-mediated TGS. An RNAi-silencing trigger can induce H3K27Me2 deposits at both episomal and chromosomal loci, mediating gene silencing. Our data support two phases of RNAi-mediated TGS: an active silencing phase where the RNAi trigger is present and both H3K27Me2 and Ago2-2 concurrently enrich at chromosomal loci; and an established silencing phase in which the RNAi trigger is removed, but gene silencing with H3K27Me2 enrichment persist independently of Ago2-2 deposition. Importantly, some genes display resistance to chromosomal silencing despite induction of functional small RNAs. In those situations, the RNAi-triggering plasmid that is maintained episomally gets partially silenced and has H3K27Me2 enrichment, but the chromosomal copy displays no repressive histone enrichment. Our data are consistent with a model in which H3K27Me2 is a repressive histone modification, which is strongly associated with transcriptional repression. This is the first example of an epigenetic histone modification that functions to mediate RNAi-mediated TGS in the deep-branching eukaryote E. histolytica. PMID:26149683

  6. Simultaneous detection and differentiation of Entamoeba histolytica, E. dispar, E. moshkovskii, Giardia lamblia and Cryptosporidium spp. in human fecal samples using multiplex PCR and qPCR-MCA.

    PubMed

    Zebardast, Nozhat; Yeganeh, Farshid; Gharavi, Mohammad Javad; Abadi, Alireza; Seyyed Tabaei, Seyyed Javad; Haghighi, Ali

    2016-10-01

    Entamoeba histolytica, Giardia lamblia and Cryptosporidium spp. are common causes of diarrheal and intestinal diseases all over the world. Microscopic methods are useful in the diagnosis of intestinal parasites (IPs), but their sensitivity was assessed approximately 60 percent. Recently, molecular techniques have been used increasingly for the identification and characterization of the parasites. Among those, in this study we have used multiplex PCR and Real-time PCR with melting curve analysis (qPCR-MCA) for simultaneous detection and differentiation of E. histolytica, E. dispar, E. moshkovskii, G. lamblia and Cryptosporidium spp. in human fecal samples. Twenty DNA samples from 12 E. histolytica and 8 E. dispar samples and twenty stool samples confirmed positive for G. lamblia and Cryptosporidium spp. were analyzed. After DNA extraction from the samples, multiplex PCR was done for detection and differentiation of above mentioned parasites. QPCR-MCA was also performed for the detection and differentiation of 11 isolates of above mentioned parasite in a cycle with a time and temperature. Multiplex PCR was able to simultaneous detect and differentiate of above mentioned parasite in a single reaction. QPCR-MCA was able to differentiate genus and species those five protozoa using melting temperature simultaneously at the same time and temperature programs. In total, qPCR-MCA diagnosed 7/11 isolation of E. histolytica, 6/8 isolation of E. dispar, 1/1 E. moshkovskii Laredo, 10/11 G. Lamblia and 6/11 Cryptosporidium spp. Application of multiplex PCR for detection of more than one species in a test in developing countries, at least in reference laboratories has accurate diagnosis and plays a critical role in differentiation of protozoan species. Multiplex PCR assay with a template and multi template had different results and it seems that using a set of primers with one template has higher diagnostic capability in compare with multi template. The results of this study

  7. Overexpression of Differentially Expressed Genes Identified in Non-pathogenic and Pathogenic Entamoeba histolytica Clones Allow Identification of New Pathogenicity Factors Involved in Amoebic Liver Abscess Formation

    PubMed Central

    Lorenzen, Stephan; Schuldt, Kathrin; Bernin, Hannah; Zaruba, Mareen; Lender, Corinna; Ittrich, Harald; Roeder, Thomas; Tannich, Egbert; Lotter, Hannelore; Bruchhaus, Iris

    2016-01-01

    We here compared pathogenic (p) and non-pathogenic (np) isolates of Entamoeba histolytica to identify molecules involved in the ability of this parasite to induce amoebic liver abscess (ALA)-like lesions in two rodent models for the disease. We performed a comprehensive analysis of 12 clones (A1–A12) derived from a non-pathogenic isolate HM-1:IMSS-A and 12 clones (B1–B12) derived from a pathogenic isolate HM-1:IMSS-B. “Non-pathogenicity” included the induction of small and quickly resolved lesions while “pathogenicity” comprised larger abscess development that overstayed day 7 post infection. All A-clones were designated as non-pathogenic, whereas 4 out of 12 B-clones lost their ability to induce ALAs in gerbils. No correlation between ALA formation and cysteine peptidase (CP) activity, haemolytic activity, erythrophagocytosis, motility or cytopathic activity was found. To identify the molecular framework underlying different pathogenic phenotypes, three clones were selected for in-depth transcriptome analyses. Comparison of a non-pathogenic clone A1np with pathogenic clone B2p revealed 76 differentially expressed genes, whereas comparison of a non-pathogenic clone B8np with B2p revealed only 19 differentially expressed genes. Only six genes were found to be similarly regulated in the two non-pathogenic clones A1np and B8np in comparison with the pathogenic clone B2p. Based on these analyses, we chose 20 candidate genes and evaluated their roles in ALA formation using the respective gene-overexpressing transfectants. We conclude that different mechanisms lead to loss of pathogenicity. In total, we identified eight proteins, comprising a metallopeptidase, C2 domain proteins, alcohol dehydrogenases and hypothetical proteins, that affect the pathogenicity of E. histolytica. PMID:27575775

  8. Identification of the galactose-adherence lectin epitopes of Entamoeba histolytica that stimulate tumor necrosis factor-alpha production by macrophages.

    PubMed Central

    Séguin, R; Mann, B J; Keller, K; Chadee, K

    1995-01-01

    The 170-kDa subunit of the galactose-adherence lectin (Gal-lectin) of Entamoeba histolytica mediates adherence to human colonic mucins and intestinal epithelium as a prerequisite to amebic invasion. The Gal-lectin is an immunodominant molecule and a protective antigen in the gerbil model of amebiasis. Tumor necrosis factor alpha (TNF-alpha) produced by activated macrophages enhances nitric oxide-dependent cytotoxicity in host defense against E. histolytica. The purpose of this study was to identify the Gal-lectin epitopes which stimulate TNF-alpha production by macrophages. Murine bone marrow-derived macrophages (BMMs) exposed to Gal-lectin (100-500 ng/ml) stimulated stable expression of TNF-alpha mRNA (8-fold increase) and TNF-alpha production similar to that of lipopolysaccharide-stimulated cells (100 ng/ml). Polyclonal anti-lectin serum specifically inhibited TNF-alpha mRNA induction in response to the Gal-lectin but not to lipopolysaccharide. Anti-lectin monoclonal antibodies 8C12, H85 and 1G7, which recognize nonoverlapping epitopes of the cysteine-rich region of the 170-kDa heavy subunit, inhibited both amebic adherence to mammalian cells and Gal-lectin-stimulated TNF-alpha mRNA expression by BMMs,but monoclonal antibody 7F4 did neither. As these inhibitory antibodies map to amino acids 596-1082 of the 170-kDa Gal-lectin, our results have identified the functional region that mediates amebic adherence and TNF-alpha mRNA induction in BMMMs; thus, this region of the Gal-lectin is a subunit vaccine candidate. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 6 PMID:8618866

  9. The 25 kDa Subunit of Cleavage Factor Im Is a RNA-Binding Protein That Interacts with the Poly(A) Polymerase in Entamoeba histolytica

    PubMed Central

    Pezet-Valdez, Marisol; Fernández-Retana, Jorge; Ospina-Villa, Juan David; Ramírez-Moreno, María Esther; Orozco, Esther; Charcas-López, Socorro; Soto-Sánchez, Jacqueline; Mendoza-Hernández, Guillermo; López-Casamicha, Mavil; López-Camarillo, César; Marchat, Laurence A.

    2013-01-01

    In eukaryotes, polyadenylation of pre-mRNA 3´ end is essential for mRNA export, stability and translation. Taking advantage of the knowledge of genomic sequences of Entamoeba histolytica, the protozoan responsible for human amoebiasis, we previously reported the putative polyadenylation machinery of this parasite. Here, we focused on the predicted protein that has the molecular features of the 25 kDa subunit of the Cleavage Factor Im (CFIm25) from other organisms, including the Nudix (nucleoside diphosphate linked to another moiety X) domain, as well as the RNA binding domain and the PAP/PAB interacting region. The recombinant EhCFIm25 protein (rEhCFIm25) was expressed in bacteria and used to generate specific antibodies in rabbit. Subcellular localization assays showed the presence of the endogenous protein in nuclear and cytoplasmic fractions. In RNA electrophoretic mobility shift assays, rEhCFIm25 was able to form specific RNA-protein complexes with the EhPgp5 mRNA 3´ UTR used as probe. In addition, Pull-Down and LC/ESI-MS/MS tandem mass spectrometry assays evidenced that the putative EhCFIm25 was able to interact with the poly(A) polymerase (EhPAP) that is responsible for the synthesis of the poly(A) tail in other eukaryotic cells. By Far-Western experiments, we confirmed the interaction between the putative EhCFIm25 and EhPAP in E. histolytica. Taken altogether, our results showed that the putative EhCFIm25 is a conserved RNA binding protein that interacts with the poly(A) polymerase, another member of the pre-mRNA 3´ end processing machinery in this protozoan parasite. PMID:23840799

  10. The 25 kDa subunit of cleavage factor Im Is a RNA-binding protein that interacts with the poly(A) polymerase in Entamoeba histolytica.

    PubMed

    Pezet-Valdez, Marisol; Fernández-Retana, Jorge; Ospina-Villa, Juan David; Ramírez-Moreno, María Esther; Orozco, Esther; Charcas-López, Socorro; Soto-Sánchez, Jacqueline; Mendoza-Hernández, Guillermo; López-Casamicha, Mavil; López-Camarillo, César; Marchat, Laurence A

    2013-01-01

    In eukaryotes, polyadenylation of pre-mRNA 3' end is essential for mRNA export, stability and translation. Taking advantage of the knowledge of genomic sequences of Entamoeba histolytica, the protozoan responsible for human amoebiasis, we previously reported the putative polyadenylation machinery of this parasite. Here, we focused on the predicted protein that has the molecular features of the 25 kDa subunit of the Cleavage Factor Im (CFIm25) from other organisms, including the Nudix (nucleoside diphosphate linked to another moiety X) domain, as well as the RNA binding domain and the PAP/PAB interacting region. The recombinant EhCFIm25 protein (rEhCFIm25) was expressed in bacteria and used to generate specific antibodies in rabbit. Subcellular localization assays showed the presence of the endogenous protein in nuclear and cytoplasmic fractions. In RNA electrophoretic mobility shift assays, rEhCFIm25 was able to form specific RNA-protein complexes with the EhPgp5 mRNA 3´ UTR used as probe. In addition, Pull-Down and LC/ESI-MS/MS tandem mass spectrometry assays evidenced that the putative EhCFIm25 was able to interact with the poly(A) polymerase (EhPAP) that is responsible for the synthesis of the poly(A) tail in other eukaryotic cells. By Far-Western experiments, we confirmed the interaction between the putative EhCFIm25 and EhPAP in E. histolytica. Taken altogether, our results showed that the putative EhCFIm25 is a conserved RNA binding protein that interacts with the poly(A) polymerase, another member of the pre-mRNA 3' end processing machinery in this protozoan parasite. PMID:23840799

  11. Identification of an Epitope on the Entamoeba histolytica 170-kD Lectin Conferring Antibody-mediated Protection against Invasive Amebiasis

    PubMed Central

    Lotter, Hannelore; Zhang, Tonghai; Seydel, Karl B.; Stanley, Samuel L.; Tannich, Egbert

    1997-01-01

    The emergence of multidrug-resistant organisms and the failure to eradicate infection by a number of important pathogens has led to increased efforts to develop vaccines to prevent infectious diseases. However, the nature of the immune response to vaccination with a given antigen can be complex and unpredictable. An example is the galactose– and N-acetylgalactosamine–inhibitable lectin, a surface antigen of Entamoeba histolytica that has been identified as a major candidate in a vaccine to prevent amebiasis. Vaccination with the lectin can induce protective immunity to amebic liver abscess in some animals, but others of the same species exhibit exacerbations of disease after vaccination. To better understand this phenomenon, we used recombinant proteins corresponding to four distinct domains of the molecule, and synthetic peptides to localize both protective and exacerbative epitopes of the heavy chain subunit of the lectin. We show that protective immunity after vaccination can be correlated with the development of an antibody response to a region of 25 amino acid residues of the lectin, and have confirmed the importance of the antibody response to this region by passive immunization studies. In addition, we show that exacerbation of disease can be linked to the development of antibodies that bind to an NH2-terminal domain of the lectin. These findings are clinically relevant, as individuals who are colonized with E. histolytica but are resistant to invasive disease have a high prevalence of antibodies to the protective epitope(s), compared to individuals with a history of invasive amebiasis. These studies should enable us to develop an improved vaccine for amebiasis, and provide a model for the identification of protective and exacerbative epitopes of complex antigens. PMID:9151705

  12. X-ray structures of thioredoxin and thioredoxin reductase from Entamoeba histolytica and prevailing hypothesis of the mechanism of Auranofin action.

    PubMed

    Parsonage, Derek; Sheng, Fang; Hirata, Ken; Debnath, Anjan; McKerrow, James H; Reed, Sharon L; Abagyan, Ruben; Poole, Leslie B; Podust, Larissa M

    2016-05-01

    The anti-arthritic gold-containing drug Auranofin is lethal to the protozoan intestinal parasite Entamoeba histolytica, the causative agent of human amebiasis, in both culture and animal models of the disease. A putative mechanism of Auranofin action proposes that monovalent gold, Au(I), released from the drug, can bind to the redox-active dithiol group of thioredoxin reductase (TrxR). Au(I) binding in the active site is expected to prevent electron transfer to the downstream substrate thioredoxin (Trx), thus interfering with redox homeostasis in the parasite. To clarify the molecular mechanism of Auranofin action in more detail, we determined a series of atomic resolution X-ray structures for E. histolytica thioredoxin (EhTrx) and thioredoxin reductase (EhTrxR), the latter with and without Auranofin. Only the disulfide-bonded form of the active site dithiol (Cys(140)-Cys(143)) was invariably observed in crystals of EhTrxR in spite of the addition of reductants in various crystallization trials, and no gold was found associated with these cysteines. Non-catalytic Cys(286) was identified as the only site of modification, but further mutagenesis studies using the C286Q mutant demonstrated that this site was not responsible for inhibition of EhTrxR by Auranofin. Interestingly, we obtained both of the catalytically-relevant conformations of this bacterial-like, low molecular weight TrxR in crystals without requiring an engineered disulfide linkage between Cys mutants of TrxR and Trx (as was originally done with Escherichia coli TrxR and Trx). We note that the -CXXC- catalytic motif, even if reduced, would likely not provide space sufficient to bind Au(I) by both cysteines of the dithiol group. PMID:26876147

  13. Overexpression of Differentially Expressed Genes Identified in Non-pathogenic and Pathogenic Entamoeba histolytica Clones Allow Identification of New Pathogenicity Factors Involved in Amoebic Liver Abscess Formation.

    PubMed

    Meyer, Martin; Fehling, Helena; Matthiesen, Jenny; Lorenzen, Stephan; Schuldt, Kathrin; Bernin, Hannah; Zaruba, Mareen; Lender, Corinna; Ernst, Thomas; Ittrich, Harald; Roeder, Thomas; Tannich, Egbert; Lotter, Hannelore; Bruchhaus, Iris

    2016-08-01

    We here compared pathogenic (p) and non-pathogenic (np) isolates of Entamoeba histolytica to identify molecules involved in the ability of this parasite to induce amoebic liver abscess (ALA)-like lesions in two rodent models for the disease. We performed a comprehensive analysis of 12 clones (A1-A12) derived from a non-pathogenic isolate HM-1:IMSS-A and 12 clones (B1-B12) derived from a pathogenic isolate HM-1:IMSS-B. "Non-pathogenicity" included the induction of small and quickly resolved lesions while "pathogenicity" comprised larger abscess development that overstayed day 7 post infection. All A-clones were designated as non-pathogenic, whereas 4 out of 12 B-clones lost their ability to induce ALAs in gerbils. No correlation between ALA formation and cysteine peptidase (CP) activity, haemolytic activity, erythrophagocytosis, motility or cytopathic activity was found. To identify the molecular framework underlying different pathogenic phenotypes, three clones were selected for in-depth transcriptome analyses. Comparison of a non-pathogenic clone A1np with pathogenic clone B2p revealed 76 differentially expressed genes, whereas comparison of a non-pathogenic clone B8np with B2p revealed only 19 differentially expressed genes. Only six genes were found to be similarly regulated in the two non-pathogenic clones A1np and B8np in comparison with the pathogenic clone B2p. Based on these analyses, we chose 20 candidate genes and evaluated their roles in ALA formation using the respective gene-overexpressing transfectants. We conclude that different mechanisms lead to loss of pathogenicity. In total, we identified eight proteins, comprising a metallopeptidase, C2 domain proteins, alcohol dehydrogenases and hypothetical proteins, that affect the pathogenicity of E. histolytica. PMID:27575775

  14. Toll-like Receptor Signaling Activation by Entamoeba histolytica Induces Beta Defensin 2 in Human Colonic Epithelial Cells: Its Possible Role as an Element of the Innate Immune Response

    PubMed Central

    Ayala-Sumuano, Jorge-Tonatiuh; Téllez-López, Victor M.; Domínguez-Robles, M. del Carmen; Shibayama-Salas, Mineko; Meza, Isaura

    2013-01-01

    Background Entamoeba histolytica, a protozoan parasite of humans, produces dysenteric diarrhea, intestinal mucosa damage and extraintestinal infection. It has been proposed that the intestinal microbiota composition could be an important regulatory factor of amebic virulence and tissue invasion, particularly if pathogenic bacteria are present. Recent in vitro studies have shown that Entamoeba histolytica trophozoites induced human colonic CaCo2 cells to synthesize TLR-2 and TLR-4 and proinflammatory cytokines after binding to the amebic Gal/GalNac lectin carbohydrate recognition domain. The magnitude of the inflammatory response induced by trophozoites and the subsequent cell damage were synergized when cells had previously been exposed to pathogenic bacteria. Methodology/Principal Findings We show here that E. histolytica activation of the classic TLR pathway in CaCo2 cells is required to induce β defensin-2 (HBD2) mRNA expression and production of a 5-kDa cationic peptide with similar properties to the antimicrobial HBD2 expressed by CaCo2 cells exposed to enterotoxigenic Escherichia coli. The induced peptide showed capacity to permeabilize membranes of bacteria and live trophozoites. This activity was abrogated by inhibition of TLR2/4-NFκB pathway or by neutralization with an anti-HBD2 antibody. Conclusions/Significance Entamoeba histolytica trophozoites bind to human intestinal cells and induce expression of HBD2; an antimicrobial molecule with capacity to destroy pathogenic bacteria and trophozoites. HDB2's possible role as a modulator of the course of intestinal infections, particularly in mixed ameba/bacteria infections, is discussed. PMID:23469306

  15. Interaction between Nbp35 and Cfd1 Proteins of Cytosolic Fe-S Cluster Assembly Reveals a Stable Complex Formation in Entamoeba histolytica

    PubMed Central

    Anwar, Shadab; Dikhit, Manas Ranjan; Singh, Krishn Pratap; Kar, Rajiv Kumar; Zaidi, Amir; Sahoo, Ganesh Chandra; Roy, Awadh Kishore; Nozaki, Tomoyoshi; Das, Pradeep; Ali, Vahab

    2014-01-01

    Iron-Sulfur (Fe-S) proteins are involved in many biological functions such as electron transport, photosynthesis, regulation of gene expression and enzymatic activities. Biosynthesis and transfer of Fe-S clusters depend on Fe-S clusters assembly processes such as ISC, SUF, NIF, and CIA systems. Unlike other eukaryotes which possess ISC and CIA systems, amitochondriate Entamoeba histolytica has retained NIF & CIA systems for Fe-S cluster assembly in the cytosol. In the present study, we have elucidated interaction between two proteins of E. histolytica CIA system, Cytosolic Fe-S cluster deficient 1 (Cfd1) protein and Nucleotide binding protein 35 (Nbp35). In-silico analysis showed that structural regions ranging from amino acid residues (P33-K35, G131-V135 and I147-E151) of Nbp35 and (G5-V6, M34-D39 and G46-A52) of Cfd1 are involved in the formation of protein-protein complex. Furthermore, Molecular dynamic (MD) simulations study suggested that hydrophobic forces surpass over hydrophilic forces between Nbp35 and Cfd1 and Van-der-Waal interaction plays crucial role in the formation of stable complex. Both proteins were separately cloned, expressed as recombinant fusion proteins in E. coli and purified to homogeneity by affinity column chromatography. Physical interaction between Nbp35 and Cfd1 proteins was confirmed in vitro by co-purification of recombinant Nbp35 with thrombin digested Cfd1 and in vivo by pull down assay and immunoprecipitation. The insilico, in vitro as well as in vivo results prove a stable interaction between these two proteins, supporting the possibility of its involvement in Fe-S cluster transfer to target apo-proteins through CIA machinery in E. histolytica. Our study indicates that initial synthesis of a Fe-S precursor in mitochondria is not necessary for the formation of Cfd1-Nbp35 complex. Thus, Cfd1 and Nbp35 with the help of cytosolic NifS and NifU proteins can participate in the maturation of non-mitosomal Fe-S proteins without any

  16. STUDIES ON THE CARBOHYDRATE METABOLISM OF A GRAM-NEGATIVE ANAEROBE (BACTEROIDES SYMBIOSUS) USED IN THE CULTURE OF ENTAMOEBA HISTOLYTICA1

    PubMed Central

    Bragg, P. D.; Reeves, Richard E.

    1962-01-01

    Bragg, P. D. (Louisiana State University, New Orleans) and R. E. Reeves. Studies on the carbohydrate metabolism of a gram-negative anaerobe (Bacteroides symbiosus) used in the culture of Entamoeba histolytica. J. Bacteriol. 83:76–84. 1962—Resting cells of Bacteroides symbiosus have been shown to utilize glucose and several other monosaccharides. The fermentation of the sugars is mediated by demonstrable kinases except in the case of mannitol. The main end products of metabolism of glucose are CO2, H2, ethanol, and acetic, butyric, succinic, and lactic acids. Changes in the thiol used in the growth media produce different enzyme complements in the cells. Thus, cells grown with cysteine as the thiol are unable to metabolize glucosamine, whereas those grown with thiomalate rapidly degrade the amino sugar. The results of the enzyme assay and the results from experiments with C14-labelled glucose suggest that glucose is metabolized by resting cells mainly by the Embden-Myerhof pathway. PMID:13872395

  17. Autophosphorylation at Thr279 of Entamoeba histolytica atypical kinase EhAK1 is required for activity and regulation of erythrophagocytosis.

    PubMed

    Mansuri, M Shahid; Babuta, Mrigya; Ali, Mohammad Sabir; Bharadwaj, Ravi; Deep jhingan, Gagan; Gourinath, Samudrala; Bhattacharya, Sudha; Bhattacharya, Alok

    2016-01-01

    Phagocytosis plays a key role in survival and pathogenicity of Entamoeba histolytica. We have recently demonstrated that an atypical kinase EhAK1 is involved in phagocytosis in this parasite. It is recruited to the phagocytic cups through interaction with EhCaBP1. EhAK1 manipulates actin dynamics by multiple mechanisms including phosphorylation of G-actin. Biochemical analysis showed that EhAK1 is a serine/threonine kinase with broad ion specificity and undergoes multiple trans-autophosphorylation. Three autophosphorylation sites were identified by mass spectrometry. Out of these Thr279 appears to be involved in both autophosphorylation as well as substrate phosphorylation. Over expression of the mutant Thr279A inhibited erythrophagocytosis showing dominant negative phenotype. Multiple alignments of different kinases including alpha kinases displayed conserved binding sites that are thought to be important for function of the protein. Mutation studies demonstrated the importance of some of these binding sites in kinase activity. Binding studies with fluorescent-ATP analogs supported our prediction regarding ATP binding site based on sequence alignment. In conclusion, EhAK1 has multiple regulatory features and enrichment of EhAK1 at the site of phagocytosis stimulates trans-autophosphorylation reaction that increases kinase activity resulting in enhanced actin dynamics and phagocytosis. Some of the properties of EhAK1 are similar to that seen in alpha kinases. PMID:26739245

  18. Biophysical studies on calcium and carbohydrate binding to carbohydrate recognition domain of Gal/GalNAc lectin from Entamoeba histolytica: insights into host cell adhesion.

    PubMed

    Yadav, Rupali; Verma, Kuldeep; Chandra, Mintu; Mukherjee, Madhumita; Datta, Sunando

    2016-09-01

    Entamoeba histolytica, an enteric parasite expresses a Gal/GalNAc-specific lectin that contributes to its virulence by establishing adhesion to host cell. In this study, carbohydrate recognition domain of Hgl (EhCRD) was purified and biophysical studies were conducted to understand the thermodynamic basis of its binding to carbohydrate and Ca(++) Here, we show that carbohydrate recognition domain (CRD) of the lectin binds to calcium through DPN motif. To decipher the role of calcium in carbohydrate binding and host cell adhesion, biophysical and cell-based studies were carried out. We demonstrated that the presence of the cation neither change the affinity of the lectin for carbohydrates nor alters its conformation. Mutation of the calcium-binding motif in EhCRD resulted in complete loss of ability to bind calcium but retained its affinity for carbohydrates. Purified EhCRD significantly diminished adhesion of the amebic trophozoites to Chinese Hamster Ovary (CHO) cells as well as triggered red blood cell agglutination. The calcium-binding defective mutant abrogated amebic adhesion to CHO cells similar to the wild-type protein, but it failed to agglutinate RBCs suggesting a differential role of the cation in these two processes. This study provides the first molecular description of the role of calcium in Gal/GalNAc mediated host cell adhesion. PMID:27008865

  19. Autophosphorylation at Thr279 of Entamoeba histolytica atypical kinase EhAK1 is required for activity and regulation of erythrophagocytosis

    PubMed Central

    Mansuri, M Shahid; Babuta, Mrigya; Ali, Mohammad Sabir; Bharadwaj, Ravi; jhingan, Gagan Deep; Gourinath, Samudrala; Bhattacharya, Sudha; Bhattacharya, Alok

    2016-01-01

    Phagocytosis plays a key role in survival and pathogenicity of Entamoeba histolytica. We have recently demonstrated that an atypical kinase EhAK1 is involved in phagocytosis in this parasite. It is recruited to the phagocytic cups through interaction with EhCaBP1. EhAK1 manipulates actin dynamics by multiple mechanisms including phosphorylation of G-actin. Biochemical analysis showed that EhAK1 is a serine/threonine kinase with broad ion specificity and undergoes multiple trans-autophosphorylation. Three autophosphorylation sites were identified by mass spectrometry. Out of these Thr279 appears to be involved in both autophosphorylation as well as substrate phosphorylation. Over expression of the mutant Thr279A inhibited erythrophagocytosis showing dominant negative phenotype. Multiple alignments of different kinases including alpha kinases displayed conserved binding sites that are thought to be important for function of the protein. Mutation studies demonstrated the importance of some of these binding sites in kinase activity. Binding studies with fluorescent-ATP analogs supported our prediction regarding ATP binding site based on sequence alignment. In conclusion, EhAK1 has multiple regulatory features and enrichment of EhAK1 at the site of phagocytosis stimulates trans-autophosphorylation reaction that increases kinase activity resulting in enhanced actin dynamics and phagocytosis. Some of the properties of EhAK1 are similar to that seen in alpha kinases. PMID:26739245

  20. mRNA Decay Proteins Are Targeted to poly(A)+ RNA and dsRNA-Containing Cytoplasmic Foci That Resemble P-Bodies in Entamoeba histolytica

    PubMed Central

    López-Rosas, Itzel; Orozco, Esther; Marchat, Laurence A.; García-Rivera, Guillermina; Guillen, Nancy; Weber, Christian; Carrillo-Tapia, Eduardo; Hernández de la Cruz, Olga; Pérez-Plasencia, Carlos; López-Camarillo, César

    2012-01-01

    In higher eukaryotes, mRNA degradation and RNA-based gene silencing occur in cytoplasmic foci referred to as processing bodies (P-bodies). In protozoan parasites, the presence of P-bodies and their putative role in mRNA decay have yet to be comprehensively addressed. Identification of P-bodies might provide information on how mRNA degradation machineries evolved in lower eukaryotes. Here, we used immunofluorescence and confocal microscopy assays to investigate the cellular localization of mRNA degradation proteins in the human intestinal parasite Entamoeba histolytica and found evidence of the existence of P-bodies. Two mRNA decay factors, namely the EhXRN2 exoribonuclease and the EhDCP2 decapping enzyme, were localized in cytoplasmic foci in a pattern resembling P-body organization. Given that amoebic foci appear to be smaller and less rounded than those described in higher eukaryotes, we have named them “P-body-like structures”. These foci contain additional mRNA degradation factors, including the EhCAF1 deadenylase and the EhAGO2-2 protein involved in RNA interference. Biochemical analysis revealed that EhCAF1 co-immunoprecipitated with EhXRN2 but not with EhDCP2 or EhAGO2-2, thus linking deadenylation to 5′-to-3′ mRNA decay. The number of EhCAF1-containing foci significantly decreased after inhibition of transcription and translation with actinomycin D and cycloheximide, respectively. Furthermore, results of RNA-FISH assays showed that (i) EhCAF1 colocalized with poly(A)+ RNA and (ii) during silencing of the Ehpc4 gene by RNA interference, EhAGO2-2 colocalized with small interfering RNAs in cytoplasmic foci. Our observation of decapping, deadenylation and RNA interference proteins within P-body-like foci suggests that these structures have been conserved after originating in the early evolution of eukaryotic lineages. To the best of our knowledge, this is the first study to report on the localization of mRNA decay proteins within P-body-like structures

  1. Prevalence of Cryptosporidium parvum/hominis, Entamoeba histolytica and Giardia lamblia among Young Children with and without Diarrhea in Dar es Salaam, Tanzania

    PubMed Central

    Tellevik, Marit G.; Moyo, Sabrina J.; Blomberg, Bjørn; Hjøllo, Torunn; Maselle, Samuel Y.; Langeland, Nina; Hanevik, Kurt

    2015-01-01

    Background Although enteroparasites are common causes of diarrheal illness, few studies have been performed among children in Tanzania. This study aimed to investigate the prevalence of Cryptosporidium parvum/hominis, Entamoeba histolytica and Giardia lamblia among young children in Dar es Salaam, Tanzania, and identify risk factors for infection. Methodology/Principal Findings We performed an unmatched case-control study among children < 2 years of age in Dar es Salaam, recruited from August 2010 to July 2011. Detection and identification of protozoans were done by PCR techniques on DNA from stool specimens from 701 cases of children admitted due to diarrhea at the three study hospitals, and 558 controls of children with no history of diarrhea during the last month prior to enrollment. The prevalence of C. parvum/hominis was 10.4% (84.7% C. hominis), and that of G. lamblia 4.6%. E. histolytica was not detected. The prevalence of Cryptosporidium was significantly higher in cases (16.3%) than in controls (3.1%; P < 0.001; OR = 6.2; 95% CI: 3.7–10.4). G. lamblia was significantly more prevalent in controls (6.1%) than in cases (3.4%; P = 0.027; OR = 1.8; 95% CI: 1.1–3.1). Cryptosporidium infection was found more often in HIV-positive (24.2%) than in HIV-negative children (3.9%; P < 0.001; OR = 7.9; 95% CI: 3.1–20.5), and was also associated with rainfall (P < 0.001; OR = 2.41; 95% CI: 1.5–3.8). Among cases, stunted children had significantly higher risk of being infected with Cryptosporidium (P = 0.011; OR = 2.12; 95% CI: 1.2–3.8). G. lamblia infection was more prevalent in the cool season (P = 0.004; OR = 2.2; 95% CI: 1.3–3.8), and more frequent among cases aged > 12 months (P = 0.003; OR = 3.5; 95% CI: 1.5–7.8). Among children aged 7–12 months, those who were breastfed had lower prevalence of G. lamblia infection than those who had been weaned (P = 0.012). Conclusions Cryptosporidium infection is common among young Tanzanian children with diarrhea

  2. Prevalent and Incident HIV Diagnoses among Entamoeba histolytica-Infected Adult Males: A Changing Epidemiology Associated with Sexual Transmission — Taiwan, 2006–2013

    PubMed Central

    Lo, Yi-Chun; Ji, Dar-Der; Hung, Chien-Ching

    2014-01-01

    Background Sexually transmitted Entamoeba histolytica infection (EHI) has been increasingly recognized among men who have sex with men (MSM). We used the National Disease Surveillance Systems (NDSS) to identify prevalent and incident HIV diagnoses among adults with EHI and to determine the associated factors. Methodology The NDSS collect demographic, clinical, and behavioral characteristics of case patients through physician reports and public health interviews. EHI was confirmed by polymerase-chain-reaction assays, histopathology, or serology with documented liver abscess. We linked NDSS databases to identify prevalent and incident HIV diagnoses among noninstitutionalized Taiwanese adults with confirmed EHI during 2006–2013. Cox proportional-hazards analysis was used to determine associated factors. Principal findings Of noninstitutionalized adults with EHI, we identified prevalent HIV diagnosis in 210 (40%) of 524 males and one (1.7%) of 59 females, and incident HIV diagnosis in 71 (23%) of 314 males. MSM accounted for 183 (87%) and 64 (90%) of prevalent and incident HIV diagnoses in males, respectively. From 2006–2009 to 2010–2013, the prevalence of HIV diagnosis increased from 32% to 45% (P = 0.001) while the incidence of HIV diagnosis increased from 5.4 to 11.3 per 100 person-years (P = 0.001) among males with EHI. Incident HIV diagnosis was independently associated with a younger age, residing in metropolitan areas, hospitalization, previous syphilis, and engagement in oral, anal, or oral–anal sex before illness onset. Conclusions/significance Prevalent and incident HIV diagnoses were increasingly identified among adult males in Taiwan, preferentially affecting younger urban MSM. Surveillance and risk-reduction interventions are recommended against the interplay of HIV epidemic and sexually transmitted EHI. PMID:25299178

  3. The NLRP3 Inflammasome Is a Pathogen Sensor for Invasive Entamoeba histolytica via Activation of α5β1 Integrin at the Macrophage-Amebae Intercellular Junction

    PubMed Central

    Mortimer, Leanne; Moreau, France; Cornick, Steve; Chadee, Kris

    2015-01-01

    Entamoeba histolytica (Eh) is an extracellular protozoan parasite of humans that invades the colon to cause life-threatening intestinal and extra-intestinal amebiasis. Colonized Eh is asymptomatic, however, when trophozoites adhere to host cells there is a considerable inflammatory response that is critical in the pathogenesis of amebiasis. The host and/or parasite factors that trigger the inflammatory response to invading Eh are not well understood. We recently identified that Eh adherence to macrophages induces inflammasome activation and in the present study we sought to determine the molecular events upon contact that coordinates this response. Here we report that Eh contact-dependent activation of α5β1 integrin is critical for activation of the NLRP3 inflammasome. Eh-macrophage contact triggered recruitment of α5β1 integrin and NLRP3 into the intercellular junction, where α5β1 integrin underwent activation by an integrin-binding cysteine protease on the parasite surface, termed EhCP5. As a result of its activation, α5β1 integrin induced ATP release into the extracellular space through opening of pannexin-1 channels that signalled through P2X7 receptors to deliver a critical co-stimulatory signal that activated the NLRP3 inflammasome. Both the cysteine protease activity and integrin-binding domain of EhCP5 were required to trigger α5β1 integrin that led to ATP release and NLRP3 inflammasome activation. These findings reveal engagement of α5β1 integrin across the parasite-host junction is a key regulatory step that initiates robust inflammatory responses to Eh. We propose that α5β1 integrin distinguishes Eh direct contact and functions with NLRP3 as pathogenicity sensor for invasive Eh infection. PMID:25955828

  4. Crystal Structure Analysis of Wild Type and Fast Hydrolyzing Mutant of EhRabX3, a Tandem Ras Superfamily GTPase from Entamoeba histolytica.

    PubMed

    Srivastava, Vijay Kumar; Chandra, Mintu; Saito-Nakano, Yumiko; Nozaki, Tomoyoshi; Datta, Sunando

    2016-01-16

    The enteric protozoan parasite, Entamoeba histolytica, is the causative agent of amoebic dysentery, liver abscess and colitis in human. Vesicular trafficking plays a key role in the survival and virulence of the protozoan and is regulated by various Rab GTPases. EhRabX3 is a catalytically inefficient amoebic Rab protein, which is unique among the eukaryotic Ras superfamily by virtue of its tandem domain organization. Here, we report the crystal structures of GDP-bound fast hydrolyzing mutant (V71A/K73Q) and GTP-bound wild type EhRabX3 at 3.1 and 2.8Å resolutions, respectively. Though both G-domains possess "phosphate binding loop containing nucleoside triphosphate hydrolases fold", only the N-terminal domain binds to guanine nucleotide. The relative orientation of the N-terminal domain and C-terminal domain is stabilized by numerous inter-domain interactions. Compared to other Ras superfamily members, both the GTPase domains displayed large deviation in switch II perhaps due to non-conservative substitutions in this region. As a result, entire switch II is restructured and moved away from the nucleotide binding pocket, providing a rationale for the diminished GTPase activity of EhRabX3. The N-terminal GTPase domain possesses unusually large number of cysteine residues. X-ray crystal structure of the fast hydrolyzing mutant of EhRabX3 revealed that C39 and C163 formed an intra-molecular disulfide bond. Subsequent mutational and biochemical studies suggest that C39 and C163 are critical for maintaining the structural integrity and function of EhRabX3. Structure-guided functional investigation of cysteine mutants could provide the physiological implications of the disulfide bond and could allow us to design potential inhibitors for the better treatment of intestinal amebiasis. PMID:26555751

  5. The knockdown of each component of the cysteine proteinase-adhesin complex of Entamoeba histolytica (EhCPADH) affects the expression of the other complex element as well as the in vitro and in vivo virulence.

    PubMed

    Ocádiz-Ruiz, Ramón; Fonseca, Wendy; Linford, Alicia S; Yoshino, Timothy P; Orozco, Esther; Rodríguez, Mario A

    2016-01-01

    Entamoeba histolytica is the protozoan parasite causative of human amoebiasis, disease responsible for 40 000-100 000 deaths annually. The cysteine proteinase-adhesin complex of this parasite (EhCPADH) is a heterodimeric protein formed by a cysteine protease (EhCP112) and an adhesin (EhADH) that plays an important role in the cytopathic mechanism of this parasite. The coding genes for EhCP112 and EhADH are adjacent in the E. histolytica genome, suggesting that their expression may be co-regulated, but this hypothesis has not yet been confirmed. Here, we performed the knockdown of EhCP112 and EhADH using gene-specific short-hairpin RNAs (shRNA), and the effect of these knockdowns on the expression of both complex components as well as on the in vitro and in vivo virulence was analysed. Results showed that the knockdown of one of the EhCPADH components produced a simultaneous downregulation of the other protein. Accordingly, a concomitant reduction in the overall expression of the complex was observed. The downregulation of each component also produced a significant decrease in the in vitro and in vivo virulence of trophozoites. These results demonstrated that the expression of EhCP112 and EhADH is co-regulated and confirmed that the EhCPADH complex plays an important role in E. histolytica virulence. PMID:26521708

  6. Laboratory diagnostic techniques for Entamoeba species.

    PubMed

    Fotedar, R; Stark, D; Beebe, N; Marriott, D; Ellis, J; Harkness, J

    2007-07-01

    The genus Entamoeba contains many species, six of which (Entamoeba histolytica, Entamoeba dispar, Entamoeba moshkovskii, Entamoeba polecki, Entamoeba coli, and Entamoeba hartmanni) reside in the human intestinal lumen. Entamoeba histolytica is the causative agent of amebiasis and is considered a leading parasitic cause of death worldwide in humans. Although recent studies highlight the recovery of E. dispar and E. moshkovskii from patients with gastrointestinal symptoms, there is still no convincing evidence of a causal link between the presence of these two species and the symptoms of the host. New approaches to the identification of E. histolytica are based on detection of E. histolytica-specific antigen and DNA in stool and other clinical samples. Several molecular diagnostic tests, including conventional and real-time PCR, have been developed for the detection and differentiation of E. histolytica, E. dispar, and E. moshkovskii in clinical samples. The purpose of this review is to discuss different methods that exist for the identification of E. histolytica, E. dispar, and E. moshkovskii which are available to the clinical diagnostic laboratory. To address the need for a specific diagnostic test for amebiasis, a substantial amount of work has been carried out over the last decade in different parts of the world. The molecular diagnostic tests are increasingly being used for both clinical and research purposes. In order to minimize undue treatment of individuals infected with other species of Entamoeba such as E. dispar and E. moshkovskii, efforts have been made for specific diagnosis of E. histolytica infection and not to treat based simply on the microscopic examination of Entamoeba species in the stool. The incorporation of many new technologies into the diagnostic laboratory will lead to a better understanding of the public health problem and measures to control the disease. PMID:17630338

  7. Laboratory Diagnostic Techniques for Entamoeba Species

    PubMed Central

    Fotedar, R.; Stark, D.; Beebe, N.; Marriott, D.; Ellis, J.; Harkness, J.

    2007-01-01

    The genus Entamoeba contains many species, six of which (Entamoeba histolytica, Entamoeba dispar, Entamoeba moshkovskii, Entamoeba polecki, Entamoeba coli, and Entamoeba hartmanni) reside in the human intestinal lumen. Entamoeba histolytica is the causative agent of amebiasis and is considered a leading parasitic cause of death worldwide in humans. Although recent studies highlight the recovery of E. dispar and E. moshkovskii from patients with gastrointestinal symptoms, there is still no convincing evidence of a causal link between the presence of these two species and the symptoms of the host. New approaches to the identification of E. histolytica are based on detection of E. histolytica-specific antigen and DNA in stool and other clinical samples. Several molecular diagnostic tests, including conventional and real-time PCR, have been developed for the detection and differentiation of E. histolytica, E. dispar, and E. moshkovskii in clinical samples. The purpose of this review is to discuss different methods that exist for the identification of E. histolytica, E. dispar, and E. moshkovskii which are available to the clinical diagnostic laboratory. To address the need for a specific diagnostic test for amebiasis, a substantial amount of work has been carried out over the last decade in different parts of the world. The molecular diagnostic tests are increasingly being used for both clinical and research purposes. In order to minimize undue treatment of individuals infected with other species of Entamoeba such as E. dispar and E. moshkovskii, efforts have been made for specific diagnosis of E. histolytica infection and not to treat based simply on the microscopic examination of Entamoeba species in the stool. The incorporation of many new technologies into the diagnostic laboratory will lead to a better understanding of the public health problem and measures to control the disease. PMID:17630338

  8. Detection of excretory Entamoeba histolytica DNA in the urine, and detection of E. histolytica DNA and lectin antigen in the liver abscess pus for the diagnosis of amoebic liver abscess

    PubMed Central

    Parija, Subhash C; Khairnar, Krishna

    2007-01-01

    Background Amoebic liver abscess (ALA) and pyogenic liver abscesses (PLA) appear identical by ultrasound and other imaging techniques. Collection of blood or liver abscess pus for diagnosis of liver abscesses is an invasive procedure, and the procedure requires technical expertise and disposable syringes. Collection of urine is a noninvasive procedure. Therefore, there has been much interest shown towards the use of urine as an alternative clinical specimen for the diagnosis of some parasitic infections. Here, we report for the first time the detection of E. histolytica DNA excreted in the urine for diagnosis of the cases of ALA. Results E. histolytica DNA was detected in liver abscess pus specimen of 80.4% of ALA patients by a nested multiplex polymerase chain reaction (PCR) targeting 16S-like r RNA gene. The nested PCR detected E. histolytica DNA in all 37 (100%) liver abscess pus specimens collected prior to metronidazole treatment, but were detected in only 53 of 75 (70.6%) pus specimens collected after therapy with metronidazole. Similarly, the PCR detected E. histolytica DNA in 21 of 53 (39.6%) urine specimens of ALA patients. The test detected E. histolytica DNA in only 4 of 23 (17.4%) urine specimens collected prior to metronidazole treatment, but were detected in 17 of 30 (56.7%) urine specimens collected after treatment with metronidazole. The enzyme-linked immunosorbent assay (ELISA) for the detection of lectin E. histolytica antigen in the liver abscess pus showed a sensitivity of 50% and the indirect haemagglutination (IHA) test for detection of amoebic antibodies in the serum showed a sensitivity of 76.8% for the diagnosis of the ALA. Conclusion The present study for the first time shows that the kidney barrier in ALA patients is permeable to E. histolytica DNA molecule resulting in excretion of E. histolytica DNA in urine which can be detected by PCR. The study also shows that the PCR for detection of E. histolytica DNA in urine of patients with ALA

  9. Heterogeneity of the serine synthetic pathway in Entamoeba species.

    PubMed

    Chiba, Yoko; Makiuchi, Takashi; Jeelani, Ghulam; Nozaki, Tomoyoshi

    2016-06-01

    Phosphoserine phosphatase (PSP) catalyzes the third step of the phosphorylated serine biosynthetic pathway, and occurred multiple times in evolution, while enzymes catalyzing the first and second steps in the pathway have single respective origins. In the present study, we examined the existence of PSP among genus Entamoeba including a human enteric parasite, Entamoeba histolytica. E. histolytica as well as majority of Entamoeba species have the first and second enzymes, but lacks PSP. In contrast, a reptilian enteric parasite, Entamoeba invadens possesses canonical PSP. Thus, there are variations in the existence of the serine biosynthetic ability among Entamoeba species. PMID:27268730

  10. Two Atypical l-Cysteine-regulated NADPH-dependent Oxidoreductases Involved in Redox Maintenance, l-Cystine and Iron Reduction, and Metronidazole Activation in the Enteric Protozoan Entamoeba histolytica*

    PubMed Central

    Jeelani, Ghulam; Husain, Afzal; Sato, Dan; Ali, Vahab; Suematsu, Makoto; Soga, Tomoyoshi; Nozaki, Tomoyoshi

    2010-01-01

    We discovered novel catalytic activities of two atypical NADPH-dependent oxidoreductases (EhNO1/2) from the enteric protozoan parasite Entamoeba histolytica. EhNO1/2 were previously annotated as the small subunit of glutamate synthase (glutamine:2-oxoglutarate amidotransferase) based on similarity to authentic bacterial homologs. As E. histolytica lacks the large subunit of glutamate synthase, EhNO1/2 were presumed to play an unknown role other than glutamine/glutamate conversion. Transcriptomic and quantitative reverse PCR analyses revealed that supplementation or deprivation of extracellular l-cysteine caused dramatic up- or down-regulation, respectively, of EhNO2, but not EhNO1 expression. Biochemical analysis showed that these FAD- and 2[4Fe-4S]-containing enzymes do not act as glutamate synthases, a conclusion which was supported by phylogenetic analyses. Rather, they catalyze the NADPH-dependent reduction of oxygen to hydrogen peroxide and l-cystine to l-cysteine and also function as ferric and ferredoxin-NADP+ reductases. EhNO1/2 showed notable differences in substrate specificity and catalytic efficiency; EhNO1 had lower Km and higher kcat/Km values for ferric ion and ferredoxin than EhNO2, whereas EhNO2 preferred l-cystine as a substrate. In accordance with these properties, only EhNO1 was observed to physically interact with intrinsic ferredoxin. Interestingly, EhNO1/2 also reduced metronidazole, and E. histolytica transformants overexpressing either of these proteins were more sensitive to metronidazole, suggesting that EhNO1/2 are targets of this anti-amebic drug. To date, this is the first report to demonstrate that small subunit-like proteins of glutamate synthase could play an important role in redox maintenance, l-cysteine/l-cystine homeostasis, iron reduction, and the activation of metronidazole. PMID:20592025

  11. Increases in Entamoeba histolytica Antibody-Positive Rates in Human Immunodeficiency Virus-Infected and Noninfected Patients in Japan: A 10-Year Hospital-Based Study of 3,514 Patients.

    PubMed

    Yanagawa, Yasuaki; Nagata, Naoyoshi; Watanabe, Koji; Tsukada, Kunihisa; Teruya, Katsuji; Kikuchi, Yoshimi; Gatanaga, Hiroyuki; Akiyama, Junichi; Uemura, Naomi; Oka, Shinichi

    2016-09-01

    Serological evidence of the epidemiological trends in Entamoeba histolytica infection is scarce, especially in nonendemic countries. We aimed to determine the antibody-positive rates over a 10-year period, and compare the trends between human immunodeficiency virus (HIV)-infected and -noninfected patients. We reviewed 3,514 patients who underwent antibody testing during the study periods, which were divided into five annual categories: 2004-2005, 2006-2007, 2008-2009, 2010-2011, and 2012-2013. Anti-E. histolytica antibody was assessed by indirect immunofluorescence assay. The antibody-positive rate increased yearly from 2004-2005 to 2012-2013 (P < 0.001), although there was no increase in the annual number of antibody tests. This trend was seen among males (18.6-28.3%; P < 0.01), females (5.4-28.2%; P < 0.01), HIV-infected patients (18.4-26.9%; P < 0.001), and non-HIV-infected patients (14.6-36.8%; P < 0.001), and HIV-infected men who have sex with men (19.4-29.1%; P < 0.001). Among antibody-positive patients, there was a significant increase in the proportion of patients with high (≥ 1,600) titers (0.7-12.9%; P < 0.001), whereas this trend was not seen in patients with low (100) or intermediate (200-800) titers (P = 0.282 and 0.409, respectively). This large hospital-based study demonstrated that positive anti-E. histolytica antibody rates increased over 10 years, even though the annual number of antibody tests remained constant. Moreover, this trend was identified in non-high-risk patients (females and non-HIV-infected patients) as well as in high-risk patients. The proportion of patients with high antibody titers significantly increased among the antibody-positive patients. PMID:27296390

  12. Mitosomes in Trophozoites and Cysts of the Reptilian Parasite Entamoeba invadens ▿

    PubMed Central

    Siegesmund, Maria A.; Hehl, Adrian B.; van der Giezen, Mark

    2011-01-01

    Heat shock protein genes led to the discovery of mitosomes in Entamoeba histolytica, but mitosomes have not been described for any other Entamoeba species, nor have they been identified in the cyst stage. Here, we show that the distantly related reptilian pathogen Entamoeba invadens contains mitosomes, in both trophozoites and cysts, suggesting all Entamoeba species contain these organelles. PMID:21965513

  13. Mitosomes in trophozoites and cysts of the reptilian parasite Entamoeba invadens.

    PubMed

    Siegesmund, Maria A; Hehl, Adrian B; van der Giezen, Mark

    2011-11-01

    Heat shock protein genes led to the discovery of mitosomes in Entamoeba histolytica, but mitosomes have not been described for any other Entamoeba species, nor have they been identified in the cyst stage. Here, we show that the distantly related reptilian pathogen Entamoeba invadens contains mitosomes, in both trophozoites and cysts, suggesting all Entamoeba species contain these organelles. PMID:21965513

  14. Diagnostic performances of antigen detection compared to conventional and nucleic acid detection of Entamoeba histolytica in a non-endemic setting.

    PubMed

    Calderaro, Adriana; Piergianni, Maddalena; Piccolo, Giovanna; Rossi, Sabina; Montecchini, Sara; Buttrini, Mirko; Arcangeletti, Maria Cristina; Medici, Maria Cristina; Chezzi, Carlo; De Conto, Flora

    2016-04-01

    This study evaluated the immunochromatographic (IC) assay "TECHLAB(®) E. HISTOLYTICA QUIK CHEK™" analysing 36 faecal samples and 7 cultured strains. This assay was compared to the methods performed in our laboratory for the diagnosis of amoebiasis. The IC assay revealed a detection limit of 103 trophozoites/g faeces and no cross-reactivity with other parasites and failed to detect E. histolytica antigen in frozen faeces. In our laboratory located in a non-endemic setting this assay could not replace the methods currently used for the diagnosis of amoebiasis. PMID:27196557

  15. Increases in Entamoeba histolytica Antibody–Positive Rates in Human Immunodeficiency Virus–Infected and Noninfected Patients in Japan: A 10-Year Hospital-Based Study of 3,514 Patients

    PubMed Central

    Yanagawa, Yasuaki; Nagata, Naoyoshi; Watanabe, Koji; Tsukada, Kunihisa; Teruya, Katsuji; Kikuchi, Yoshimi; Gatanaga, Hiroyuki; Akiyama, Junichi; Uemura, Naomi; Oka, Shinichi

    2016-01-01

    Serological evidence of the epidemiological trends in Entamoeba histolytica infection is scarce, especially in nonendemic countries. We aimed to determine the antibody-positive rates over a 10-year period, and compare the trends between human immunodeficiency virus (HIV)–infected and –noninfected patients. We reviewed 3,514 patients who underwent antibody testing during the study periods, which were divided into five annual categories: 2004–2005, 2006–2007, 2008–2009, 2010–2011, and 2012–2013. Anti-E. histolytica antibody was assessed by indirect immunofluorescence assay. The antibody-positive rate increased yearly from 2004–2005 to 2012–2013 (P < 0.001), although there was no increase in the annual number of antibody tests. This trend was seen among males (18.6–28.3%; P < 0.01), females (5.4–28.2%; P < 0.01), HIV-infected patients (18.4–26.9%; P < 0.001), and non-HIV-infected patients (14.6–36.8%; P < 0.001), and HIV-infected men who have sex with men (19.4–29.1%; P < 0.001). Among antibody-positive patients, there was a significant increase in the proportion of patients with high (≥ 1,600) titers (0.7–12.9%; P < 0.001), whereas this trend was not seen in patients with low (100) or intermediate (200–800) titers (P = 0.282 and 0.409, respectively). This large hospital-based study demonstrated that positive anti-E. histolytica antibody rates increased over 10 years, even though the annual number of antibody tests remained constant. Moreover, this trend was identified in non-high-risk patients (females and non-HIV-infected patients) as well as in high-risk patients. The proportion of patients with high antibody titers significantly increased among the antibody-positive patients. PMID:27296390

  16. Detection and Identification of Entamoeba Species in Stool Samples by a Reverse Line Hybridization Assay

    PubMed Central

    Verweij, Jaco J.; Laeijendecker, Daphne; Brienen, Eric A. T.; van Lieshout, Lisette; Polderman, Anton M.

    2003-01-01

    Classically, detection of Entamoeba histolytica is performed by microscopic examination for characteristic cysts and/or trophozoites in fecal preparations. Differentiation of E. histolytica cysts and those of nonpathogenic amoebic species is made on the basis of the appearance and the size of the cysts. However, by classical means objective tools for confirmation and quality control do not exist. Therefore, a reverse line blot hybridization assay was developed to detect a variety of Entamoeba species and genetic variants known to infect humans. The assay was performed after amplification with general Entamoeba-specific primers. The assay could identify four genetic variants of Entamoeba polecki-like cysts as well as E. histolytica, Entamoeba dispar, Entamoeba hartmanni, Entamoeba moshkovskii and Entamoeba coli and even mixed infections in a range of controls and fecal samples. This technique can be used as an additional standard for diagnosis, epidemiology, and quality control for amoebic infections. PMID:14605136

  17. Detection and identification of entamoeba species in stool samples by a reverse line hybridization assay.

    PubMed

    Verweij, Jaco J; Laeijendecker, Daphne; Brienen, Eric A T; van Lieshout, Lisette; Polderman, Anton M

    2003-11-01

    Classically, detection of Entamoeba histolytica is performed by microscopic examination for characteristic cysts and/or trophozoites in fecal preparations. Differentiation of E. histolytica cysts and those of nonpathogenic amoebic species is made on the basis of the appearance and the size of the cysts. However, by classical means objective tools for confirmation and quality control do not exist. Therefore, a reverse line blot hybridization assay was developed to detect a variety of Entamoeba species and genetic variants known to infect humans. The assay was performed after amplification with general Entamoeba-specific primers. The assay could identify four genetic variants of Entamoeba polecki-like cysts as well as E. histolytica, Entamoeba dispar, Entamoeba hartmanni, Entamoeba moshkovskii and Entamoeba coli and even mixed infections in a range of controls and fecal samples. This technique can be used as an additional standard for diagnosis, epidemiology, and quality control for amoebic infections. PMID:14605136

  18. Insights into endosomal maturation of human holo-transferrin in the enteric parasite Entamoeba histolytica: essential roles of Rab7A and Rab5 in biogenesis of giant early endocytic vacuoles.

    PubMed

    Verma, Kuldeep; Saito-Nakano, Yumiko; Nozaki, Tomoyoshi; Datta, Sunando

    2015-12-01

    The pathogenic amoeba Entamoeba histolytica is one of the causative agents of health hazards in tropical countries. It causes amoebic dysentery, colitis and liver abscesses in human. Iron is one of the essential nutritional resources for survival and chronic infection caused by the amoeba. The parasite has developed multiple ways to import, sequester and utilize iron from various iron-binding proteins from its host. In spite of its central role in pathogenesis, the mechanism of iron uptake by the parasite is largely unknown. Here, we carried out a systematic study to understand the role of some of the amoebic homologues of mammalian endocytic Rab GTPases (Rab5 and Rab21, Rab7A and Rab7B) in intracellular transport of human holo-transferrin by the parasite. Flow cytometry and quantitative microscopic image analysis revealed that Rab5 and Rab7A are required for the biogenesis of amoebic giant endocytic vacuoles (GEVs) and regulate the early phase of intracellular trafficking of transferrin. Rab7B is involved in the late phase, leading to the degradation of transferrin in the amoebic lysosome-like compartments. Using time-lapse fluorescence imaging in fixed trophozoites, we determined the kinetics of the vesicular transport of transferrin through Rab5-, Rab7A- and Rab7B-positive compartments. The involvement of Rab7A in the early phase of endocytosis by the parasite marks a significant divergence from its host in terms of spatiotemporal regulation by the Rab GTPases. PMID:26096601

  19. Molecular identification of Entamoeba species in savanna woodland chimpanzees (Pan troglodytes schweinfurthii).

    PubMed

    Jirků-Pomajbíková, Kateřina; Čepička, Ivan; Kalousová, Barbora; Jirků, Milan; Stewart, Fiona; Levecke, Bruno; Modrý, David; Piel, Alex K; Petrželková, Klára J

    2016-05-01

    To address the molecular diversity and occurrence of pathogenic species of the genus Entamoeba spp. in wild non-human primates (NHP) we conducted molecular-phylogenetic analyses on Entamoeba from wild chimpanzees living in the Issa Valley, Tanzania. We compared the sensitivity of molecular [using a genus-specific polymerase chain reaction (PCR)] and coproscopic detection (merthiolate-iodine-formaldehyde concentration) of Entamoeba spp. We identified Entamoeba spp. in 72 chimpanzee fecal samples (79%) subjected to species-specific PCRs for six Entamoeba species/groups (Entamoeba histolytica, Entamoeba nuttalli, Entamoeba dispar, Entamoeba moshkovskii, Entamoeba coli and Entamoeba polecki ST2). We recorded three Entamoeba species: E. coli (47%), E. dispar (16%), Entamoeba hartmanni (51%). Coproscopically, we could only distinguish the cysts of complex E. histolytica/dispar/moshkovskii/nuttalli and E. coli. Molecular prevalence of entamoebas was higher than the prevalence based on the coproscopic examination. Our molecular phylogenies showed that sequences of E. dispar and E. coli from Issa chimpanzees are closely related to sequences from humans and other NHP from GenBank. The results showed that wild chimpanzees harbour Entamoeba species similar to those occurring in humans; however, no pathogenic species were detected. Molecular-phylogenetic methods are critical to improve diagnostics of entamoebas in wild NHP and for determining an accurate prevalence of Entamoeba species. PMID:26935395

  20. Molecular identification of Entamoeba spp. in captive nonhuman primates.

    PubMed

    Levecke, B; Dreesen, Leentje; Dorny, Pierre; Verweij, Jaco J; Vercammen, Francis; Casaert, Stijn; Vercruysse, Jozef; Geldhof, Peter

    2010-08-01

    This study describes the molecular identification of 520 Entamoeba-positive fecal samples from a large and diverse population of captive nonhuman primates (NHP). The results revealed the presence of Entamoeba histolytica (NHP variant only), E. dispar, E. moshkovskii, E. hartmanni, E. coli, and E. polecki-like organisms. PMID:20573870

  1. Is real-time PCR-based diagnosis similar in performance to routine parasitological examination for the identification of Giardia intestinalis, Cryptosporidium parvum/Cryptosporidium hominis and Entamoeba histolytica from stool samples? Evaluation of a new commercial multiplex PCR assay and literature review.

    PubMed

    Laude, A; Valot, S; Desoubeaux, G; Argy, N; Nourrisson, C; Pomares, C; Machouart, M; Le Govic, Y; Dalle, F; Botterel, F; Bourgeois, N; Cateau, E; Leterrier, M; Le Pape, P; Morio, F

    2016-02-01

    Microscopy is the reference standard for routine laboratory diagnosis in faecal parasitology but there is growing interest in alternative methods to overcome the limitations of microscopic examination, which is time-consuming and highly dependent on an operator's skills and expertise. Compared with microscopy, DNA detection by PCR is simple and can offer a better turnaround time. However, PCR performances remain difficult to assess as most studies have been conducted on a limited number of positive clinical samples and used in-house PCR methods. Our aim was to evaluate a new multiplex PCR assay (G-DiaParaTrio; Diagenode Diagnostics), targeting Giardia intestinalis, Cryptosporidium parvum/Cryptosporidium hominis and Entamoeba histolytica. To minimize the turnaround time, PCR was coupled with automated DNA extraction (QiaSymphony; Qiagen). The PCR assay was evaluated using a reference panel of 185 samples established by routine microscopic examination using a standardized protocol including Ziehl-Neelsen staining and adhesin detection by ELISA (E. histolytica II; TechLab). This panel, collected from 12 French parasitology laboratories, included 135 positive samples for G. intestinalis (n = 38), C. parvum/C. hominis (n = 26), E. histolytica (n = 5), 21 other gastrointestinal parasites, together with 50 negative samples. In all, the G-DiaParaTrio multiplex PCR assay identified 38 G. intestinalis, 25 C. parvum/C. hominis and five E. histolytica leading to sensitivity/specificity of 92%/100%, 96%/100% and 100%/100% for G. intestinalis, C. parvum/C. hominis and E. histolytica, respectively. This new multiplex PCR assay offers fast and reliable results, similar to microscopy-driven diagnosis for the detection of these gastrointestinal protozoa, allowing its implementation in routine clinical practice. PMID:26548509

  2. Entamoeba moshkovskii and Entamoeba dispar-associated infections in pondicherry, India.

    PubMed

    Parija, Subhash Chandra; Khairnar, Krishna

    2005-09-01

    The prevalence of Laredo strain--Entamoeba moshkovskii--and non-pathogenic E. dispar in patients attending the Jawaharlal Institute of Postgraduate Medical Education and Research hospital, Pondicherry, India, is reported here. E. moshkovskii is reported for the first time in India. The species are morphologically indistinguishable from pathogenic E. histolytica. Of 746 stool samples screened, 68 showing cyst or trophozoite stage of E. histolytica, E. dispar, or E. moshkovskii were subjected to small subunit (SSU) rRNA gene-based polymerase chain reaction, which revealed a higher prevalence of E. dispar (8.8%) and E. moshkovskii (2.2%) compared to E. histolytica (1.7%) in patients. Only 19% of the 68 stool samples, resembling E. histolytica by microscopy, were actually E. histolytica, implying that 81% of suspected infections were misdiagnosed and would have been treated unnecessarily with anti-amoebic drugs. PMID:16262027

  3. Molecular Identification of Entamoeba spp. in Captive Nonhuman Primates ▿ †

    PubMed Central

    Levecke, B.; Dreesen, Leentje; Dorny, Pierre; Verweij, Jaco J.; Vercammen, Francis; Casaert, Stijn; Vercruysse, Jozef; Geldhof, Peter

    2010-01-01

    This study describes the molecular identification of 520 Entamoeba-positive fecal samples from a large and diverse population of captive nonhuman primates (NHP). The results revealed the presence of Entamoeba histolytica (NHP variant only), E. dispar, E. moshkovskii, E. hartmanni, E. coli, and E. polecki-like organisms. PMID:20573870

  4. Heat Shock Protein 90 regulates encystation in Entamoeba

    PubMed Central

    Singh, Meetali; Sharma, Shalini; Bhattacharya, Alok; Tatu, Utpal

    2015-01-01

    Enteric protozoan Entamoeba histolytica is a major cause of debilitating diarrheal infection worldwide with high morbidity and mortality. Even though the clinical burden of this parasite is very high, this infection is categorized as a neglected disease. Parasite is transmitted through feco-oral route and exhibit two distinct stages namely – trophozoites and cysts. Mechanism and regulation of encystation is not clearly understood. Previous studies have established the role of Heat shock protein 90 (Hsp90) in regulating stage transition in various protozoan parasites like Giardia, Plasmodium, Leishmania, and Toxoplasma. Our study for the first time reports that Hsp90 plays a crucial role in life cycle of Entamoeba as well. We identify Hsp90 to be a negative regulator of encystation in Entamoeba. We also show that Hsp90 inhibition interferes with the process of phagocytosis in Entamoeba. Overall, we show that Hsp90 plays an important role in virulence and transmission of Entamoeba. PMID:26528271

  5. QSAR studies of benzofuran/benzothiophene biphenyl derivatives as inhibitors of PTPase-1B

    PubMed Central

    Kaushik, D.; Kumar, R.; Saxena, A. K.

    2010-01-01

    Objectives: Insulin resistance is associated with a defect in protein tyrosine phosphorylation in the insulin signal transduction cascade. The PTPase enzyme dephosphorylates the active form of the insulin receptor and thus attenuates its tyrosine kinase activity, therefore, the need for a potent PTPase inhibitor exists, with the intention of which the QSAR was performed. Materials and Methods: Quantitative structure-activity relationship (QSAR) has been established on a series of 106 compounds considering 27 variables, for novel biphenyl analogs, using the SYSTAT (Version 7.0) software, for their protein tyrosine phosphatase (PTPase-1B) inhibitor activity, in order to understand the essential structural requirement for binding with the receptor. Results: Among several regression models, one per series was selected on the basis of a high correlation coefficient (r, 0.86), least standard deviation (s, 0.234), and a high value of significance for the maximum number of subjects (n, 101). Conclusions: The influence of the different physicochemical parameters of the substituents in various positions has been discussed by generating the best QSAR model using multiple regression analysis, and the information thus obtained from the present study can be used to design and predict more potent molecules as PTPase-1B inhibitors, prior to their synthesis. PMID:21814427

  6. Molecular Epidemiology of Entamoeba spp.: Evidence of a Bottleneck (Demographic Sweep) and Transcontinental Spread of Diploid Parasites

    PubMed Central

    Ghosh, Sudip; Frisardi, Marta; Ramirez-Avila, Lynn; Descoteaux, Steven; Sturm-Ramirez, Katherine; Newton-Sanchez, Oscar Alberto; Santos-Preciado, Jose Ignacio; Ganguly, Chaiti; Lohia, Anuradha; Reed, Sharon; Samuelson, John

    2000-01-01

    Entamoeba histolytica causes amebic colitis and liver abscess in developing countries such as Mexico and India. Entamoeba dispar is morphologically identical but is not associated with disease. Here we determined the ploidy of E. histolytica and developed PCR-based methods for distinguishing field isolates of E. histolytica or E. dispar. Fluorescence in situ hybridization showed that E. histolytica trophozoites are diploid for five “single-copy” probes tested. Intergenic sequences between superoxide dismutase and actin 3 genes of clinical isolates of E. histolytica from the New and Old Worlds were identical, as were those of E. dispar. These results suggest a bottleneck or demographic sweep in entamoebae which infect humans. In contrast, E. histolytica and E. dispar genes encoding repeat antigens on the surface of trophozoites (Ser-rich protein) or encysting parasites (chitinase) were highly polymorphic. chitinase alleles suggested that the early axenized strains of E. histolytica, HM-1 from Mexico City, Mexico, and NIH-200 from Calcutta, India, are still present and that similar E. dispar parasites can be identified in both the New and Old Worlds. Ser-rich protein alleles, which suggested the presence of the HM-1 strain in Mexico City, included some E. histolytica genes that predicted Ser-rich proteins with very few repeats. These results, which suggest diversifying selection at chitinase and Ser-rich protein loci, demonstrate the usefulness of these alleles for distinguishing clinical isolates of E. histolytica and E. dispar. PMID:11015408

  7. Molecular Epidemiology of Entamoeba: First Description of Entamoeba moshkovskii in a Rural Area from Central Colombia

    PubMed Central

    León, Cielo M.; Fonseca, Jairo; Reyes, Patricia; Moncada, Ligia; Olivera, Mario J.

    2015-01-01

    Background Entamoeba histolytica, E. dispar and E. moshkovskii are the most frequent species described in human infection where E. histolytica is the only true pathogen. The epidemiology of this infection is complex due to the absence of a routine exam that allows a correct discrimination of the Entamoeba species complex. Therefore, molecular methods appear as the unique epidemiological tool to accomplish the species discrimination. Herein, we conducted a cross-sectional study to determine the frequency of Entamoeba species infections in a group of asymptomatic individuals from a rural area in central Colombia. Methodology/Principal Findings A total of 181 fecal samples from asymptomatic children under 16 years old from the hamlet La Vírgen, Cundinamarca (Colombia) that voluntarily accepted to participate in the study were collected. The fecal samples were examined by light microscopy and DNA-extracted, subsequently submitted to molecular discrimination of E. dispar/E. histolytica/E. moshkovskii infection based on a multiplex PCR assay targeting the 18S rRNA fragment. To confirm the species description, twenty samples were randomly submitted to DNA sequencing of the aforementioned fragment. By direct microscopic examination, frequency of the complex E. histolytica/E. dispar/E. moshkovskii was 18.8% (34/181). PCR showed a frequency of 49.1% (89/181), discriminated as 23.2% (42/181) that were positive for E. dispar, 25.4% (46/181) for E. moshkovskii and 0.55% (1/ 181) for E. histolytica. Also, mixed infections were detected between E. dispar and E. moshkovskii at 4.42% (8/181) of the samples. Molecular barcoding confirmed the diagnosis depicted by the multiplex PCR assay. Conclusions/Significance This is the first description of E. moshkovskii in Colombia and the second report in South-America to our knowledge. Our results suggest the need to unravel the true epidemiology of Entamoeba infections around the world, including the real pathogenic role that E

  8. LUMINEX®: a new technology for the simultaneous identification of five Entamoeba spp. commonly found in human stools

    PubMed Central

    2013-01-01

    Background Six species of the genus Entamoeba, i.e., E. histolytica, E. dispar, E. moshkovskii, E. polecki, E. coli, and E. hartmanii can be found in human stools. Among these, only E. histolytica is considered to be pathogenic, causing intestinal and extra-intestinal disease, but it is morphologically identical to E. dispar and E. moshkovskii. In general, E. polecki, E. coli, and E. hartmanii can be differentiated morphologically from E. histolytica, but some of their diagnostic morphologic features may overlap creating issues for the differential diagnosis. Moreover, the previous inability to differentiate among Entamoeba species has limited epidemiologic information on E histolytica. The objective of this study was to develop a rapid, high-throughput screening method using Luminex technique for the simultaneous detection and differentiation of Entamoeba species. Methods PCR amplification was performed with biotinylated Entamoeba sp 18S rRNA gene primers, designed to amplify a fragment ranging from 382 to 429 bp of the Entamoeba spp studied. Regions of this fragment that could differentiate among E. histolytica, E. moshkovskii, E. dispar, E. hartmanii and E. coli were selected to design hybridization probes to link to Luminex beads. The assay was standardized with cloned DNA samples of each species and evaluated with 24 DNA extracts from samples obtained from individuals diagnosed with these amebas in their stools. Results Using this approach we were able to correctly identify E. histoltyica, E. dispar, E hartmanni, E. coli and E. moshkovskii in all specimens studied. From twenty four samples tested by microscopy, PCR/DNA Sequencing and real-time PCR, 100% agreed with PCR-Luminex assay for identification of E. dispar, E. moshkovskii, E. hartmanni, E. histolytica, and E. coli. Conclusion These results show that this method could be used in the diagnostic detection of Entamoeba spp in fecal samples. This diagnostic test was useful to clearly distinguish E

  9. Histopathological and immunohistochemical study of the hepatic lesions experimentally induced by Entamoeba dispar

    PubMed Central

    Costa, C.A.X.; Brito, K.N.O.; Gomes, M.A.; Caliari, M.V.

    2010-01-01

    The sequence of hepatic necrotic-inflammatory events produced by Entamoeba dispar are originally described in this work. For the first time the experimental lesions produced by E. dispar were described in details, as well as the distribution of the trophozoites detected by the immunohistochemistry. Animals experimentally infected with E. dispar presented necrosis, thrombosis and chronic granulomatous inflammation. Immunoreactive products derived from trofozoites were observed close or associated with trophozoites, epithelioid cells, leucocytes and hepatocytes. Few are the articles on the literature about virulence of E. dispar, which is approximately 9 times more frequent than to Entamoeba histolytica (E. histolytica). Variation in the virulence is therefore expected and signalizing the need of the continuity of studies with E. dispar strains from different places in the world. Taking into account that E. dispar is a closely related species to E. histolytica, these studies could determine new elements involved with E. histolytica pathogenesis, helping us to better understand the disease. PMID:20819776

  10. A PCR-RFLP method for the simultaneous differentiation of three Entamoeba species.

    PubMed

    Fontecha, Gustavo A; García, Kimberly; Rueda, María Mercedes; Sosa-Ochoa, Wilfredo; Sánchez, Ana Lourdes; Leiva, Byron

    2015-01-01

    Amoebiasis caused by Entamoeba histolytica continues to be one of the most common parasitic diseases in the developing world. Despite its relevance, due to the lack of accurate diagnostic methods, the true clinical and public health importance of this parasite remains uncertain. The aim of this study was to develop a new diagnostic tool to differentiate E.histolytica from the morphologically undistinguishable E.dispar and E.moshkovskii. We developed a specific, fast and simple PCR-RFLP method that was able to accurately differentiate experimentally-obtained restriction patterns from the three Entamoeba species. This new method could prove useful for clinical and epidemiological purposes. PMID:25680279

  11. Entamoeba moshkovskii Infections in Children in Bangladesh

    PubMed Central

    Ali, Ibne Karin M.; Hossain, Mohammad Bakhtiar; Roy, Shantanu; Ayeh-Kumi, Patrick F.; Petri, William A.; Haque, Rashidul

    2003-01-01

    Entamoeba moshkovskii cysts are morphologically indistinguishable from those of the disease-causing species E. histolytica and the nonpathogenic E. dispar. Although sporadic cases of human infection with E. moshkovskii have been reported, the organism is considered primarily a free-living ameba. No simple molecular detection tool is available for diagnosing E. moshkovskii infections. We used polymerase chain reaction (PCR) to detect E. moshkovskii directly in stool. We tested 109 stool specimens from preschool children in Bangladesh by PCR; 17 were positive for E. histolytica (15.6%) and 39 were positive for E. dispar (35.8%). In addition, we found that 23 (21.1%) were positive for E. moshkovskii infection, and 17 (73.9%) of these also carried E. histolytica or E. dispar. The high association of E. moshkovskii with E. histolytica and E. dispar may have obscured its identification in previous studies. The high prevalence found in this study suggests that humans may be a true host for this ameba. PMID:12737742

  12. Identification of Differentially Expressed Genes in Virulent and Nonvirulent Entamoeba Species: Potential Implications for Amebic Pathogenesis †

    PubMed Central

    MacFarlane, Ryan C.; Singh, Upinder

    2006-01-01

    Entamoeba histolytica is a protozoan parasite that causes colitis and liver abscesses. Several Entamoeba species and strains with differing levels of virulence have been identified. E. histolytica HM-1:IMSS is a virulent strain, E. histolytica Rahman is a nonvirulent strain, and Entamoeba dispar is a nonvirulent species. We used an E. histolytica DNA microarray consisting of 2,110 genes to assess the transcriptional differences between these species/strains with the goal of identifying genes whose expression correlated with a virulence phenotype. We found 415 genes expressed at lower levels in E. dispar and 32 genes with lower expression in E. histolytica Rahman than in E. histolytica HM-1:IMSS. Overall, 29 genes had decreased expression in both the nonvirulent species/strains than the virulent E. histolytica HM-1:IMSS. Interestingly, a number of genes with potential roles in stress response and virulence had decreased expression in either one or both nonvirulent Entamoeba species/strains. These included genes encoding Fe hydrogenase (9.m00419), peroxiredoxin (176.m00112), type A flavoprotein (6.m00467), lysozyme (6.m00454), sphingomyelinase C (29.m00231), and a hypothetical protein with homology to both a Plasmodium sporozoite threonine-asparagine-rich protein (STARP) and a streptococcal hemagglutinin (238.m00054). The function of these genes in Entamoeba and their specific roles in parasite virulence need to be determined. We also found that a number of the non-long-terminal-repeat retrotransposons (EhLINEs and EhSINEs), which have been shown to modulate gene expression and genomic evolution, had lower expression in the nonvirulent species/strains than in E. histolytica HM-1:IMSS. Our results, identifying expression profiles and patterns indicative of a virulence phenotype, may be useful in characterizing the transcriptional framework of virulence. PMID:16368989

  13. Prevalence of Entamoeba species in captive primates in zoological gardens in the UK

    PubMed Central

    Regan, Carl S.; Yon, Lisa; Hossain, Maqsud

    2014-01-01

    The aim of this study was to determine the prevalence of amoebic infection in non-human primates (NHPs) from six Zoological gardens in the United Kingdom. Initially, 126 faecal samples were collected from 37 individually identified NHPs at Twycross Zoo, UK, and were subjected to microscopic examination. A subsequent, nationwide experiment included 350 faecal samples from 89 individually identified NHPs and 73 unidentified NHPs from a number of UK captive wildlife facilities: Twycross Zoo (n = 60), Colchester Zoo (n = 3), Edinburgh Zoo (n = 6), Port Lympne Wild Animal Park (n = 58), Howletts Wild Animal Park (n = 31), and Cotswold Wildlife Park (n = 4). Samples were examined by PCR and sequencing using four specific primer sets designed to differentiate between the pathogenic E. histolytica, the non-pathogenic E. dispar, and non-pathogenic uninucleate cyst-producing Entamoeba species. In the first experiment, Entamoeba was detected in 30 primates (81.1%). Six (16.2%) primates were infected with E. histolytica species complex. The highest carriage of Entamoeba species was found in Old World Colobinae primates. In the nationwide experiment, molecular analysis of faecal samples revealed notable rates of Entamoeba infection (101 samples, 28.9%), including one sample infected with E. histolytica, 14 samples with E. dispar, and 86 samples with uninucleated-cyst producing Entamoeba species. Sequences of positive uninucleated-cyst producing Entamoeba samples from Twycross Zoo clustered with the E. polecki reference sequences ST4 reported in Homo sapiens, and are widely separated from other Entamoeba species. These findings suggest a low prevalence of the pathogenic Entamoeba infection, but notable prevalence of non-pathogenic E. polecki infection in NHPs in the UK. PMID:25097822

  14. Genotyping of Giardia lamblia and Entamoeba spp from river waters in Iran.

    PubMed

    Mahmoudi, Mohammad Reza; Nazemalhosseini-Mojarad, Ehsan; Karanis, Panagiotis

    2015-12-01

    In this study, DNA from 55 surface and river water samples, which were collected from some water sources of Tehran and the Guilan Province, Iran, were extracted and examined for Entamoeba spp. and Giardia lamblia by PCR and genotyping. Twenty-seven samples, which were concentrated using the immunomagnetic separation technology (IMS) method, were examined for Giardia alone. Twenty-eight samples, which were concentrated using the sucrose flotation (SF) method, were examined for both Giardia and Entamoeba species. The results showed that 27/55 (17/27 and 10/28) (49 %), 4 /28 (14.28 %) and 3/28 (10.7 %) of the samples were positive for Giardia lamblia, Entamoeba spp and mixed infections (Entamoeba spp. and Giardia spp.), respectively. Sixteen out of 55 samples were negative. Entamoeba genus-specific PCR primers in single-round PCR were used to differentiate between the Entamoeba spp. (E. histolytica, E. dispar and E. moshkovskii). With respect to the 7 samples that were positive for Entamoeba, (14.28 %) 4 out of 28 were positive for E. moshkovskii, (7.14 %), 2 out of 28 were positive for E. histolytica and (3.57 %) 1 out of 28 was positive for E. dispar. Genus-specific PCR primers in a semi-nested PCR assay was performed to genotype Giardia species. Of the 27 samples that were positive for Giardia, 10 samples were sequences. All 10 successfully sequenced samples contained assemblage B of Giardia lamblia.This is first study to investigate the G. lamblia genotypes in the water supply of the Tehran and Guilan provinces, and it is the first study to investigate Entamoeba species in the water supplies of Iran. The investigated river water supplies, which are used for agriculture, camping and animal farming, were heavily contaminated by the human pathogenic Entamoeba and Giardia parasites. There is a potential risk of waterborne outbreaks in humans and animals. PMID:26350378

  15. Entamoeba moshkovskii Is Associated With Diarrhea in Infants and Causes Diarrhea and Colitis in Mice

    PubMed Central

    Shimokawa, Chikako; Kabir, Mamun; Taniuchi, Mami; Mondal, Dinesh; Kobayashi, Seiki; Ali, Ibne Karim M.; Sobuz, Shihab U.; Senba, Masachika; Houpt, Eric; Haque, Rashidul; Petri, William A.; Hamano, Shinjiro

    2012-01-01

    Background.Entamoeba moshkovskii is prevalent in developing countries and morphologically indistinguishable from pathogenic Entamoeba histolytica and nonpathogenic Entamoeba dispar. It is not known if E. moshkovskii is pathogenic. Methods.Mice were intracecally challenged with the trophozoites of each Entamoeba spp. to test the ability to cause diarrhea, and infants in Bangladesh were prospectively observed to see if newly acquired E. moshkovskii infection was associated with diarrhea. Results.E. moshkovskii and E. histolytica caused diarrhea and weight loss in susceptible mice. E. dispar infected none of the mouse strains tested. In Mirpur, Dhaka, Bangladesh, E. moshkovskii, E. histolytica, and E. dispar were identified in 42 (2.95%), 66 (4.63%), and 5 (0.35%), respectively, of 1426 diarrheal episodes in 385 children followed prospectively from birth to one year of age. Diarrhea occurred temporally with acquisition of a new E. moshkovskii infection: in the 2 months preceding E. moshkvskii-associated diarrhea, 86% (36 of 42) of monthly surveillance stool samples were negative for E. moshkovskii. Conclusions.E. moshkovskii was found to be pathogenic in mice. In children, the acquisition of E. moshkovskii infection was associated with diarrhea. These data are consistent with E. moshkovskii causing disease, indicating that it is important to reexamine its pathogenicity. PMID:22723640

  16. Differential diagnosis of Entamoeba spp. in clinical stool samples using SYBR green real-time polymerase chain reaction.

    PubMed

    Gomes, Thiago Dos Santos; Garcia, Mariana Coimbra; de Souza Cunha, Flavia; Werneck de Macedo, Heloisa; Peralta, José Mauro; Peralta, Regina Helena Saramago

    2014-01-01

    Amoebiasis, a disease caused by Entamoeba histolytica, is usually diagnosed by microscopic examination, which does not differentiate the morphologically identical species of the E. histolytica/E. dispar complex. Furthermore, morphologically similar species such as Entamoeba hartmanni contribute to misidentification. Therefore, there is a need for more sensitive and specific methods. This study standardized a multiplex real-time PCR system for E. histolytica and E. dispar and a single real-time PCR for E. hartmanni. The multiplex protocol detected up to 0.0143 pg of E. histolytica DNA and 0.5156 pg of E. dispar DNA, and the average melting temperature (T(m)) was 73 °C and 70 °C, respectively. For E. hartmanni, the T(m) was 73 °C and the amplification was successful down to 0.03 fg of plasmid DNA. Negative controls and other intestinal parasites presented no amplification. Among the 48 samples tested, E. dispar DNA was detected in 37; none exhibited E. histolytica DNA and 11 were negative in the multiplex protocol. In 4 of these 11 samples, however, E. hartmanni DNA was amplified. SYBR Green is demonstrated to be an interesting option and these combined PCR reactions can improve laboratory diagnosis of amoebiasis in developing countries. PMID:24693242

  17. Differential Diagnosis of Entamoeba spp. in Clinical Stool Samples Using SYBR Green Real-Time Polymerase Chain Reaction

    PubMed Central

    Gomes, Thiago dos Santos; Garcia, Mariana Coimbra; de Souza Cunha, Flavia; Peralta, José Mauro; Peralta, Regina Helena Saramago

    2014-01-01

    Amoebiasis, a disease caused by Entamoeba histolytica, is usually diagnosed by microscopic examination, which does not differentiate the morphologically identical species of the E. histolytica/E. dispar complex. Furthermore, morphologically similar species such as Entamoeba hartmanni contribute to misidentification. Therefore, there is a need for more sensitive and specific methods. This study standardized a multiplex real-time PCR system for E. histolytica and E. dispar and a single real-time PCR for E. hartmanni. The multiplex protocol detected up to 0.0143 pg of E. histolytica DNA and 0.5156 pg of E. dispar DNA, and the average melting temperature (Tm) was 73°C and 70°C, respectively. For E. hartmanni, the Tm was 73°C and the amplification was successful down to 0.03 fg of plasmid DNA. Negative controls and other intestinal parasites presented no amplification. Among the 48 samples tested, E. dispar DNA was detected in 37; none exhibited E. histolytica DNA and 11 were negative in the multiplex protocol. In 4 of these 11 samples, however, E. hartmanni DNA was amplified. SYBR Green is demonstrated to be an interesting option and these combined PCR reactions can improve laboratory diagnosis of amoebiasis in developing countries. PMID:24693242

  18. Genetic identification of Entamoeba polecki subtype 3 from pigs in Japan and characterisation of its pathogenic role in ulcerative colitis.

    PubMed

    Matsubayashi, Makoto; Murakoshi, Naoko; Komatsu, Tetsuya; Tokoro, Masaharu; Haritani, Makoto; Shibahara, Tomoyuki

    2015-12-01

    To date, three Entamoeba spp. (E. suis, zoonotic E. polecki and E. histolytica) have been identified in pigs, but their pathogenicity and molecular classification have not been fully determined. Examination and pathological analysis of pigs (n=3) with diarrhoea was conducted and revealed the presence of Entamoeba organisms. We performed a genetic analysis of the isolate using the small-subunit ribosomal RNA (SSU rRNA) gene region to identify the species. A severe ulcerative colitis was observed histopathologically with inflammatory cells, including macrophages and neutrophils, infiltrating the mucous membranes of the cecum and colon. Many Entamoeba trophozoites were found at the erosion site or at ulcerative lesions. Pathogenic viruses or bacteria were not detected. The SSU rRNA sequence of the Entamoeba isolate was found to be completely homologous to that of E. polecki subtype 3. PMID:26318541

  19. Horizontal Gene Transfers from Bacteria to Entamoeba Complex: A Strategy for Dating Events along Species Divergence.

    PubMed

    Romero, Miguel; Cerritos, R; Ximenez, Cecilia

    2016-01-01

    Horizontal gene transfer has proved to be relevant in eukaryotic evolution, as it has been found more often than expected and related to adaptation to certain niches. A relatively large list of laterally transferred genes has been proposed and evaluated for the parasite Entamoeba histolytica. The goals of this work were to elucidate the importance of lateral gene transfer along the evolutionary history of some members of the genus Entamoeba, through identifying donor groups and estimating the divergence time of some of these events. In order to estimate the divergence time of some of the horizontal gene transfer events, the dating of some Entamoeba species was necessary, following an indirect dating strategy based on the fossil record of plausible hosts. The divergence between E. histolytica and E. nuttallii probably occurred 5.93 million years ago (Mya); this lineage diverged from E. dispar 9.97 Mya, while the ancestor of the latter separated from E. invadens 68.18 Mya. We estimated times for 22 transferences; the most recent occurred 31.45 Mya and the oldest 253.59 Mya. Indeed, the acquisition of genes through lateral transfer may have triggered a period of adaptive radiation, thus playing a major role in the evolution of the Entamoeba genus. PMID:27239333

  20. Horizontal Gene Transfers from Bacteria to Entamoeba Complex: A Strategy for Dating Events along Species Divergence

    PubMed Central

    Romero, Miguel; Ximenez, Cecilia

    2016-01-01

    Horizontal gene transfer has proved to be relevant in eukaryotic evolution, as it has been found more often than expected and related to adaptation to certain niches. A relatively large list of laterally transferred genes has been proposed and evaluated for the parasite Entamoeba histolytica. The goals of this work were to elucidate the importance of lateral gene transfer along the evolutionary history of some members of the genus Entamoeba, through identifying donor groups and estimating the divergence time of some of these events. In order to estimate the divergence time of some of the horizontal gene transfer events, the dating of some Entamoeba species was necessary, following an indirect dating strategy based on the fossil record of plausible hosts. The divergence between E. histolytica and E. nuttallii probably occurred 5.93 million years ago (Mya); this lineage diverged from E. dispar 9.97 Mya, while the ancestor of the latter separated from E. invadens 68.18 Mya. We estimated times for 22 transferences; the most recent occurred 31.45 Mya and the oldest 253.59 Mya. Indeed, the acquisition of genes through lateral transfer may have triggered a period of adaptive radiation, thus playing a major role in the evolution of the Entamoeba genus. PMID:27239333

  1. Entamoeba thiol-based redox metabolism: A potential target for drug development.

    PubMed

    Jeelani, Ghulam; Nozaki, Tomoyoshi

    2016-01-01

    Amebiasis is an intestinal infection widespread throughout the world caused by the human pathogen Entamoeba histolytica. Metronidazole has been a drug of choice against amebiasis for decades despite its low efficacy against asymptomatic cyst carriers and emergence of resistance in other protozoa with similar anaerobic metabolism. Therefore, identification and characterization of specific targets is urgently needed to design new therapeutics for improved treatment against amebiasis. Toward this goal, thiol-dependent redox metabolism is of particular interest. The thiol-dependent redox metabolism in E. histolytica consists of proteins including peroxiredoxin, rubrerythrin, Fe-superoxide dismutase, flavodiiron proteins, NADPH: flavin oxidoreductase, and amino acids including l-cysteine, S-methyl-l-cysteine, and thioprolines (thiazolidine-4-carboxylic acids). E. histolytica completely lacks glutathione and its metabolism, and l-cysteine is the major intracellular low molecular mass thiol. Moreover, this parasite possesses a functional thioredoxin system consisting of thioredoxin and thioredoxin reductase, which is a ubiquitous oxidoreductase system with antioxidant and redox regulatory roles. In this review, we summarize and highlight the thiol-based redox metabolism and its control mechanisms in E. histolytica, in particular, the features of the system unique to E. histolytica, and its potential use for drug development against amebiasis. PMID:26775086

  2. An Annotated Checklist of the Human and Animal Entamoeba (Amoebida: Endamoebidae) Species- A Review Article

    PubMed Central

    HOOSHYAR, Hossein; ROSTAMKHANI, Parvin; REZAEIAN, Mostafa

    2015-01-01

    Background: The number of valid of pathogen and non-pathogen species of Entamoeba has continuously increased in human and animals. This review is performed to provide an update list and some summarized information on Entamoeba species, which were identified up to the 2014. Methods: We evaluated the Entamoeba genus with a broad systematic review of the literature, books and electronic databases until February 2014. The synonyms, hosts, pathogenicity and geographical distribution of valid species were considered and recorded. Repeated and unrelated cases were excluded. Results: Totally 51 defined species of Entamoeba were found and arranged by the number of nuclei in mature cyst according to Levin’s grouping. Seven of these species within the 4 nucleate mature cysts group and 1 species with one nucleate mature cyst are pathogen. E. histolytica, E. invadence, E. rananrum and E. anatis causes lethal infection in human, reptiles, amphibians and brides respectively, four species causes non-lethal mild dysentery. The other species were non-pathogen and are important to differential diagnosis of amoebiasis. Conclusion: There are some unknown true species of Entamoeba that available information on the morphology, hosts, pathogenicity and distribution of them are still very limited and more considerable investigation will be needed in order to clarify the status of them. PMID:26246811

  3. A Novel Mitosomal β-Barrel Outer Membrane Protein in Entamoeba

    PubMed Central

    Santos, Herbert J.; Imai, Kenichiro; Makiuchi, Takashi; Tomii, Kentaro; Horton, Paul; Nozawa, Akira; Ibrahim, Mohamed; Tozawa, Yuzuru; Nozaki, Tomoyoshi

    2015-01-01

    Entamoeba possesses a highly divergent mitochondrion-related organelle known as the mitosome. Here, we report the discovery of a novel protein in Entamoeba, which we name Mitosomal β-barrel Outer Membrane Protein of 30 kDa (MBOMP30). Initially identified through in silico analysis, we experimentally confirmed that MBOMP30 is indeed a β-barrel protein. Circular dichroism analysis showed MBOMP30 has a predominant β-sheet structure. Localization to Entamoeba histolytica mitosomes was observed through Percoll-gradient fractionation and immunofluorescence assay. Mitosomal membrane integration was demonstrated by carbonate fractionation, proteinase K digestion, and immunoelectron microscopy. Interestingly, the deletion of the putative β-signal, a sequence believed to guide β-barrel outer membrane protein (BOMP) assembly, did not affect membrane integration, but abolished the formation of a ~240 kDa complex. MBOMP30 represents only the seventh subclass of eukaryotic BOMPs discovered to date and lacks detectable homologs outside Entamoeba, suggesting that it may be unique to Entamoeba mitosomes. PMID:25711150

  4. Zinc in Entamoeba invadens.

    NASA Technical Reports Server (NTRS)

    Morgan, R. S.; Sattilaro, R. F.

    1972-01-01

    Atomic absorption spectroscopy, electron microprobe analysis, and dithizone staining of trophozoites and cysts of Entamoeba invadens demonstrate that these cells have a high concentration of zinc (approximately one picogram per cell or 1% of their dry weight). In the cysts of this organism, the zinc is confined to the chromatoid bodies, which previous work has shown to contain crystals of ribosomes. The chemical state and function of this zinc are unknown.

  5. The genome and transcriptome of the enteric parasite Entamoeba invadens, a model for encystation

    PubMed Central

    2013-01-01

    Background Several eukaryotic parasites form cysts that transmit infection. The process is found in diverse organisms such as Toxoplasma, Giardia, and nematodes. In Entamoeba histolytica this process cannot be induced in vitro, making it difficult to study. In Entamoeba invadens, stage conversion can be induced, but its utility as a model system to study developmental biology has been limited by a lack of genomic resources. We carried out genome and transcriptome sequencing of E. invadens to identify molecular processes involved in stage conversion. Results We report the sequencing and assembly of the E. invadens genome and use whole transcriptome sequencing to characterize changes in gene expression during encystation and excystation. The E. invadens genome is larger than that of E. histolytica, apparently largely due to expansion of intergenic regions; overall gene number and the machinery for gene regulation are conserved between the species. Over half the genes are regulated during the switch between morphological forms and a key signaling molecule, phospholipase D, appears to regulate encystation. We provide evidence for the occurrence of meiosis during encystation, suggesting that stage conversion may play a key role in recombination between strains. Conclusions Our analysis demonstrates that a number of core processes are common to encystation between distantly related parasites, including meiosis, lipid signaling and RNA modification. These data provide a foundation for understanding the developmental cascade in the important human pathogen E. histolytica and highlight conserved processes more widely relevant in enteric pathogens. PMID:23889909

  6. Ethanol and isopropanol in concentrations present in hand sanitizers sharply reduce excystation of Giardia and Entamoeba and eliminate oral infectivity of Giardia cysts in gerbils.

    PubMed

    Chatterjee, Aparajita; Bandini, Giulia; Motari, Edwin; Samuelson, John

    2015-11-01

    Enteric protozoan parasites, which are spread by the fecal-oral route, are important causes of diarrhea (Giardia duodenalis) and amebic dysentery (Entamoeba histolytica). Cyst walls of Giardia and Entamoeba have a single layer composed of fibrils of β-1,3-linked GalNAc and β-1,4-linked GlcNAc (chitin), respectively. The goal here was to determine whether hand sanitizers that contain ethanol or isopropanol as the active microbicide might reduce transmission of these parasites. We found that treatment with these alcohols with or without drying in a rotary evaporator (to model rapid evaporation of sanitizers on hands) kills 85 to 100% of cysts of G. duodenalis and 90 to 100% of cysts of Entamoeba invadens (a nonpathogenic model for E. histolytica), as shown by nuclear labeling with propidium iodide and failure to excyst in vitro. Alcohols with or without drying collapsed the cyst walls of Giardia but did not collapse the cyst walls of Entamoeba. To validate the in vitro results, we showed that treatment with alcohols eliminated oral infection of gerbils by 1,000 G. duodenalis cysts, while a commercial hand sanitizer (Purell) killed E. invadens cysts that were directly applied to the hands. These results suggest that expanded use of alcohol-based hand sanitizers might reduce the transmission of Giardia and Entamoeba. PMID:26282413

  7. Ethanol and Isopropanol in Concentrations Present in Hand Sanitizers Sharply Reduce Excystation of Giardia and Entamoeba and Eliminate Oral Infectivity of Giardia Cysts in Gerbils

    PubMed Central

    Chatterjee, Aparajita; Bandini, Giulia; Motari, Edwin

    2015-01-01

    Enteric protozoan parasites, which are spread by the fecal-oral route, are important causes of diarrhea (Giardia duodenalis) and amebic dysentery (Entamoeba histolytica). Cyst walls of Giardia and Entamoeba have a single layer composed of fibrils of β-1,3-linked GalNAc and β-1,4-linked GlcNAc (chitin), respectively. The goal here was to determine whether hand sanitizers that contain ethanol or isopropanol as the active microbicide might reduce transmission of these parasites. We found that treatment with these alcohols with or without drying in a rotary evaporator (to model rapid evaporation of sanitizers on hands) kills 85 to 100% of cysts of G. duodenalis and 90 to 100% of cysts of Entamoeba invadens (a nonpathogenic model for E. histolytica), as shown by nuclear labeling with propidium iodide and failure to excyst in vitro. Alcohols with or without drying collapsed the cyst walls of Giardia but did not collapse the cyst walls of Entamoeba. To validate the in vitro results, we showed that treatment with alcohols eliminated oral infection of gerbils by 1,000 G. duodenalis cysts, while a commercial hand sanitizer (Purell) killed E. invadens cysts that were directly applied to the hands. These results suggest that expanded use of alcohol-based hand sanitizers might reduce the transmission of Giardia and Entamoeba. PMID:26282413

  8. Pyp1 and Pyp2 PTPases dephosphorylate an osmosensing MAP kinase controlling cell size at division in fission yeast.

    PubMed

    Millar, J B; Buck, V; Wilkinson, M G

    1995-09-01

    Simultaneous inactivation of pyp1 and pyp2 PTPases in fission yeast leads to aberrant cell morphology and growth arrest. Spontaneous recessive mutations that bypass the requirement for pyp1 and pyp2 and reside in two complementation groups were isolated, sty1 and sty2. sty1- and sty2- mutant cells are substantially delayed in the timing of mitotic initiation. We have isolated the sty1 gene, which encodes a MAP kinase that is closely related to a subfamily of MAP kinases regulated by osmotic stress including Saccharomyces cervisiae HOG1 and human CSBP1. We find that sty2 is allelic to the wis1 MAP kinase kinase and that delta sty1 and delta wis1 cells are unable to grow in high osmolarity medium. Osmotic stress induces both tyrosine phosphorylation of Sty1 and a reduction in cell size at division. Pyp2 associates with and tyrosine dephosphorylates Sty1 in vitro. We find that wis1-dependent induction of pyp2 mRNA is responsible for tyrosine dephosphorylation of Sty1 in vivo on prolonged exposure to osmotic stress. We conclude that Pyp1 and Pyp2 are tyrosine-specific MAP kinase phosphatases that inactivate an osmoregulated MAP kinase, Sty1, which acts downstream of the Wis1 MAP kinase kinase to control cell size at division in fission yeast. PMID:7657164

  9. The ribosomal RNA transcription unit of Entamoeba invadens: accumulation of unprocessed pre-rRNA and a long non coding RNA during encystation.

    PubMed

    Ojha, Sandeep; Singh, Nishant; Bhattacharya, Alok; Bhattacharya, Sudha

    2013-01-01

    The ribosomal RNA genes in Entamoeba spp. are located on extrachromosomal circular molecules. Unlike model organisms where rRNA transcription stops during growth stress, Entamoeba histolytica continues transcription; but unprocessed pre-rRNA accumulates during stress, along with a novel class of circular transcripts from the 5'-external transcribed spacer (ETS). To determine the fate of rRNA transcription during stage conversion between trophozoite to cyst we analyzed Entamoeba invadens, a model system for differentiation studies in Entamoeba. We characterized the complete rDNA transcription unit by mapping the ends of pre-rRNA and mature rRNAs. The 3' end of mature 28S rRNA was located 321 nt downstream of the end predicted by sequence homology with E. histolytica. The major processing sites were mapped in external and internal transcribed spacers. The promoter located within 146 nt upstream of 5' ETS was used to transcribe the pre-rRNA. On the other hand, a second promoter located at the 3' end of 28S rDNA was used to transcribe almost the entire intergenic spacer into a long non coding (nc) RNA (>10 kb). Interestingly we found that the levels of pre-rRNA and long ncRNA, measured by northern hybridization, decreased initially in cells shifted to encystation medium, after which they began to increase and reached high levels by 72 h when mature cysts were formed. Unlike E. histolytica, no circular transcripts were found in E. invadens. E. histolytica and E. invadens express fundamentally different ncRNAs from the rDNA locus, which may reflect their adaptation to different hosts (human and reptiles, respectively). This is the first description of rDNA organization and transcription in E. invadens, and provides the framework for further studies on regulation of rRNA synthesis during cyst formation. PMID:24200639

  10. First molecular identification of Entamoeba polecki in a piglet in Japan and implications for aggravation of ileitis by coinfection with Lawsonia intracellularis.

    PubMed

    Matsubayashi, Makoto; Kanamori, Kenta; Sadahiro, Masayuki; Tokoro, Masaharu; Abe, Niichiro; Haritani, Makoto; Shibahara, Tomoyuki

    2015-08-01

    Parasitic Entamoeba spp. are found in many vertebrate species including humans, as well as many livestock including pigs. In pigs, three Entamoeba spp., E. suis, and E. polecki and E. histolytica as zoonotic species, have been identified, but their pathogenicity has not been fully characterized. Here, we report the bacteriological, virological, and histopathological examination of three piglets with chronic diarrhea. Two animals appeared to be additionally infected with Lawsonia intracellularis, which caused a characteristic proliferative ileitis. In the piglet infected with Entamoeba spp., the trophozoites (approximately 10-15 μm with one nucleus in their cytoplasm) invaded into the lamina propria and the disease was worsened by the formation of ulcers and pseudomembranes. Genetic analysis identified the parasite as E. polecki (99.5% identity). Although E. polecki in humans or animals might be less pathogenic in the case of a single infection, coinfections with other pathogens including L. intracellularis may increase the severity of the disease. PMID:25963884

  11. Development of RNA Interference Trigger-Mediated Gene Silencing in Entamoeba invadens.

    PubMed

    Suresh, Susmitha; Ehrenkaufer, Gretchen; Zhang, Hanbang; Singh, Upinder

    2016-04-01

    Entamoeba histolytica, a protozoan parasite, is an important human pathogen and a leading parasitic cause of death. The organism has two life cycle stages, trophozoites, which are responsible for tissue invasion, and cysts, which are involved in pathogen transmission. Entamoeba invadens is the model system to study Entamoeba developmental biology, as high-grade regulated encystation and excystation are readily achievable. However, the lack of gene-silencing tools in E. invadens has limited the molecular studies that can be performed. Using the endogenous RNA interference (RNAi) pathway in Entamoeba, we developed an RNAi-based trigger gene-silencing approach inE. invadens We demonstrate that a gene's coding region that has abundant antisense small RNAs (sRNAs) can trigger silencing of a gene that is fused to it. The trigger fusion leads to the generation of abundant antisense sRNAs that map to the target gene, with silencing occurring independently of trigger location at the 5' or 3' end of a gene. Gene silencing is stably maintained during development, including encystation and excystation. We have used this approach to successfully silence two E. invadens genes: a putative rhomboid protease gene and a SHAQKY family Myb gene. The Myb gene is upregulated during oxidative stress and development, and its downregulation led, as predicted, to decreased viability under oxidative stress and decreased cyst formation. Thus, the RNAi trigger silencing method can be used to successfully investigate the molecular functions of genes inE. invadens Dissection of the molecular basis of Entamoeba stage conversion is now possible, representing an important technical advance for the system. PMID:26787723

  12. Magnetic removal of Entamoeba cysts from water using chitosan oligosaccharide-coated iron oxide nanoparticles

    PubMed Central

    Shukla, Sudeep; Arora, Vikas; Jadaun, Alka; Kumar, Jitender; Singh, Nishant; Jain, Vinod Kumar

    2015-01-01

    Amebiasis, a major health problem in developing countries, is the second most common cause of death due to parasitic infection. Amebiasis is usually transmitted by the ingestion of Entamoeba histolytica cysts through oral–fecal route. Herein, we report on the use of chitosan oligosaccharide-functionalized iron oxide nanoparticles for efficient capture and removal of pathogenic protozoan cysts under the influence of an external magnetic field. These nanoparticles were synthesized through a chemical synthesis process. The synthesized particles were characterized by transmission electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction, and zeta potential analysis. The particles were found to be well dispersed and uniform in size. The capture and removal of pathogenic cysts were demonstrated by fluorescent microscopy, transmission electron microscopy, and scanning electron microscopy (SEM). Three-dimensional modeling of various biochemical components of cyst walls, and thereafter, flexible docking studies demonstrate the probable interaction mechanism of nanoparticles with various components of E. histolytica cyst walls. Results of the present study suggest that E. histolytica cysts can be efficiently captured and removed from contaminated aqueous systems through the application of synthesized nanoparticles. PMID:26261417

  13. Comparative analysis of genotypic diversity in Entamoeba nuttalli isolates from Tibetan macaques and rhesus macaques in China.

    PubMed

    Guan, Yue; Feng, Meng; Cai, Junlong; Min, Xiangyang; Zhou, Xingyu; Xu, Qing; Tan, Ning; Cheng, Xunjia; Tachibana, Hiroshi

    2016-03-01

    We have recently demonstrated the potentially virulent species Entamoeba nuttalli as one of the highly prevalent parasites in rhesus macaques (Macaca mulatta) in Mount Long-hu and Gui-yang in China. Tibetan macaque (Macaca thibetana) is a unique species living in China. To evaluate the prevalence of Entamoeba species in wild Tibetan macaques, we obtained 89 stool samples in Mount E-mei of Si-chuan Province in China. PCR analysis detected E. nuttalli, Entamoeba coli, and Entamoeba polecki ST2 in 17%, 42%, and 66% of the samples, respectively, whereas Entamoeba histolytica and Entamoeba dispar were undetected. This study is the first to report on the detection of E. nuttalli from Tibetan macaques. Six E. nuttalli isolates were obtained, 18S rRNA gene and six tRNA-linked short tandem repeat (STR) loci of the isolates were sequenced. The Mantel test results gave an r value of 0.97 of relationships between geographical distance and genetic diversity of Chinese E. nuttalli populations, indicating a significant isolation-by-distance effect in Chinese E. nuttalli according to the tRNA-STR loci sequences. Structural analysis of E. nuttalli isolates based on tRNA-linked STR loci demonstrated three Chinese E. nuttalli populations with their respective features, but the Gui-yang population was located in the middle. In the distance-based NJ tree, E. nuttalli isolates were divided into five different branches, and E-mei isolates were attributed to an independent branch to distinguish them from Gui-yang and Long-hu isolates. Genetic analysis in this study provided clues of the genetic differences between E. nuttalli isolates from Tibetan macaques and rhesus macaques in China. PMID:26723919

  14. Entamoeba lysyl-tRNA Synthetase Contains a Cytokine-Like Domain with Chemokine Activity towards Human Endothelial Cells

    PubMed Central

    Han, Jung Min; Kim, Sunghoon; Celada, Antonio; Ribas de Pouplana, Lluís

    2011-01-01

    Immunological pressure encountered by protozoan parasites drives the selection of strategies to modulate or avoid the immune responses of their hosts. Here we show that the parasite Entamoeba histolytica has evolved a chemokine that mimics the sequence, structure, and function of the human cytokine HsEMAPII (Homo sapiens endothelial monocyte activating polypeptide II). This Entamoeba EMAPII-like polypeptide (EELP) is translated as a domain attached to two different aminoacyl-tRNA synthetases (aaRS) that are overexpressed when parasites are exposed to inflammatory signals. EELP is dispensable for the tRNA aminoacylation activity of the enzymes that harbor it, and it is cleaved from them by Entamoeba proteases to generate a standalone cytokine. Isolated EELP acts as a chemoattractant for human cells, but its cell specificity is different from that of HsEMAPII. We show that cell specificity differences between HsEMAPII and EELP can be swapped by site directed mutagenesis of only two residues in the cytokines' signal sequence. Thus, Entamoeba has evolved a functional mimic of an aaRS-associated human cytokine with modified cell specificity. PMID:22140588

  15. Isolation and Molecular Characterization of Entamoeba nuttalli Strains Showing Novel Isoenzyme Patterns from Wild Toque Macaques in Sri Lanka.

    PubMed

    Tachibana, Hiroshi; Yanagi, Tetsuo; Feng, Meng; Bandara, K B Anura T; Kobayashi, Seiki; Cheng, Xunjia; Hirayama, Kenji; Rajapakse, R P V Jayanthe

    2016-03-01

    We have proposed the revival of the name Entamoeba nuttalli for a virulent ameba strain, P19-061405, from a rhesus macaque and located it phylogenetically between E. histolytica and E. dispar. As E. nuttalli was originally described for an ameba found in a toque macaque in Sri Lanka, the prevalence and characteristics of Entamoeba species in wild toque macaques were examined. PCR analysis of 227 stool samples from six locations showed positive rates for E. nuttalli, E. dispar, and E. histolytica of 18.5%, 0.4%, and 0%, respectively. Fifteen E. nuttalli strains were cultured successfully from five locations. The 18S ribosomal RNA gene showed only three nucleotide differences in comparison with P19-061405 strain. In isoenzyme analysis, the pattern of hexokinase in Sri Lankan strains was different from that of P19-061405 strains and the difference was confirmed by analysis of the genes. Hepatic inoculation of one of the Sri Lankan E. nuttalli strains in hamsters resulted in amebic abscess formation and body weight loss. These results demonstrate that E. nuttalli is prevalent in wild toque macaques and that several characteristics of the strains are unique. We conclude that use of the name E. nuttalli is appropriate for the new Entamoeba species found in nonhuman primates. PMID:26333681

  16. Identification of EhTIF-IA: The putative E. histolytica orthologue of the human ribosomal RNA transcription initiation factor-IA.

    PubMed

    Srivastava, Ankita; Bhattacharya, Alok; Bhattacharya, Sudha; Jhingan, Gagan Deep

    2016-03-01

    Initiation of rDNA transcription requires the assembly of a specific multi-protein complex at the rDNA promoter containing the RNA Pol I with auxiliary factors. One of these factors is known as Rrn3P in yeast and Transcription Initiation Factor IA (TIF-IA) in mammals. Rrn3p/TIF-IA serves as a bridge between RNA Pol I and the pre-initiation complex at the promoter. It is phosphorylated at multiple sites and is involved in regulation of rDNA transcription in a growth-dependent manner. In the early branching parasitic protist Entamoeba histolytica, the rRNA genes are present exclusively on circular extra chromosomal plasmids. The protein factors involved in regulation of rDNA transcription in E. histolytica are not known. We have identified the E. histolytica equivalent of TIF-1A (EhTIF-IA) by homology search within the database and was further cloned and expressed. Immuno-localization studies showed that EhTIF-IA co-localized partially with fibrillarin in the peripherally localized nucleolus. EhTIF-IA was shown to interact with the RNA Pol I-specific subunit RPA12 both in vivo and in vitro. Mass spectroscopy data identified RNA Pol I-specific subunits and other nucleolar proteins to be the interacting partners of EhTIF-IA. Our study demonstrates for the first time a conserved putative RNA Pol I transcription factor TIF-IA in E. histolytica. PMID:26949087

  17. Viruses of Entamoeba histolytica. IV. Studies on the nucleic acids of the filamentous and polyhedral viruses.

    PubMed

    Hruska, J F; Mattern, C F; Diamond, L S

    1974-01-01

    The nucleic acids of two amoebal viruses were studied by several independent methods. The filamentous virus, V(ABRM), was shown to be inhibited by bromodeoxyuridine, iododeoxyuridine, and cytosine arabinoside. With acridine orange staining, V(ABRM) inclusions appeared greenish-yellow, indicating that these contained double-stranded nucleic acid. The polyhedral virus, V(301), was also inhibited by bromodeoxyuridine, iododeoxyuridine, and cytosine arabinoside. In addition, nucleic acid hybridization showed that a new DNA species was synthesized in infected amoebal cultures. The intracellular localization of this new DNA was consistent with previous electron microscope studies of the cytoplasmic maturation of V(301). PMID:4129841

  18. Proteases from Entamoeba spp. and Pathogenic Free-Living Amoebae as Virulence Factors

    PubMed Central

    Serrano-Luna, Jesús; Piña-Vázquez, Carolina; Reyes-López, Magda; Ortiz-Estrada, Guillermo

    2013-01-01

    The standard reference for pathogenic and nonpathogenic amoebae is the human parasite Entamoeba histolytica; a direct correlation between virulence and protease expression has been demonstrated for this amoeba. Traditionally, proteases are considered virulence factors, including those that produce cytopathic effects in the host or that have been implicated in manipulating the immune response. Here, we expand the scope to other amoebae, including less-pathogenic Entamoeba species and highly pathogenic free-living amoebae. In this paper, proteases that affect mucin, extracellular matrix, immune system components, and diverse tissues and cells are included, based on studies in amoebic cultures and animal models. We also include proteases used by amoebae to degrade iron-containing proteins because iron scavenger capacity is currently considered a virulence factor for pathogens. In addition, proteases that have a role in adhesion and encystation, which are essential for establishing and transmitting infection, are discussed. The study of proteases and their specific inhibitors is relevant to the search for new therapeutic targets and to increase the power of drugs used to treat the diseases caused by these complex microorganisms. PMID:23476670

  19. Identification of Entamoeba moshkovskii in Treated Waste Water Used for Agriculture.

    PubMed

    Fonseca, Jairo Andres; Heredia, Rubén Darío; Ortiz, Carolina; Mazo, Martín; Clavijo-Ramírez, Carlos Arturo; Lopez, Myriam Consuelo

    2016-03-01

    We conducted an observational study to determine the prevalence of Entamoeba spp., in samples collected in a waste water treatment plant that provides water for agricultural irrigation. Samples were collected weekly over a period of 10 weeks at representative contamination stages from within the treatment plant. Protozoan identification was performed via light microscopy and culture. PCR amplification of small subunit rRNA gene sequences of E. histolytica/dispar/moshkovskii was performed in culture positive samples. Light microscopy revealed the presence of Entamoeba spp., in 70% (14/20) of the raw waste water samples and in 80% (8/10) of the treated water samples. PCR amplification after culture at both 24 and 37°C revealed that 100% (29/29) of the raw waste water samples and 78.6% (11/14) of the treated waste water were positive for E. moshkovskii. We report the first isolation of E. moshkovskii in Colombia, confirmed by PCR. Recent reports of E. moshkovskii pathogenic potential suggest this finding could constitute a public health risk for people exposed to this water. PMID:26732073

  20. Entamoeba Clone-Recognition Experiments: Morphometrics, Aggregative Behavior, and Cell-Signaling Characterization.

    PubMed

    Espinosa, Avelina; Paz-Y-Miño-C, Guillermo; Hackey, Meagan; Rutherford, Scott

    2016-05-01

    Studies on clone- and kin-discrimination in protists have proliferated during the past decade. We report clone-recognition experiments in seven Entamoeba lineages (E. invadens IP-1, E. invadens VK-1:NS, E. terrapinae, E. moshkovskii Laredo, E. moshkovskii Snake, E. histolytica HM-1:IMSS and E. dispar). First, we characterized morphometrically each clone (length, width, and cell-surface area) and documented how they differed statistically from one another (as per single-variable or canonical-discriminant analyses). Second, we demonstrated that amebas themselves could discriminate self (clone) from different (themselves vs. other clones). In mix-cell-line cultures between closely-related (E. invadens IP-1 vs. E. invadens VK-1:NS) or distant-phylogenetic clones (E. terrapinae vs. E. moshkovskii Laredo), amebas consistently aggregated with same-clone members. Third, we identified six putative cell-signals secreted by the amebas (RasGap/Ankyrin, coronin-WD40, actin, protein kinases, heat shock 70, and ubiquitin) and which known functions in Entamoeba spp. included: cell proliferation, cell adhesion, cell movement, and stress-induced encystation. To our knowledge, this is the first multi-clone characterization of Entamoeba spp. morphometrics, aggregative behavior, and cell-signaling secretion in the context of clone-recognition. Protists allow us to study cell-cell recognition from ecological and evolutionary perspectives. Modern protistan lineages can be central to studies about the origins and evolution of multicellularity. PMID:26990199

  1. Amoebiasis vaccine development: A snapshot on E. histolytica with emphasis on perspectives of Gal/GalNAc lectin.

    PubMed

    Singh, Ram Sarup; Walia, Amandeep Kaur; Kanwar, Jagat Rakesh; Kennedy, John F

    2016-10-01

    Amoebiasis/amebiasis is a gastrointestinal infection caused by an enteric dwelling protozoan, Entamoeba histolytica. The disease is endemic in the developing world and is transmitted mainly via the faecal-oral route (e.g., in water or food) and may or may not be symptomatic. This disease of socio-economic importance worldwide involves parasite adherence and cytolysis of human cells followed by invasion that is mediated by galactose-binding (Gal/GalNAc) surface lectin. Disruption of the mucus layer leads to invasive intestinal and extraintestinal infection. Gal-lectin based vaccinations have conferred protection in various animal models against E. histolytica infections. Keeping in view the pivotal role of Gal/GalNAc lectin in amoebiasis vaccine development, its regulation, genomic view of the parasite involving gene conversion in lectin gene families, current knowledge about involvement of Gal/GalNAc lectin in adherence, pathogenicity, signalling, encystment, generating host immune response, and in turn protozoa escape strategies, and finally its role as effective vaccine candidate has been described. This review will help researchers to explore pathogenesis mechanism along with genomic studies and will also provide a framework for future amoebiasis vaccine development studies. PMID:27181579

  2. Solution structure of the calmodulin-like C-terminal domain of Entamoeba α-actinin2.

    PubMed

    Karlsson, Göran; Persson, Cecilia; Mayzel, Maxim; Hedenström, Mattias; Backman, Lars

    2016-04-01

    Cell motility is dependent on a dynamic meshwork of actin filaments that is remodelled continuously. A large number of associated proteins that are severs, cross-links, or caps the filament ends have been identified and the actin cross-linker α-actinin has been implied in several important cellular processes. In Entamoeba histolytica, the etiological agent of human amoebiasis, α-actinin is believed to be required for infection. To better understand the role of α-actinin in the infectious process we have determined the solution structure of the C-terminal calmodulin-like domain using NMR. The final structure ensemble of the apo form shows two lobes, that both resemble other pairs of calcium-binding EF-hand motifs, connected with a mobile linker. PMID:26800385

  3. Novel protein-protein interactions between Entamoeba histolyticad-phosphoglycerate dehydrogenase and phosphoserine aminotransferase.

    PubMed

    Mishra, Vibhor; Kumar, Ashutosh; Ali, Vahab; Nozaki, Tomoyoshi; Zhang, Kam Y J; Bhakuni, Vinod

    2012-08-01

    Physical interactions between d-phosphoglycerate dehydrogenase (EhPGDH) and phosphoserine aminotransferase (EhPSAT) from an enteric human parasite Entamoeba histolytica was observed by pull-down assay, gel filtration chromatography, chemical cross-linking, emission anisotropy, molecular docking and molecular dynamic simulations. The protein-protein complex had a 1:1 stochiometry with a dissociation constant of 3.453 × 10(-7) M. Ionic interactions play a significant role in complex formation and stability. Analysis of the energy minimized average simulated model of the protein complex show that the nucleotide binding domain of EhPGDH specifically interacts with EhPSAT. Denaturation studies suggest that the nucleotide binding domain (Nbd) and substrate binding domain (Sbd) of EhPGDH are independent folding/unfolding units. Thus the Nbd-EhPGDH was separately cloned over-expressed and purified to homogeneity. Fluorescence anisotropy study show that the purified Nbd interacts with EhPSAT. Forward enzyme catalyzed reaction for the EhPGDH-PSAT complex showed efficient Km values for 3-phosphoglyceric acid as compared to only EhPGDH suggesting a possibility of substrate channelling in the protein complex. PMID:22386871

  4. Diphtheroid colitis in a Boa constrictor infected with amphibian Entamoeba sp.

    PubMed

    Richter, Barbara; Kübber-Heiss, Anna; Weissenböck, Herbert

    2008-05-01

    A female boa (Boa constrictor) from a zoological collection was submitted for necropsy after sudden death. Prominent pathological findings included a diphtheroid colitis, endoparasitism, focal pneumonia and inclusion bodies typical for inclusion body disease (IBD). In the colon entamoebae were identified, which differed in size and distribution from Entamoeba invadens. Gene sequence analysis of the 18S ribosomal RNA revealed 100% similarity with an Entamoeba species from the African bullfrog (Pyxicephalus adspersus), probably Entamoeba ranarum. The snake was possibly immunosuppressed, and the source of infection remains unclear. This is the first report of an infection with an amphibian Entamoeba species associated with colitis in a snake. PMID:18291588

  5. Entamoeba marina n. sp.; a New Species of Entamoeba Isolated from Tidal Flat Sediment of Iriomote Island, Okinawa, Japan.

    PubMed

    Shiratori, Takashi; Ishida, Ken-Ichiro

    2016-05-01

    The genus Entamoeba includes anaerobic lobose amoebae, most of which are parasites of various vertebrates and invertebrates. We report a new Entamoeba species, E. marina n. sp. that was isolated from a sample of tidal flat sediment collected at Iriomote Island, Okinawa, Japan. Trophozoites of E. marina were 12.8-32.1 μm in length and 6.8-15.9 μm in width, whereas the cysts were 8.9-15.8 μm in diam. and contained four nuclei. The E. marina cells contained a rounded nucleus with a small centric karyosome and uniformly arranged peripheral chromatin. Although E. marina is morphologically indistinguishable from other tetranucleated cyst-forming Entamoeba species, E. marina can be distinguished from them based on the combination of molecular phylogenetic analyses using SSU rDNA gene and the difference of collection sites. Therefore, we propose E. marina as a new species of the genus Entamoeba. PMID:26452446

  6. Monocyte Locomotion Inhibitory Factor Produced by E. histolytica Improves Motor Recovery and Develops Neuroprotection after Traumatic Injury to the Spinal Cord

    PubMed Central

    Bermeo, Gabriela; García, Elisa; Flores-Romero, Adrian; Rico-Rosillo, Guadalupe; Marroquín, Rubén; Flores, Carmina; Blanco-Favela, Francisco; Silva-García, Raúl

    2013-01-01

    Monocyte locomotion inhibitory factor (MLIF) is a pentapeptide produced by Entamoeba histolytica that has a potent anti-inflammatory effect. Either MLIF or phosphate buffered saline (PBS) was administered directly onto the spinal cord (SC) immediately after injury. Motor recovery was evaluated. We also analyzed neuroprotection by quantifying the number of surviving ventral horn motor neurons and the persistence of rubrospinal tract neurons. To evaluate the mechanism through which MLIF improved the outcome of SC injury, we quantified the expression of inducible nitric oxide synthase (iNOS), interleukin-10 (IL-10), and transforming growth factor-β (TGF-β) genes at the site of injury. Finally, the levels of nitric oxide and of lipid peroxidation were also determined in peripheral blood. Results showed that MLIF improved the rate of motor recovery and this correlated with an increased survival of ventral horn and rubrospinal neurons. These beneficial effects were in turn associated with a reduction in iNOS gene products and a significant upregulation of IL-10 and TGF-β expression. In the same way, MLIF reduced the concentration of nitric oxide and the levels of lipid peroxidation in systemic circulation. The present results demonstrate for the first time the neuroprotective effects endowed by MLIF after SC injury. PMID:24294606

  7. A simple fibril and lectin model for cyst walls of Entamoeba and perhaps Giardia

    PubMed Central

    Samuelson, John; Robbins, Phillips

    2010-01-01

    Cyst walls of Entamoeba and Giardia protect them from environmental insults, stomach acids, and intestinal proteases. Each cyst wall contains a sugar homopolymer: chitin in Entamoeba and a unique N-acetylgalactosamine (GalNAc) homopolymer in Giardia. Entamoeba cyst wall proteins include Jacob lectins (carbohydrate-binding proteins) that cross-link chitin, chitinases that degrade chitin, and Jessie lectins that make walls impermeable. Giardia cyst wall proteins are also lectins that bind fibrils of the GalNAc homopolymer. While many of the details remain to be determined for the Giardia cyst wall, current data suggests a relatively simple fibril and lectin model for the Entamoeba cyst wall. PMID:20934911

  8. Genetic Variation among Human Isolates of Uninucleated Cyst-Producing Entamoeba Species

    PubMed Central

    Verweij, Jaco J.; Polderman, Anton M.; Clark, C. Graham

    2001-01-01

    Twelve human infections with Entamoeba spp. producing uninucleated cysts were studied. DNA was extracted from infected feces and used to amplify part of the ameba small-subunit rRNA gene. Sequence analysis identified four distinct types of Entamoeba, all of which are related to Entamoeba polecki and E. chattoni and two of which have not been reported previously. Whether these genetic types represent different species is unclear. We propose that the agent of all human infections with uninucleated cyst-producing Entamoeba species be reported as “E. polecki-like.” PMID:11283106

  9. Entamoeba sp. (Sarcomastigophora: Endamoebidae) from ostriches (Struthio camelus) (Aves: Struthionidae).

    PubMed

    Martínez-Díaz, R A; Herrera, S; Castro, A; Ponce, F

    2000-10-01

    The first case of Entamoeba of the 1-nucleate mature cyst group in birds is described. Trophozoites and cysts of Entamoeba have been found in ostriches (Struthio camelus) from farms located all over Spain. The cysts are large (13.47microm mean diameter); they possess one nucleus when mature, with a large endosome and peripheral chromatine arranged in small granules; chromatoid bodies, when present, are large and elongated. Trophozoites are large (19. 88microm mean diameter), with a clear differentiation between ecto- and endoplasm, this containing numerous vacuoles; the nucleus is large and diffuse. The characteristics of this amoeba resembles but do not completely fulfill those of E. suis and E. chattoni; also, these species are from mammals. PMID:10962154

  10. Increased sampling reveals novel lineages of Entamoeba: consequences of genetic diversity and host specificity for taxonomy and molecular detection.

    PubMed

    Stensvold, C Rune; Lebbad, Marianne; Victory, Emma L; Verweij, Jaco J; Tannich, Egbert; Alfellani, Mohammed; Legarraga, Paulette; Clark, C Graham

    2011-07-01

    To expand the representation for phylogenetic analysis, ten additional complete Entamoeba small-subunit rRNA gene sequences were obtained from humans, non-human primates, cattle and a tortoise. For some novel sequences no corresponding morphological data were available, and we suggest that these organisms should be referred to as ribosomal lineages (RL) rather than being assigned species names at present. To investigate genetic diversity and host specificity of selected Entamoeba species, a total of 91 new partial small subunit rRNA gene sequences were obtained, including 49 from Entamoeba coli, 18 from Entamoeba polecki, and 17 from Entamoeba hartmanni. We propose a new nomenclature for significant variants within established Entamoeba species. Based on current data we propose that the uninucleated-cyst-producing Entamoeba infecting humans is called Entamoeba polecki and divided into four subtypes (ST1-ST4) and that Entamoeba coli is divided into two subtypes (ST1-ST2). New hosts for several species were detected and, while host specificity and genetic diversity of several species remain to be clarified, it is clear that previous reliance on cultivated material has given us a misleading and incomplete picture of variation within the genus Entamoeba. PMID:21295520

  11. Chitin synthetase in encysting Giardia lamblia and Entamoeba invadens

    SciTech Connect

    Das, S.; Gillin, F.D.

    1987-05-01

    Giardia lamblia (Gl) and Entamoeba invadens (Ei) are protozoan parasites with two morphologic stages in their life cycles. Motile trophozoites colonize the intestine of humans and reptiles respectively. Water resistant cysts, which can survive outside the host, transmit infection. In vitro cyst formation of Ei from trophozoites has been reported, and the authors have recently induced in vitro encystation of Gl. Although the cyst walls of both parasites contain chitin, it synthesis by encysting trophozoites has not been reported. The authors now show that encystation conditions greatly increase chitin synthetase (CS) specific activity (incorporation of /sup 3/H GlcNAc from UDP-GlcNAc into TCA-or alcohol-precipitable material). Extracts of encysting Gl incorporated 3.6 nmol/mg protein in 5 hr compared to < 0.005 in controls. Extracts of encysting Fi incorporated 4.8 n mol/mg protein, compared to 1.7 in the control. CS activity of both parasites requires preformed chitin. The Gl enzyme requires a reducing agent, is inhibited by digitonin and the CS inhibitors, polyoxin D and Nikkomycin, but not by tunicamycin. The product is digested by chitinase. Ei enzyme does not require a reducing agent and is stimulated by 1 mg/ml digitonin, but inhibited by higher concentrations. These studies demonstrate CS enzymes which may play important roles in encystation of Gl and Ei.

  12. Detection of the amoeba Entamoeba gingivalis in periodontal pockets

    PubMed Central

    Bonner, Mark; Amard, Véronique; Bar-Pinatel, Charlotte; Charpentier, Frédéric; Chatard, Jean-Michel; Desmuyck, Yvan; Ihler, Serge; Rochet, Jean-Pierre; Roux de La Tribouille, Véronique; Saladin, Luc; Verdy, Marion; Gironès, Núria; Fresno, Manuel; Santi-Rocca, Julien

    2014-01-01

    Periodontitis is a public health issue, being one of the most prevalent diseases worldwide. However, the aetiology of the disease is still unclear: genetics of patients cannot explain the dispersed or isolated localisation of gingival pockets, while bacteria-based models are insufficient to distinguish gingivitis and periodontitis. The possible role of parasites in the establishment of periodontitis has been poorly studied until now. The aim of this project was to study a potential link between colonisation of gingival crevices by the amoeba Entamoeba gingivalis and periodontitis. In eight different dental clinics in France, samples were taken in periodontal pockets (72) or healthy sites (33), and submitted to microscopic observation and molecular identification by PCR with a new set of primers designed to specifically detect E. gingivalis. This blind sample analysis showed the strong sensitivity of PCR compared with clinical diagnosis (58/72 = 81%), and microscopy (51/65 = 78%). The results of this work show that the parasites detected by microscopy mainly – if not exclusively – belong to the species E. gingivalis and that the presence of the parasite is correlated with periodontitis. PMID:24983705

  13. Observations on the intestinal protozoa infecting man in Rhodesia.

    PubMed

    Goldsmid, J M; Rogers, S; Mahomed, K

    1976-09-18

    Humans in Rhodesia harbour a wide range of intestinal protozoa. Of the species included, Entamoeba histolytica, Entamoeba coli and Giardia lamblia have previously been recorded. Other species which are either rarely reported or which have previously never been reported from this country, include Trichomonas hominis, Chilomastix mesnili, Enteromonas hominis, Retortamonas intestinalis, Balantidum coli,Entamoeba hartmanni,Entamoeba histolytica Laredo. Endolimax nana, Dientamoeba fragilis and Isospora belli. The importance in Rhodesia of these species, and especially of E. histolytica, is discussed. PMID:988643

  14. Evaluation of five commercial methods for the extraction and purification of DNA from human faecal samples for downstream molecular detection of the enteric protozoan parasites Cryptosporidium spp., Giardia duodenalis, and Entamoeba spp.

    PubMed

    Paulos, Silvia; Mateo, Marta; de Lucio, Aida; Hernández-de Mingo, Marta; Bailo, Begoña; Saugar, José M; Cardona, Guillermo A; Fuentes, Isabel; Mateo, María; Carmena, David

    2016-08-01

    High quality, pure DNA is required for ensuring reliable and reproducible results in molecular diagnosis applications. A number of in-house and commercial methods are available for the extraction and purification of genomic DNA from faecal material, each one offering a specific combination of performance, cost-effectiveness, and easiness of use that should be conveniently evaluated in function of the pathogen of interest. In this comparative study the marketed kits QIAamp DNA stool mini (Qiagen), SpeedTools DNA extraction (Biotools), DNAExtract-VK (Vacunek), PowerFecal DNA isolation (MoBio), and Wizard magnetic DNA purification system (Promega Corporation) were assessed for their efficacy in obtaining DNA of the most relevant enteric protozoan parasites associated to gastrointestinal disease globally. A panel of 113 stool specimens of clinically confirmed patients with cryptosporidiosis (n=29), giardiasis (n=47) and amoebiasis by Entamoeba histolytica (n=3) or E. dispar (n=10) and apparently healthy subjects (n=24) were used for this purpose. Stool samples were aliquoted in five sub-samples and individually processed by each extraction method evaluated. Purified DNA samples were subsequently tested in PCR-based assays routinely used in our laboratory. The five compared methods yielded amplifiable amounts of DNA of the pathogens tested, although performance differences were observed among them depending on the parasite and the infection burden. Methods combining chemical, enzymatic and/or mechanical lysis procedures at temperatures of at least 56°C were proven more efficient for the release of DNA from Cryptosporidium oocysts. PMID:27241828

  15. Compounds of the upper gastrointestinal tract induce rapid and efficient excystation of Entamoeba invadens

    PubMed Central

    Mitra, Biswa Nath; Pradel, Gabriele; Frevert, Ute; Eichinger, Daniel

    2010-01-01

    The infective stage of Entamoeba parasites is an encysted form. This stage can be readily generated in vitro, which has allowed identification of stimuli that trigger the differentiation of the parasite trophozoite stage into the cyst stage. Studies of the second differentiation event, emergence of the parasite from the cyst upon infection of a host, have been hampered by the lack of an efficient means to excyst the parasite and complete the life cycle in vitro. We have determined that a combination of exposures to water, bicarbonate and bile induces rapid excystment of Entamoeba invadens cysts. The high efficiency of this method has allowed the visualization of the dynamics of the process by electron and confocal microscopy, and should permit the analysis of stage-specific gene expression and high through-put screening of inhibitory compounds. PMID:20018192

  16. Variation in Rural African Gut Microbiota Is Strongly Correlated with Colonization by Entamoeba and Subsistence.

    PubMed

    Morton, Elise R; Lynch, Joshua; Froment, Alain; Lafosse, Sophie; Heyer, Evelyne; Przeworski, Molly; Blekhman, Ran; Ségurel, Laure

    2015-11-01

    The human gut microbiota is impacted by host nutrition and health status and therefore represents a potentially adaptive phenotype influenced by metabolic and immune constraints. Previous studies contrasting rural populations in developing countries to urban industrialized ones have shown that industrialization is strongly correlated with patterns in human gut microbiota; however, we know little about the relative contribution of factors such as climate, diet, medicine, hygiene practices, host genetics, and parasitism. Here, we focus on fine-scale comparisons of African rural populations in order to (i) contrast the gut microbiota of populations inhabiting similar environments but having different traditional subsistence modes and either shared or distinct genetic ancestry, and (ii) examine the relationship between gut parasites and bacterial communities. Characterizing the fecal microbiota of Pygmy hunter-gatherers as well as Bantu individuals from both farming and fishing populations in Southwest Cameroon, we found that the gut parasite Entamoeba is significantly correlated with microbiome composition and diversity. We show that across populations, colonization by this protozoa can be predicted with 79% accuracy based on the composition of an individual's gut microbiota, and that several of the taxa most important for distinguishing Entamoeba absence or presence are signature taxa for autoimmune disorders. We also found gut communities to vary significantly with subsistence mode, notably with some taxa previously shown to be enriched in other hunter-gatherers groups (in Tanzania and Peru) also discriminating hunter-gatherers from neighboring farming or fishing populations in Cameroon. PMID:26619199

  17. Variation in Rural African Gut Microbiota Is Strongly Correlated with Colonization by Entamoeba and Subsistence

    PubMed Central

    Morton, Elise R.; Lynch, Joshua; Froment, Alain; Lafosse, Sophie; Heyer, Evelyne; Przeworski, Molly; Blekhman, Ran; Ségurel, Laure

    2015-01-01

    The human gut microbiota is impacted by host nutrition and health status and therefore represents a potentially adaptive phenotype influenced by metabolic and immune constraints. Previous studies contrasting rural populations in developing countries to urban industrialized ones have shown that industrialization is strongly correlated with patterns in human gut microbiota; however, we know little about the relative contribution of factors such as climate, diet, medicine, hygiene practices, host genetics, and parasitism. Here, we focus on fine-scale comparisons of African rural populations in order to (i) contrast the gut microbiota of populations inhabiting similar environments but having different traditional subsistence modes and either shared or distinct genetic ancestry, and (ii) examine the relationship between gut parasites and bacterial communities. Characterizing the fecal microbiota of Pygmy hunter-gatherers as well as Bantu individuals from both farming and fishing populations in Southwest Cameroon, we found that the gut parasite Entamoeba is significantly correlated with microbiome composition and diversity. We show that across populations, colonization by this protozoa can be predicted with 79% accuracy based on the composition of an individual's gut microbiota, and that several of the taxa most important for distinguishing Entamoeba absence or presence are signature taxa for autoimmune disorders. We also found gut communities to vary significantly with subsistence mode, notably with some taxa previously shown to be enriched in other hunter-gatherers groups (in Tanzania and Peru) also discriminating hunter-gatherers from neighboring farming or fishing populations in Cameroon. PMID:26619199

  18. The Effect of Nonsurgical Periodontal Therapy on Trichomonas Tenax and Entamoeba Gingivalis in Patients with Chronic Periodontitis

    PubMed Central

    Rashidi Maybodi, Fahimeh; Haerian Ardakani, Ahmad; Fattahi Bafghi, Ali; Haerian Ardakani, Alireza; Zafarbakhsh, Akram

    2016-01-01

    Statement of the Problem Trichomonas tenax and Entamoeba gingivalis are commensal protozoa which inhabit the human oral cavity. These parasites are found in patients with poor oral hygiene and might be a reason for progressive periodontal diseases. Purpose The aim of this study was to evaluate the effect of nonsurgical periodontal treatment on the frequency of these protozoa in saliva and plaque samples. Materials and Method In this clinical trial, samples of saliva and dental plaque were collected from 46 patients with moderate to severe chronic periodontitis before and after periodontal therapy. The samples were assessed for the frequency of parasites. Results The frequency of Entamoeba gingivalis was reduced in saliva (p= 0.007) and plaque (p= 0.027) three weeks after the treatment. Likewise, the frequency of Trichomonas tenax reduced in saliva (p= 0.030); however, the decrease was not significant in plaque (p= 0.913). Trichomonas tenax frequency in dental plaque directly related to the severity of periodontitis (r= 0.565, p≤ 0.000). In contrast, the number of Entamoeba gingivalis in both saliva (r= -0.405, p≤ 0.005) and plaque (r= -0.304, p= 0.040) was inversely related with the severity of the periodontal disease. Conclusion Nonsurgical periodontal treatment could reduce the number of Trichomonas Tenax and Entamoeba gingivalis in the oral environment of patients with chronic periodontitis. PMID:27602391

  19. Amebic liver abscess

    MedlinePlus

    Hepatic amebiasis; Extraintestinal amebiasis; Abscess - amebic liver ... Amebic liver abscess is caused by Entamoeba histolytica. This parasite causes amebiasis , an intestinal infection that is also called ...

  20. Viruses of Entamoeba histolytica. II. Morphogenesis of the polyhedral particle (ABRM 2 leads to HK-9) leads to HB-301 and the filamentous agent (ABRM) 2 leads to HK-9.

    PubMed

    Mattern, C F; Diamond, L S; Daniel, W A

    1972-02-01

    The intracellular development of two morphologically different amoebal viruses has been studied by electron microscopy. One is a polyhedral agent which was observed as early as 24 hr after infection in the perinuclear cytoplasm. Subsequently, cell lysis occurred and particles were found in large number bound to membranes of disrupted amoebae. Other particles were found in phagocytic vacuoles suggesting a possible portal of entry into amoebae. The other virus is a filamentous particle which is first seen in small clusters in the nucleus after 24 hr of infection. The number of particles increases such that by 72 hr massive whorls of particles occupy a substantial part of the nucleus. After rupture of the nuclear membrane, clusters of filaments are widely dispersed throughout the cytoplasm. Still later, the cytoplasmic membrane disintegrates and clusters of filaments are found extracellularly, but free of cell membranes. The morphology of these agents is discussed in comparison with a variety of plant, animal, and bacterial viruses. PMID:4335523

  1. Site-specific free energy changes in proteins upon ligand binding by nuclear magnetic resonance: Ca2+ -displacement by Ln3+ in a Ca2+ -binding protein from Entamoeba histolytica.

    PubMed

    Chandra, Kousik; Mustafi, Sourajit M; Muthukumar, Subramanian; Chary, Kandala V R

    2011-04-01

    The study of protein-ligand interaction has been of a great interest in contemporary structural biology. The understanding of the nature of such interaction and determining the associated binding affinities are of utmost importance. Nuclear magnetic resonance has become a powerful tool in deriving information related to such interactions in proteins. Nuclear magnetic resonance data provide the site-specific information even in the case of proteins having multiple-binding sites and populations of respective species. In this communication, we set out to use such information to derive the associated microscopic binding constants. PMID:21235730

  2. Amebiasis

    MedlinePlus

    ... article/000298.htm Amebiasis To use the sharing features on this page, please enable JavaScript. Amebiasis is an infection of the intestines caused by the parasite Entamoeba histolytica . Causes Entamoeba histolytica can live in the large intestine (colon) without causing damage ...

  3. Establishment of a continuous culture system for Entamoeba muris and analysis of the small subunit rRNA gene.

    PubMed

    Kobayashi, S; Suzuki, J; Takeuchi, T

    2009-06-01

    We established a culture system for Entamoeba muris (MG-EM-01 strain isolated from a Mongolian gerbil) using a modified Balamuth's egg yolk infusion medium supplemented with 4% adult bovine serum and Bacteroides fragilis cocultured with Escherichia coli. Further, encystation was observed in the culture medium. The morphological characteristics of E. muris are similar to those of Entamoeba coli (E. coli); moreover, the malic isoenzyme electrophoretic band, which shows species-specific electrophoretic mobility, of E. muris had almost the same mobility as that observed with the malic isoenzyme electrophorectic band of E. coli (UZG-EC-01 strain isolated from a gorilla). We determined the small subunit rRNA (SSU-rRNA) gene sequence of the MG-EM-01 strain, and this sequence was observed to show 82.7% homology with that of the UZG-EC-01 strain. Further, the resultant phylogenetic tree for molecular taxonomy based on the SSU-rRNA genes of the 21 strains of the intestinal parasitic amoeba species indicated that the MG-EM-01 strain was most closely related to E. coli. PMID:19585892

  4. Giardia duodenalis assemblages and Entamoeba species infecting non-human primates in an Italian zoological garden: zoonotic potential and management traits

    PubMed Central

    2011-01-01

    Background Giardia duodenalis and Entamoeba spp. are among the most common intestinal human protozoan parasites worldwide and they are frequently reported in captive non-human primates (NHP). From a public health point of view, infected animals in zoos constitute a risk for animal caretakers and visitors. In this study we carried out the molecular identification of G. duodenalis and Entamoeba spp. from nine species of primates housed in the zoological garden of Rome, to better ascertain their occurrence and zoonotic potential. Results G. duodenalis was found only in Lemur catta (47.0%). Entamoeba spp. were detected in all species studied, with the exception of Eulemur macaco and Varecia rubra. The number of positive pools ranged from 5.9% in L. catta to 81.2% in Mandrillus sphinx; in Pan troglodytes the observed prevalence was 53.6%. A mixed Entamoeba-Giardia infection was recorded only in one sample of L. catta. All G. duodenalis isolates belonged to the zoonotic assemblage B, sub assemblage BIV. Three Entamoeba species were identified: E. hartmanni, E. coli and E. dispar. Conclusions Our results highlight the importance of regularly testing animals kept in zoos for the diagnosis of zoonotic parasites, in order to evaluate their pathogenic role in the housed animals and the zoonotic risk linked to their presence. A quick detection of the arrival of pathogens into the enclosures could also be a prerequisite to limit their spread into the structure via the introduction of specific control strategies. The need for molecular identification of some parasite species/genotype in order to better define the zoonotic risk is also highlighted. PMID:21988762

  5. Coincidence of deficient delayed hypersensitivity and intestinal protozoa in homosexual men.

    PubMed

    Jokipii, L; Sargeaunt, P G; Jokipii, A M

    1989-01-01

    Stool samples of 134 subjectively and apparently healthy male homosexual volunteers were studied, and in 86 men (64.2%) 190 parasite infestations, up to 6 per host, were identified. The most frequent species were Endolimax nana (40), Entamoeba coli (38), Entamoeba histolytica (34), Entamoeba hartmanni (32), and Iodamoeba bütschlii (22); Cryptosporidium was not found. All 11 isolates of Entamoeba histolytica analysed belonged to a noninvasive zymodeme: 10 were of zymodeme I and I of zymodeme III. In 172 healthy control adults 6 parasite infestations were found. In the homosexual men, a negative tuberculin test was more frequent in the presence than in the absence of parasites. Entamoeba histolytica (p = 0.005) Entamoeba coli (p = 0.013) and Iodamoeba bütschlii (p = 0.004) were associated with the anergy. PMID:2555912

  6. Molecular Epidemiology of Amebiasis

    PubMed Central

    Ali, Ibne Karim M.; Clark, C. Graham; Petri, William A.

    2008-01-01

    Entamoeba histolytica, the causative agent of human amebiasis, remains a significant cause of morbidity and mortality in developing countries and is responsible for up to 100,000 deaths worldwide each year. Entamoeba dispar, morphologically indistinguishable from E. histolytica, is more common in humans in many parts of the world. Similarly Entamoeba moshkovskii, which was long considered to be a free-living ameba, is also morphologically identical to E. histolytica and E. dispar, and is highly prevalent in some E. histolytica endemic countries. However, the only species to cause disease in humans is E. histolytica. Most old epidemiological data on E. histolytica are unusable as the techniques employed do not differentiate between the above three Entamoeba species. Molecular tools are now available not only to diagnose these species accurately but also to study intra-species genetic diversity. Recent studies suggest that only a minority of all E. histolytica infections progress to development of clinical symptoms in the host and there exist population level differences between the E. histolytica strains isolated from the asymptomatic and symptomatic individuals. Nevertheless the underlying factors responsible for variable clinical outcome of infection by E. histolytica remain largely unknown. We anticipate that the recently completed E. histolytica genome sequence and new molecular techniques will rapidly advance our understanding of the epidemiology and pathogenicity of amebiasis. PMID:18571478

  7. Safety and efficacy of diphetarsone in the treatment of amoebiasis, non-pathogenic amoebiasis and trichuriasis.

    PubMed

    Keystone, J S; Proctor, E; Glenn, C; McIntyre, L

    1983-01-01

    Eighty-nine patients infected with Entamoeba histolytica, non-pathogenic amoebae or Trichuris trichiura were studied prospectively to determine the safety and efficacy of diphetarsone therapy. An additional 75 patients were studied retrospectively to assess further the efficacy of diphetarsone in the treatment of E. histolytica cyst passers. Side effects were noted in 9% and included gastrointestinal upset, lightheadedness and headache. Transient liver function abnormalities were recorded in 5.6%. diphetarsone was completely effective in the treatment of Dientamoeba fragilis, Entamoeba hartmanni, Iodamoeba buetschlii and Trichuris trichiura. 99% of the patients with E. histolytica, 97% of those with E. coli and 98% of those with Endolimax nana were cured. PMID:6304952

  8. [Investigation of intestinal parasites in residents of the Kayseri Karpuzsekisi basin].

    PubMed

    Sahin, Izzet; Yazar, Süleyman; Yaman, Ozan; Gözkenç, Niğmet

    2006-01-01

    In this study, we collected stool and cellophane tape samples from residents in four villages located in the Kayseri-Karpuzsekisi basin in order to investigate the distribution of intestinal parasites. A total of 240 samples collected from Hanyeri (40), Sariküklü (20), Dokuzpinar (80) and Karpuzsekisi (120) were investigated. The parasites detected were as follows: Blastocystis hominis, 82 (34.16%); Enterobius vermicularis, 37 (15.41%); Entamoeba coli 26, (10.83%); Entamoeba hartmanni, 10 (4.16%); Giardia intestinalis, 7 (2.91%); Endolimax nana, 5 (2.08%); Entamoeba histolytica/E. dispar, 4 (1.66%); and Taenia saginata, 1 (0.41%). PMID:17160846

  9. Human cysticercosis and intestinal parasitism amongst the Ekari people of Irian Jaya.

    PubMed

    Muller, R; Lillywhite, J; Bending, J J; Catford, J C

    1987-12-01

    A random sample of 242 people showed that 42 had palpable cysts of Taenia solium. Faecal examination recovered eggs of T. solium in seven (3%), while Trichuris (83%), Ascaris (83%), hookworms (76%), Strongyloides stercoralis (10%) and Strongyloides sp. (29%), Entamoeba histolytica (14%), Entamoeba coli (22%), Entamoeba hartmanni (7%), Entamoeba polecki (7%), Balantidium coli (9%) and Dientamoeba fragilis (21%) were the most common other intestinal parasites encountered. ELISA tests, using antigens prepared from adults and eggs of T. solium and from cysticerci of T. saginata were not very sensitive, the last diagnosing less than half of known positives while still retaining good specificity. PMID:3430662

  10. Evaluation of the BD Max Enteric Parasite Panel for Clinical Diagnostics.

    PubMed

    Mölling, Paula; Nilsson, Peter; Ennefors, Theresa; Ögren, Jessica; Florén, Kerstin; Thulin Hedberg, Sara; Sundqvist, Martin

    2016-02-01

    We compared the performance of the BD Max enteric parasite panel to routine microscopy and an in-house PCR for the detection of Giardia intestinalis, Entamoeba histolytica, and Cryptosporidium spp. The enteric parasite panel showed good specificity for all targets and good sensitivity for E. histolytica and Cryptosporidium spp. Sensitivity for G. intestinalis with the BD Max enteric parasite panel was equivalent to that with microscopy. PMID:26582832

  11. Comparison of Stool Antigen Detection Kits to PCR for Diagnosis of Amebiasis ▿

    PubMed Central

    Stark, D.; van Hal, S.; Fotedar, R.; Butcher, A.; Marriott, D.; Ellis, J.; Harkness, J.

    2008-01-01

    The present study was conducted to compare two stool antigen detection kits with PCR for the diagnosis of Entamoeba histolytica infections by using fecal specimens submitted to the Department of Microbiology at St. Vincent's Hospital, Sydney, and the Institute of Medical and Veterinary Science, Adelaide, Australia. A total of 279 stool samples containing the E complex (E. histolytica, Entamoeba dispar, and Entamoeba moshkovskii) were included in this study. The stool specimens were tested by using two commercially produced enzyme immunoassays (the Entamoeba CELISA PATH and TechLab E. histolytica II kits) to detect antigens of E. histolytica. DNA was extracted from all of the samples with a Qiagen DNA stool mini kit (Qiagen, Hilden, Germany), and a PCR targeting the small-subunit ribosomal DNA was performed on all of the samples. When PCR was used as a reference standard, the CELISA PATH kit showed 28% sensitivity and 100% specificity. The TechLab ELISA (enzyme-linked immunosorbent assay) kit did not prove to be useful in detecting E. histolytica, as it failed to identify any of the E. histolytica samples which were positive by PCR. With the TechLab kit, cross-reactivity was observed for three specimens, one of which was positive for both E. dispar and E. moshkovskii while the other two samples contained E. moshkovskii. Quantitative assessment of the PCR and ELISA results obtained showed that the ELISA kits were 1,000 to 10,000 times less sensitive, and our results show that the CELISA PATH kit and the TechLab ELISA are not useful for the detection of E. histolytica in stool samples from patients in geographical regions where this parasite is not endemic. PMID:18367563

  12. Microscopic overdiagnosis of intestinal amoebiasis.

    PubMed

    Rayan, Hanan Z E

    2005-12-01

    To determine the misdiagnosis of intestinal amoebiasis associated to microscopic examination of faeces, 50 stool samples of patients infected with Entamoeba histolytica were collected from different Primary Health Care Centers, hospitals and private laboratories in Ismailia G. The samples were examined using Wheatley's trichrome staining technique to differrentiate E. histolytica E. dispar complex from other non-pathogenic intestinal amoebae and multiplex polymerase chain reaction (PCR). PCR differentiated between the two morphologic identical species (E. histolytica and E. dispar) and had the advantage to save time and resources. E. histolytica was detected in only 5 (10%) samples and in association with E. dispar in 8 (16%) samples. On the other hand, 20 samples (40%) were E. dispar. The other 17 samples were negative. E. coli, E. hartmanni and polymorphs were commonly misdiagnosed as E. histolytica. PMID:16333901

  13. Plasmids containing small subunit ribosomal RNA gene fragments from Babesia bovis and Babesia bigemina

    Technology Transfer Automated Retrieval System (TEKTRAN)

    BEI Resources was developed by NIAID as a centralized biological resource center for research reagents to the scientific community (http://www.beiresources.org/). They have a considerable amount of reagents and isolates for parasitologists working with Entamoeba histolytica, Giardia, Toxoplasma, and...

  14. Bruceantin, a potent amoebicide from a plant, Brucea antidysenterica.

    PubMed Central

    Gillin, F D; Reiner, D S; Suffness, M

    1982-01-01

    Bruceantin, purified from an Ethiopian plant used to treat dysentery, killed Entamoeba histolytica in vitro (IC50 [the concentration of drug which decreased the number of colonies to half that of controls] = 0.018 microgram/ml). Six related quassinoids of 17 tested were also amoebicidal. No relationship between quassinoid structure and amoebicidal activity was apparent. PMID:6100431

  15. Comparison of Real-Time PCR Protocols for Differential Laboratory Diagnosis of Amebiasis

    PubMed Central

    Qvarnstrom, Yvonne; James, Cleve; Xayavong, Maniphet; Holloway, Brian P.; Visvesvara, Govinda S.; Sriram, Rama; da Silva, Alexandre J.

    2005-01-01

    Specific identification of Entamoeba spp. in clinical specimens is an important confirmatory diagnostic step in the management of patients who may be infected with Entamoeba histolytica, the species that causes clinical amebiasis. Distinct real-time PCR protocols have recently been published for identification of E. histolytica and differentiation from the morphologically identical nonpathogenic Entamoeba dispar. In this study, we compared three E. histolytica real-time PCR techniques published by December 2004. The limits of detection and efficiency of each real-time PCR assay were determined using DNA extracted from stool samples spiked with serially diluted cultured E. histolytica trophozoites. The ability of each assay to correctly distinguish E. histolytica from E. dispar was evaluated with DNA extracted from patients' stools and liver aspirates submitted for confirmatory diagnosis. Real-time PCR allowed quantitative analysis of the spiked stool samples, but major differences in detection limits and assay performance were observed among the evaluated tests. These results illustrate the usefulness of comparative evaluations of diagnostic assays. PMID:16272475

  16. Heat-labile enterotoxigenic Escherichia coli and intestinal protozoa in asymptomatic travellers.

    PubMed

    Echeverria, P; Cross, J H

    1977-12-01

    Thirty-two asymptomatic travellers who had recently journeyed in the Near, Middle, and Far East and had experienced a high incidence of diarrhoeal disease were screened for heat-labile enterotoxigenic Escherichia coli (ent+ E. coli) and other bacterial and parasitic pathogens. Six percent were colonized with ent+ E. coli and while other bacterial pathogens were not found, the intestinal protozoa Giardia lamblia (13%), Entamoeba histolytica (6%), Entamoeba coli (6%), Endolimax nana (6%), and Entamoeba hartmanni (3%) were detected in the stools. Ent+ E. coli, G. lamblia and E. histolytica should be considered in the differential diagnosis of gastrointestinal disease in travellers returning from the Orient. Furthermore, these travellers may be a potential source for the introduction of ent+ E. coli into communities where such organisms are relatively rare. PMID:351820

  17. [Distribution of intestinal parasites among patients who presented at the Department of Parasitology of the Erciyes University Medical School.].

    PubMed

    Yazar, Süleyman; Yaman, Ozan; Gözkenç, Niğmet; Sahın, Izzet

    2005-01-01

    In this study carried out from 2000-2004, a total of 34,883 stool samples were examined using native-Lugol and flotation/sedimentation methods and 9,879 cellophane tape preparations were examined directly. Intestinal parasites were found in 9,704 (27.8%) of the specimens. The parasites that were found and their prevalence is as follows: Blastocystis hominis, 6,723 (19.3%); Entamoeba coli, 1,007 (2.9%); Giardia intestinalis, 892 (2.6%); Entamoeba histolytica/E. dispar, 798 (2.3%); Endolimax nana, 486 (1.4%); Entamoeba hartmanni, 252 (0.7%); Enterobius vermicularis, 242 (0.7%); Iodamoeba bütschlii, 109 (0.3%); Taenia saginata, 92 (0.3%); Chilomastix mesnili, 67 (0.2%); Ascaris lumbricoides, 55 (0.2%); and Hymenolepis nana, 40 (0.1%). PMID:17124684

  18. [Prevalence of intestinal parasites among workers in food sector in Van region].

    PubMed

    Kurtoğlu, Muhammet Güzel; Körkoca, Hanifi; Ciçek, Mutalip; Cengiz, Zeynep Taş

    2007-01-01

    Stool and cellophane tape specimens were taken for parasitological examination from 739 people who work in food sector and applied to the public health lab of the Van Health Administration for porter examination. Parasites were determined at 131 people (17.71%) of 739 worker whom samples were investigated. Ninety-five people had one, 30 people had two, 5 people had three and one person had four parasite species. Parasites determined in the study were 19.08% helminthes and 80.91% protozoon. In this study, 1.21% Ascaris lumbricoides, 0.81% Enterobius vermicularis, 0.67% Hymenolepis nana, 0.40% Trichuris trichiura, 0.27% Taenia saginata, 4.87% Blastocystis hominis, 3.24% Entamoeba coli, 2.84% Giardia intestinalis, 2.02% Iodamoeba bütschlii, 0.67% Endolimax nana, 0.27% Entamoeba histolytica/Entamoeba dispar, 0.27% Chilomastix mesnili, 0.13% Entamoeba hartmanni were found. PMID:18224624

  19. The presence of antiamoebic constituents in psyllium husk.

    PubMed

    Zaman, Viqar; Manzoor, Syed Munazza; Zaki, Mehreen; Aziz, Nauman; Gilani, Anwar-ul-Hassan

    2002-02-01

    The crude extract of psyllium husk (ispaghula) and its active constituent (petroleum fraction) caused varying degrees of growth inhibition in three different species of Entamoeba, i.e. Entamoeba histolytica, E. invadens and E. dispar. The inhibitory effect of the crude extract was in the dose range of 1-10 mg/mL, whereas a similar inhibitory effect was obtained with the petroleum fraction at a much lower dose (0.1-1.0 mg/mL), indicating that the active chemical(s) is/are concentrated in the petroleum fraction. These data support the traditional use of psyllium husk in amoebic dysentery. PMID:11807972

  20. Intestinal parasitic infections in the residents of an emigration camp in Tijuana, Mexico.

    PubMed

    Huh, S; Ahn, C; Chai, J Y

    1995-03-01

    We examined stool specimens of the residents in the emigration camp in Tijuana, Mexico for helminth eggs or protozoan cysts with formalin-ethyl acetate concentration method in February and July 1992. Out of 92 examined samples, number of positive was 49 (53.3%). While number of cumulative positive was 66 (71.7%). Cysts of Entamoeba coli (29.3%) Giardia lamblia (9.8%), Entamoeba histolytica (7.6%), and eggs of Taenia spp. (6.5) were most frequently observed. Filtered water supply and chemotherapy were required in this camp. PMID:7735788

  1. [Enteropathogens associated with diarrheal disease in infants (< 5 years old) in a population sample in Greater Metropolitan Criciúma, Santa Catarina State, Brazil].

    PubMed

    Schnack, Felice Jaqueline; Fontana Ld, Lidiani de Medeiros; Barbosa, Paulo Roberto; Silva, Loraine Storch Meyer da; Baillargeon, Clair Maria Martinello; Barichello, Tatiana; Póvoa, Marinete Marins; Cavasini, Carlos Eug nio; Machado, Ricardo Luiz Dantas

    2003-01-01

    Enteropathogens were investigated in 94 children with diarrhea and 45 age-matched controls, 0 to 5 years old, attending an outpatient unit in Criciúma, Santa Catarina State, Brazil. Cryptosporidium (85.1%) topped the list of parasite isolates, followed by Entamoeba histolytica (56.4%) and Giardia lamblia (4.3%). Four samples contained enteropathogenic Escherichia coli (4.3%). Samonella and Shiguella were not detected. Only one sample contained rotavirus (1.1%). PMID:12973585

  2. Sesquiterpene lactones from neurolaena oaxacana

    PubMed

    Passreiter; Sandoval-Ramirez; Wright

    1999-08-01

    Twelve sesquiterpene lactones, two new (1 and 2) and 10 known neurolenin-type germacranolides and furanoheliangolides (3-12) were isolated from Neurolaena oaxacana, and their structures were elucidated by NMR and GC-MS analysis. The chemotaxonomic importance of these findings is discussed. As N. lobata is used against dysenteries, neurolenin B (4) and a mixture of the neurolenins C (5) and D (6) were tested against Entamoeba histolytica and Giardia intestinalis. PMID:10479310

  3. Methods for Cultivation of Luminal Parasitic Protists of Clinical Importance

    PubMed Central

    Clark, C. Graham; Diamond, Louis S.

    2002-01-01

    Cultivation of luminal protistan parasites has a long history. In this review we discuss the methods and media that are most widely used for the establishment and maintenance of the following organisms in culture: Entamoeba histolytica, Giardia intestinalis, Trichomonas vaginalis, Dientamoeba fragilis, Blastocystis hominis, and Balantidium coli. While cultivation is of limited importance in the diagnostic laboratory, it is essential to most research laboratories, and it is toward the latter that this review is primarily aimed. PMID:12097242

  4. Intestinal parasites in man in Labrador, Canada.

    PubMed

    Sole, T D; Croll, N A

    1980-05-01

    Labrador, a previously unsurveyed area of Canada, has been sampled for human intestinal parasites. Four hundred and one asymptomatic volunteers between 1 and 72 years of age, including Inuit, Naskapi and whites, were examined during the summer of 1977. They harboured: Entamoeba coli, E. histolytica, E. hartmanni, Giardia lamblia and Diphyllobothrium sp. The infection rates are considerably lower than those found in other studies of Northern Canadian communities. PMID:6966896

  5. Parasitological and microbiological evaluation of Mixe Indian medicinal plants (Mexico).

    PubMed

    Heinrich, M; Kuhnt, M; Wright, C W; Rimpler, H; Phillipson, J D; Schandelmaier, A; Warhurst, D C

    1992-02-01

    Medicinal plants are an important health resource in many regions of the Americas and are of particular importance to many Indian communities. Based on a recent ethnobotanical study in Mexico, we investigated the activity of 29 plant extracts against Entamoeba histolytica, three bacteria (Bacillus subtilis, Escherichia coli, and Micrococcus luteus) and two fungi (Cladosporium cucumerinum and Penicillium oxalicum). After separation of these extracts between CH2Cl2 and H2O the resulting phases were also evaluated. PMID:1501496

  6. Amebiasis presenting as acute appendicitis.

    PubMed

    Andrade, Javier E; Mederos, Raul; Rivero, Haidy; Sendzischew, Morgan A; Soaita, Mauela; Robinson, Morton J; Sendzischew, Harry; Danielpour, Payman

    2007-11-01

    Amebiasis presenting as acute appendicitis is extremely rare. The case of a 38-year-old Hispanic man who presented to the hospital with symptoms and signs suggestive of acute appendicitis is reported. He underwent laparoscopic appendectomy and the pathologic examination of the appendix revealed multiple trophozoites of Entamoeba histolytica. The patient was treated postoperatively with metronidazole for amebiasis, and follow-up stool studies showed no sign of residual infection. The patient has remained asymptomatic. PMID:17984748

  7. Cellular immune responses to amoebic liver abcess in the guinea-pig.

    PubMed Central

    Bray, R S; Harris, W G

    1977-01-01

    Guinea-pigs infected in the liver with the Biswas strain of Entamoeba histolytica showed no dermal hypersensitivity but showed positive lymphocyte transformation and macrophage-migration inhibition. The time sequence showed an activated response at 4 days after infection, a full response at 8 days when the liver abscesses were resolving and a waning response at 12 days when the abscesses had healed. PMID:891028

  8. Frequency of enteric protozoan parasites among patients with gastrointestinal complaints in medical centers of Zahedan, Iran.

    PubMed

    Haghighi, Ali; Khorashad, Alireza Salimi; Nazemalhosseini Mojarad, Ehsan; Kazemi, Bahram; Rostami Nejad, Mohammad; Rasti, Sima

    2009-05-01

    We investigated the prevalence of intestinal protozoan parasites in patients with gastrointestinal complaints in medical centers in Zahedan, Iran. A total of 1562 stool samples was examined from July 2004 to January 2006 using microscopy (direct smear, formalin-ether concentration), xenic culture and PCR techniques. Four hundred and twenty-seven (27.3%) of the patients were infected with one or more intestinal parasites. Giardia lamblia (10.1%), Entamoeba coli (10%), E. hartmanni (1.7%), Blastocystis hominis (2.2%), Chilomastix mesnili (1.7%), Trichomonas hominis (0.7%), E. histolytica/E. dispar (0.51%) and Iodamoeba butschlii (0.45%) were the most prevalent protozoa detected with microscopy. Of the eight microscopy-positive E. histolytica/E. dispar samples, six were identified as E. dispar by PCR/gel electrophoresis, whereas E. histolytica was not detected at all. Although Zahedan is an area with poor hygiene located in a tropical area near the border of Pakistan and Afghanistan, the prevalence of E. histolytica and E. dispar here compared with other parasites and infectious diseases is unexpectedly low. PMID:19084249

  9. Analysis of the Bacterial Diversity in Liver Abscess: Differences Between Pyogenic and Amebic Abscesses.

    PubMed

    Reyna-Fabián, Miriam E; Zermeño, Valeria; Ximénez, Cecilia; Flores, Janin; Romero, Miguel F; Diaz, Daniel; Argueta, Jesús; Moran, Patricia; Valadez, Alicia; Cerritos, René

    2016-01-01

    Several recent studies have demonstrated that virulence in Entamoeba histolytica is triggered in the presence of both pathogenic and nonpathogenic bacteria species using in vitro and in vivo experimental animal models. In this study, we examined samples aspirated from abscess material obtained from patients who were clinically diagnosed with amebic liver abscess (ALA) or pyogenic liver abscess (PLA). To determine the diversity of bacterial species in the abscesses, we performed partial 16S rRNA gene sequencing. In addition, the E. histolytica and Entamoeba dispar species were genotyped using tRNA-linked short tandem repeats as specific molecular markers. The association between clinical data and bacterial and parasite genotypes were examined through a correspondence analysis. The results showed the presence of numerous bacterial groups. These taxonomic groups constitute common members of the gut microbiota, although all of the detected bacterial species have a close phylogenetic relationship with bacterial pathogens. Furthermore, some patients clinically diagnosed with PLA and ALA were coinfected with E. dispar or E. histolytica, which suggests that the virulence of these parasites increased in the presence of bacteria. However, no specific bacterial groups were associated with this effect. Together, our results suggest a nonspecific mechanism of virulence modulation by bacteria in Entamoeba. PMID:26572872

  10. The Dynamic Interdependence of Amebiasis, Innate Immunity, and Undernutrition1

    PubMed Central

    Verkerke, Hans P.; Petri, William A.; Marie, Chelsea S.

    2012-01-01

    Entamoeba histolytica, the protozoan parasite that causes amebic dysentery, greatly contributes to disease burden in the developing world. Efforts to exhaustively characterize the pathogenesis of amebiasis have increased our understanding of the dynamic host-parasite interaction and the process by which E. histolytica trophozoites transition from gut commensals to invaders of the intestinal epithelium. Mouse models of disease continue to be instrumental in this area. At the same time, large-scale studies in human populations have identified genetic and environmental factors that influence susceptibility to amebiasis. Nutritional status has long been known to globally influence immune function. So it is not surprising that undernutrition has emerged as a critical risk factor. A better understanding of how nutritional status affects immunity to E. histolytica will have dramatic implications in the development of novel treatments. Future work should continue to characterize the fascinating host-parasite arms race that occurs at each stage of infection. PMID:23114864

  11. Biomedical survey in Irian Jaya (West Irian), Indonesia.

    PubMed

    Cross, J H; Irving, G S; Anderson, K E; Gunawan, S; Saroso, J S

    1977-12-01

    A biomedical survey was conducted in several areas of Irian Jaya, Indonesia in July 1972 in association with an investigation of reports of a cholera outbreak. Stool specimens, blood smears and sera were collected and examined for evidence of parasitic as well as other infectious diseases. A total of 114 stools were examined and the most commonly found intestinal parasites were Trichuris trichiura (94%), Ascaris lumbricoides (74%), hookworm (58%), Entamoeba coli (15%), Endolimax nana (8%), Entamoeba histolytica (7), Entamoeba hartmanni (4%), Giardia lamblia (3%) and Chilomastix mesnili (3%). A total of 513 blood smears were examined and Wucheria bancrofti microfilariae were detected in 4% and malaria in 4% (Plasmodium falciparum 3%, Plasmodium vivax 2%). The malaria and filarial positive individuals lived in Beeuw, Waigeo and Arar, Sorong. These parasitic infections were not detected in people from Biak City and Sburia, Biak. Sera were collected from 357 persons and significant antibody titers were found for Entamoeba histolytica (4%) Toxoplasma gondii (7%), Influenza A2 Hong Kong 68 (65%), Influenza B Taiwan 68 (78%), Japanese encephalitis virus (87%) and Dengue 1 virus (79%). PMID:208184

  12. Parasitology survey in northern Sumatra, Indonesia.

    PubMed

    Cross, J H; Clarke, M D; Cole, W C; Lien, J C; Partono, F; Joesoef, A; Kosin, E H

    1976-06-01

    A parasitology survey was conducted in five villages in North Sumatra, Indonesia. A total of 3,207 blood smears, 2,066 stool specimens and 969 sera were examined. Sixty (1.9%) inhabitants had malaria (Plasmodium vivax 41, P. falciparum 19), and 20 had Brugia malayi microfilaraemia. The most common intestinal helminths were Trichuris trichiura (87%), Ascaris lumbricoides (75%) and hookworm (58%). Other helminths found in low numbers were Enterobius vermicularis, Strongyloides stercoralis, Taenia sp., Fasciolid, Dicrocoeliid and Echinostoma sp. eggs. Entamoeba coli (25%) was the most common intestinal protozoa followed by Endolimax nana (8%), Entamoeba histolytica (7%), Giardia lamblia (6%), Iodamoeba bütschlii (5%), Entamoeba hartmanni (1%) and Chilomastix mesnili (1%). The amoeba prevalence rate was 31 per cent. Testing of sera for Entamoeba histolytica and Toxoplasma gondii antibodies by the indirect haemagglutination test demonstrated positive reactors in 13 per cent and nine per cent of the population respectively. The greatest number of seropositives for Toxoplasma gondii was at elevations of sea level to five meters and the lowest number at elevations of 5OO-1,000 meters. PMID:950682

  13. Prevalence of intestinal parasites among primary schoolchildren in Derna District, Libya.

    PubMed

    Sadaga, Gazala A; Kassem, Hamed H

    2007-04-01

    The prevalence of intestinal parasites and some hygienic factors was evaluated in primary schoolchildren in Derna District. A total of 1039 stool specimens were examined by direct smear and formaline-ether concentration methods. The results showed that 31% of the children were infected with at least one or two parasites. These parasites were Giardia lamblia (12.7%), Blastocystis hominis (6.7%), Entamoeba histolytica/ Entamoeba dispar (6.6%), Entamoeba coli (3.2%), E. hartmanni (1.0%), Enterobius vermicularis (0.6%), Ascaris lumbricoides (0.1%) and Hymenolepis nana (0.1%). A significant difference was between infection rate and parent's education (P = 0.000), socio-economic status of the family (P = 0.000), family size and number of rooms in houses (P = 0.000). Also, there was a significant differences between infection rate and source of water for human consumption (P = 0.05). PMID:17580578

  14. Intestinal parasites of man in Bukidnon, Philippines, with emphasis on schistosomiasis.

    PubMed

    Carney, W P; de Veyra, V U; Cala, E M; Cross, J H

    1981-03-01

    A total of 831 fecal specimens from Malay-balay, Mindanao, Philippines were examined for schistosomiasis and common intestinal parasites. Schistosoma japonicum eggs were found in 16% of the samples. Infections were more common in males (20%) than in females (12%). Age-wise, infections were infrequently diagnosed in children less than 10 years and infections in older age groups varied from 12-27%. Hookworm, chiefly Necator americanus, was the most common parasite found (44%) followed by Ascaris lumbricoides (32%) Trichuris trichiura was found in only 12%. Enterobius vermicularis and eggs of Taenia sp., echinostome, heterophyid and dicrocoelid trematodes were identified in less than 1% of the samples. Entamoeba histolytica, Entamoeba coli, Entamoeba hartmanni, Endolimax nana, Giardia lamblia, Iodamoeba butschlii, Trichomonas hominis and Chilomastix mesnili were also found to parasitize man in this region of Mindanao. PMID:7256354

  15. Intestinal and blood parasites of man in Bireuen and Takengon, Aceh Province, Sumatra, Indonesia.

    PubMed

    Stafford, E E; Joesoef, A

    1976-12-01

    A survey for blood and intestinal parasites was carried out in Aceh Province of North Sumatra, Indonesia. A total of 348 stool specimens were obtained from 167 males and 181 females ranging in age from 6 months to 70 years. Over 98% of the population sampled were found infected with at least one intestinal parasite. Ascaris lumbricoides, Trichuris trichiura, hookworm and Entamoeba coli, in that order, were the most common parasites detected. Other intestinal parasites found less frequently were Entamoeba histolytica, Iodamoeba bütschlii, Entamoeba hartmanni, Endolimax nana and Giardia lamblia, Brugia malayi microfilaraemias were detected in 2% of those examined and only in the coastal villages of Cot Ketapang and Rusip Dayah. No malaria was found. PMID:1030850

  16. Intestinal parasites of man in Northern Bohol, Philippines, with emphasis on schistosomiasis.

    PubMed

    Carney, W P; Banzon, T; De Veyra, V; Daña, E; Cross, J H

    1980-12-01

    A survey for intestinal parasites with emphasis on Oriental schistosomiasis was conducted in the townships of Trinidad and Talibon, Bohol Province, Philippines and approximately 1,700 stool samples were examined. Schistosoma japonicum is still endemic to these areas of northern Bohol but infection rates were much lower than expected, 5% rather than 30%. Soil-transmitted helminths were the most common parasitic infections. Hookworms were found in 71% of the samples tested, most infections were due to Necator americanus 97% and Ancylostoma duodenale accounted for only 3%. Trichuris trichiura and Ascaris lumbricoides eggs were diagnosed in 58% and 45% respectively of the fecal samples examined. Other helminths and protozoan parasites detected were Enterobius vermicularis, Stronglyoides stercoralis, Entamoeba histolytica, Entamoeba hartmanni, Entamoeba coli, Endolimax nana, Iodamoeba bütschlii, Giardia lamblia and Chilomastix mesnili. PMID:7221688

  17. Human intestinal parasites in Karakuak, West Flores, Indonesia and the effect of treatment with mebendazole and pyrantel pamoate.

    PubMed

    Purnomo; Partono, F; Soewarta, A

    1980-09-01

    A survey for intestinal parasites and mass-treatment with a combination of mebendazole and pyrantel pamoate were conducted in Karakuak, West Flores in 1977. A total of 198 stool specimens from 104 males and 94 females ranging in age from less than 1 to 70 years were examined and 72% harbored one or more intestinal parasites. Ascaris lumbricoides (43%) and Entamoeba histolytica (21%) were the most common, followed by Entamoeba coli (19%), hookworm (18%), Iodamoeba bütschlii (8%), Giardia lamblia (5%) and Trichuris trichiura (4%). Other intestinal parasites infrequently found were: Entamoeba hartmanni (2%), Chilomastix mesnili (2%), Endolimax nana (1%), Enterobius vermicularis (1%) and a heterophyid sp. (1%). A combination of mebendazole base at 200 mg/day and pyrantel pamoate salt at 60 mg/day for three consecutive days was 100% effective. PMID:7444573

  18. Biomedical survey in North Samar Province, Philippine Islands.

    PubMed

    Cross, J H; Banzon, T; Wheeling, C H; Cometa, H; Lien, J C; Clarke, R; Petersen, H; Sevilla, J; Basaca-Sevilla, V

    1977-12-01

    A biomedical survey was carried out in North Samar Province, Philipines to update information on the prevalence of parasitic and other infectious diseases. A total of 1,394 stool specimens, 1,859 blood smears and 1,274 sera were collected from persons living in 8 barrios. Stools were examined for intestinal parasites, bloods smears for malaria and filariasis and sera tested for antibodies to Schistosoma japonicum, Entamoeba histolytica, Toxoplasma gondii, influenza A and B, and Japanese encephalitis virus. The prevalence rates for intestinal parasites were: Trichuris trichiura 90%, Ascaris lumbricoides 78%, hookworm 65%, Schistosoma japonicum 15%, Strongyloides stercoralis 1%, Entamoeba coli 16%, Endolimax nana 6%, entamoeba histolytica 5%, Giardia lamblia 3%, Entaemoeba hartmanii 1%, Chilomastix mesnili 1%. No malaria was found but microfilariae of Wuchereria bancrofti were detected in 4% of the blood smears; the MfD50 was 12.9. The circumoval precipitin test (COPT) was used to detect antibodies to Schistosoma japonicum and 65% of 994 sera was considered positive. The indirect hemagglutination test (IHA) was used for detecting antibodies to Entamoeba histolytica and Toxoplasma gondii and 5% and 3% of 1,274 sera tested were positive at titers equal to or greater than 1:128 and 1:256, respectively. Hemagglutination inhibition tests (HI) were used to detect antibodies to Influenza A2HK68, Influenza A2HK68, Influenza B2T62 and Japanese encephalitis virus and 72%, 12% and 78%, respectively, of 1201 sera were considered positive at titers equal to or greater than 1:20. PMID:614707

  19. In vitro and in vivo studies of macrophage functions in amebiasis.

    PubMed Central

    Denis, M; Chadee, K

    1988-01-01

    Experimental intrahepatic inoculation of the gerbil with Entamoeba histolytica trophozoites was used as a model of liver amebiasis to study the cellular immune response elicited by the parasite. It was shown that abscess-derived macrophages (5 to 20 days old) were deficient in their capacity to develop a respiratory burst, to secrete and express membrane-bound interleukin-1-like activity, and to kill E. histolytica trophozoites as well as to respond to lymphokines in vitro. However, macrophages isolated from the spleen and peritoneal cavities from the same infected animals were not significantly down regulated in these functions. Splenocytes from infected gerbils were shown to develop a strong responsiveness to amebic antigen, whereas their response to concanavalin A was suppressed. Crude E. histolytica extracts or conditioned medium down regulated murine BALB/c macrophage accessory and effector cell functions in vitro in a manner similar to abscess-derived macrophages, whereas crude extracts of the nonvirulent E. histolytica-like Laredo strain did not. Our results indicate that intrinsic or secreted products or both from E. histolytica are actively regulating macrophage functions at the abscess site and can possibly mediate other immunoregulatory mechanisms at distant targets. PMID:2903124

  20. [Carriers and excretors of protozoa].

    PubMed

    Eckert, J

    1993-02-01

    Causative agents of intestinal infections in humans are about 10 pathogenic or facultative pathogenic species of protozoa of which Giardia lamblia, Entamoeba histolytica, Cryptosporidium parvum and Enterozytozoon bieneusi are discussed. G. lamblia from humans is morphologically indistinguishable from Giardia isolates originating from several species of domestic and wild mammals. Swiss Giardia isolates of human, sheep, cattle and dog origin could be transmitted to Giardia-free rodents and were rather homogenous in biochemical parameters. These data support the hypothesis that zoonotic transmission of Giardia may occur. Routine faecal examinations in Zürich in 1991 revealed 3.61% of Giardia excretors among 5017 examined patients. Of the same group of persons 3.95% excreted E. histolytica. At present it is anticipated that the species E. histolytica consists of invasive and non-invasive strains which can be differentiated by isoenzyme electrophoresis. The confirmation of this assumption could deeply influence the opinion about medical and epidemiological significance of Entamoeba infections. Cryptosporidium parvum is an important cause of diarrhea in HIV-infected patients. Person-to-person, zonnotic and waterborne transmission may play a role. A short review of new data on Enterozytozoon bieneusi and other microsporidia is presented. E. bieneusi appears to be an important causative agent of diarrhea in HIV-infected persons. PMID:8333895

  1. Prevalence of enteric protozoa in human immunodeficiency virus (HIV)-positive and HIV-negative men who have sex with men from Sydney, Australia.

    PubMed

    Stark, Damien; Fotedar, Rashmi; van Hal, Sebastian; Beebe, Nigel; Marriott, Deborah; Ellis, John T; Harkness, John

    2007-03-01

    A prospective, comparative study of the prevalence of enteric protozoa was determined among human immunodeficiency virus (HIV)- positive and HIV-negative men who have sex with men (MSM) in Sydney, Australia. A total of 1,868 patients submitted stool specimens; 1,246 were from MSM (628 HIV positive and 618 HIV positive) and 622 from non-MSM were examined over a 36-month period. A total of 651 (52.2%) stool specimens from MSM were positive for protozoa compared with 85 (13%) from non-MSM. There was a significant difference in the prevalence of Blastocystis hominis, Endolimax nana, Entamoeba histolytica/dispar complex, Entamoeba hartmanni, Iodamoeba butschlii, and Enteromonas hominis detected between MSM and non-MSM (P<0.001). The only notable difference between HIV-negative and HIV-positive MSM was that HIV-infected MSM were found to more likely have a Cryptosporidium parvum infection. Entamoeba histolytica was found in 3 patients, E. dispar in 25, and E. moshkovskii in 17, all of whom were MSM. When compared with a control group, MSM were significantly more likely to harbor intestinal protozoa and have multiple parasites present. The results of this study show high rates of enteric parasites persist in MSM and highlight the importance of testing for intestinal parasites in MSM. This is the first report of E. moshkovskii from MSM. PMID:17360882

  2. Hymenolepis nana: a common cause of paediatric diarrhoea in urban slum dwellers in India.

    PubMed

    Mirdha, Bijay Ranjan; Samantray, Jyotish Chandra

    2002-12-01

    The prevalence of intestinal parasitic infections was studied for a period of 5 years (April 1996-April 2001) among urban slum dwellers. All age groups were represented in the study. Parasitological examinations were performed on 939 faecal specimens collected on a household basis. The total prevalence of pathogenic parasites was 33.6 per cent. No significant age and sex differences in pathogenic parasites were observed. The prevalence of intestinal helminths and pathogenic protozoa was as follows: Hymenolepis nana (9.9 per cent), Ascaris lumbricoides (8.5 per cent), Giardia lamblia (8.4 per cent) and Entamoeba histolyticaldispar (3.7 per cent). Thirty-four E. histolytica/dispar positive samples were cultured and speciation was done using polymerase chain reaction (PCR). The predominant isolate was E. dispar compared to E. histolytica. The notable finding of the present study was high prevalence of Hymenolepis nana compared with other parasitic infections in slum dwellers. PMID:12521273

  3. Primary Pulmonary Amebiasis Complicated with Multicystic Empyema

    PubMed Central

    Al-Share, Bayan; Al Asad, Khaled

    2016-01-01

    Amebiasis is a parasitic infection caused by the protozoan Entamoeba histolytica. While most infections are asymptomatic, the disease could manifest clinically as amebic dysentery and/or extraintestinal invasion in the form of amebic liver abscess or other more rare manifestations such as pulmonary, cardiac, or brain involvement. Herein we are reporting a case of a 24-year-old male with history of Down syndrome who presented with severe right side pneumonia complicated with multicystic empyema resistant to regular medical therapy. Further investigation revealed a positive pleural fluid for E. histolytica cysts and trophozoites. The patient was diagnosed with primary pleuropulmonary amebiasis and he responded promptly to surgical drainage and metronidazole therapy. In patients from endemic areas all physicians should keep a high index of suspicion of amebiasis as a cause of pulmonary disease. PMID:27478673

  4. Structure and biochemical characterization of proliferating cellular nuclear antigen from a parasitic protozoon

    SciTech Connect

    Cardona-Felix, Cesar S.; Lara-Gonzalez, Samuel; Brieba, Luis G.

    2012-02-08

    Proliferating cellular nuclear antigen (PCNA) is a toroidal-shaped protein that is involved in cell-cycle control, DNA replication and DNA repair. Parasitic protozoa are early-diverged eukaryotes that are responsible for neglected diseases. In this work, a PCNA from a parasitic protozoon was identified, cloned and biochemically characterized and its crystal structure was determined. Structural and biochemical studies demonstrate that PCNA from Entamoeba histolytica assembles as a homotrimer that is able to interact with and stimulate the activity of a PCNA-interacting peptide-motif protein from E. histolytica, EhDNAligI. The data indicate a conservation of the biochemical mechanisms of PCNA-mediated interactions between metazoa, yeast and parasitic protozoa.

  5. Primary Pulmonary Amebiasis Complicated with Multicystic Empyema.

    PubMed

    Zakaria, Ali; Al-Share, Bayan; Al Asad, Khaled

    2016-01-01

    Amebiasis is a parasitic infection caused by the protozoan Entamoeba histolytica. While most infections are asymptomatic, the disease could manifest clinically as amebic dysentery and/or extraintestinal invasion in the form of amebic liver abscess or other more rare manifestations such as pulmonary, cardiac, or brain involvement. Herein we are reporting a case of a 24-year-old male with history of Down syndrome who presented with severe right side pneumonia complicated with multicystic empyema resistant to regular medical therapy. Further investigation revealed a positive pleural fluid for E. histolytica cysts and trophozoites. The patient was diagnosed with primary pleuropulmonary amebiasis and he responded promptly to surgical drainage and metronidazole therapy. In patients from endemic areas all physicians should keep a high index of suspicion of amebiasis as a cause of pulmonary disease. PMID:27478673

  6. Current Therapeutics, Their Problems, and Sulfur-Containing-Amino-Acid Metabolism as a Novel Target against Infections by “Amitochondriate” Protozoan Parasites

    PubMed Central

    Ali, Vahab; Nozaki, Tomoyoshi

    2007-01-01

    The “amitochondriate” protozoan parasites of humans Entamoeba histolytica, Giardia intestinalis, and Trichomonas vaginalis share many biochemical features, e.g., energy and amino acid metabolism, a spectrum of drugs for their treatment, and the occurrence of drug resistance. These parasites possess metabolic pathways that are divergent from those of their mammalian hosts and are often considered to be good targets for drug development. Sulfur-containing-amino-acid metabolism represents one such divergent metabolic pathway, namely, the cysteine biosynthetic pathway and methionine γ-lyase-mediated catabolism of sulfur-containing amino acids, which are present in T. vaginalis and E. histolytica but absent in G. intestinalis. These pathways are potentially exploitable for development of drugs against amoebiasis and trichomoniasis. For instance, l-trifluoromethionine, which is catalyzed by methionine γ-lyase and produces a toxic product, is effective against T. vaginalis and E. histolytica parasites in vitro and in vivo and may represent a good lead compound. In this review, we summarize the biology of these microaerophilic parasites, their clinical manifestation and epidemiology of disease, chemotherapeutics, the modes of action of representative drugs, and problems related to these drugs, including drug resistance. We further discuss our approach to exploit unique sulfur-containing-amino-acid metabolism, focusing on development of drugs against E. histolytica. PMID:17223627

  7. Development of a multiplex PCR assay to detect gastroenteric pathogens in the feces of Mexican children.

    PubMed

    Tolentino-Ruiz, R; Montoya-Varela, D; García-Espitia, M; Salas-Benito, M; Gutiérrez-Escolano, A; Gómez-García, C; Figueroa-Arredondo, P; Salas-Benito, J; De Nova-Ocampo, M

    2012-10-01

    Acute gastroenteritis (AGE) is a major cause of childhood morbidity and mortality worldwide; the etiology of AGE includes viruses, bacteria, and parasites. A multiplex PCR assay to simultaneously identify human Astrovirus (HAstV), Calicivirus (HuCVs), Entamoeba histolytica (E. histolytica), and enteroinvasive Escherichia coli (EIEC) in stool samples is described. A total of 103 samples were individually analyzed by ELISA (enzyme-linked immunosorbent assays) and RT-PCR/PCR. HAstV and HuCVs were detected in four out of 103 samples (3.8 %) by RT-PCR, but ELISAs found only one sample as positive for HuCVs (2.5 %). E. histolytica was identified in two out of 19 samples (10.5 %) and EIEC in 13 out of 20 samples (70 %) by PCR, and all PCR products were sequenced to verify their identities. Our multiplex PCR results demonstrate the simultaneous amplification of different pathogens such as HAstV, EIEC, and E. histolytica in the same reaction, though the HuCVs signal was weak in every replicate. Regardless, this multiplex PCR protocol represents a novel tool for the identification of distinct pathogens and may provide support for the diagnosis of AGE in children. PMID:22711331

  8. Laboratory Diagnosis of Amebiasis

    PubMed Central

    Tanyuksel, Mehmet; Petri, William A.

    2003-01-01

    The detection of Entamoeba histolytica, the causative agent of amebiasis, is an important goal of the clinical microbiology laboratory. To assess the scope of E. histolytica infection, it is necessary to utilize accurate diagnostic tools. As more is discovered about the molecular and cell biology of E. histolytica, there is great potential for further understanding the pathogenesis of amebiasis. Molecular biology-based diagnosis may become the technique of choice in the future because establishment of these protozoa in culture is still not a routine clinical laboratory process. In all cases, combination of serologic tests with detection of the parasite (by antigen detection or PCR) offers the best approach to diagnosis, while PCR techniques remain impractical in many developing country settings. The detection of amebic markers in serum in patients with amebic colitis and liver abscess appears promising but is still only a research tool. On the other hand, stool antigen detection tests offer a practical, sensitive, and specific way for the clinical laboratory to detect intestinal E. histolytica. All the current tests suffer from the fact that the antigens detected are denatured by fixation of the stool specimen, limiting testing to fresh or frozen samples. PMID:14557296

  9. Amebiasis.

    PubMed Central

    Bruckner, D A

    1992-01-01

    Entamoeba histolytica, the causative agent of amebiasis, was first described in 1875. Although a large number of people throughout the world are infected with this organism, only a small percentage will develop clinical symptoms. Morbidity and mortality due to E. histolytica vary from area to area and person to person. Recent findings have suggested that there are pathogenic and nonpathogenic strains of E. histolytica that can be differentiated by isoenzyme (zymodeme) analysis, monoclonal antibodies, and DNA probes. Whether pathogenicity is a genotypic trait or can be changed by environmental influences has not been resolved. Exchange of genetic material between strains of amebae can influence zymodeme patterns. Currently, detection of E. histolytica infections depends on examinations for ova and parasites and on serologic tests; however, the development of monoclonal antibodies and DNA probes specific for pathogenic zymodemes may be beneficial for clinical laboratory testing and therapeutic decisions when approved tests become available. A better understanding of the mechanisms of pathogenicity at the molecular level is evolving and should promote the development of vaccines and better target selection for therapeutic agents. PMID:1423215

  10. Comparison of microscopic and immunoassay examination in the diagnosis of intestinal protozoa of humans in Mansoura, Egypt.

    PubMed

    Elswaifi, Shaadi F; Palmieri, James R; El-Tantawy, Nora; El-Hussiny, Mona; Besheer, Tarek; Abohashem, Ekbal

    2016-09-01

    Protozoal diseases are prevalent globally and especially in developing countries that have relatively lower socioeconomic populations such as Egypt. Direct microscopic examination (DME) is used for the detection and identification of protozoa but lacks sufficient reliability, and thus may be detrimental in obtaining accurate diagnostic or epidemiological data. In this study, we determine the prevalence of infections by Giardia intestinalis, Cryptosporidium sp., and Entamoeba histolytica in humans in Egypt. Furthermore, we determine the reliability of DME in determining infections caused by these protozoa and compare the results to enzyme linked Immunosorbent assays (ELISA). Our results indicate that the prevalence of giardiasis, cryptosporidiosis, and entamoebiasis is 38, 22, and 16 %, respectively. The sensitivity and specificity of DME for detection of G. intestinalis is 45 and 99 %, for Cryptosporidium 66 and 99 %, and for Entamoeba 45 and 100 %, respectively. Our findings demonstrate that ELISA is more reliable for diagnostic and epidemiologic study purposes. PMID:27605751

  11. Socio-economic factors associated with intestinal parasites among children living in Gombak, Malaysia.

    PubMed

    Rajeswari, B; Sinniah, B; Hussein, H

    1994-01-01

    Fecal specimens collected from 456 school children in Gombak, Malaysia, revealed an overall prevalence rate of 62.9%. The most common parasite found was Trichuris trichiura (47.1%) followed by Giardia intestinalis (14.7%), Entamoeba coli (11.4%), Entamoeba histolytica (9.9%) and Ascaris lumbricoides (7.9%). Infection rates were high among the Indonesian immigrant workers' children (90%) followed by the Orang Asli (79.5%), Malay (59.4%) and Indians (36.4%). Females (66.3%) had a higher prevalence rate than the males (58.5%). The prevalence of infection was found to be associated with the socio-economic status, water supply, sanitary disposal of feces and family size. Albendazole administered as a single dose (400 mg) was found to be effective against Ascaris (100%) and hookworm (92.3%) but was not effective against Trichuris (39.2%). PMID:8074940

  12. Enteric pathogenic protozoa in homosexual men from San Francisco.

    PubMed

    Ortega, H B; Borchardt, K A; Hamilton, R; Ortega, P; Mahood, J

    1984-01-01

    The prevalence of enteric protozoa was studied in a survey conducted among 150 male homosexual patients in San Francisco. All patients were from a private practice in internal medicine. Each was asked to complete a questionnaire and to submit multiple stool specimens for examination. Of this group, 47% were positive for one or more potentially pathogenic intestinal protozoa; Entamoeba histolytica was found in 36%, Entamoeba hartmanni in 35%, Giardia lamblia in 5%, and Dientamoeba fragilis in 1.3%. Symptoms were unreliable as a diagnostic index of intestinal protozoan infection. Colonization rates could not be correlated with any specific sexual technique. The large number of homosexuals at risk, combined with the potential for difficulties in diagnosis and contact-tracing, indicate the possibility that enteric pathogenic protozoa will cause future health problems in this population. PMID:6087479

  13. Epidemiological survey of the prevalence of parasites among children in Khan Younis governorate, Palestine.

    PubMed

    Astal, Z

    2004-12-01

    The prevalence of intestinal parasites was determined for 1,370 children in Khan Younis Governorate, Gaza Strip. The age of the children ranged from 6 to 11 years. For stool samples, inspection, direct smear microscopy, flotation and sedimentation techniques were used. The general prevalence of intestinal parasites was 34.2%. Different types of intestinal parasites were detected during this survey: Ascaris lumbricoides seemed to be the most common parasite (12.8%), whereas Giardia lamblia had a prevalence of 8.0%, Entamoeba histolytica 7.0%, Entamoeba coli 3.6%, Trichuris trichiura 1.6% and Hymenolepis nana 1.0%. The prevalence of enterobiasis was determined using a scotch tape preparation. A total of 20.9% of the children examined were infected and there was sex variation in the prevalence of enterobiasis. PMID:15517386

  14. Cockroaches and flies in mechanical transmission of medical important parasites in Khaldyia Village, El-Fayoum, Governorate, Egypt.

    PubMed

    El-Sherbini, Gehad T; Gneidy, Morsy Rateb

    2012-04-01

    The role of non-blood sucking insects in dissemination of human parasites was investigated in Khaldyia Village, Al-Fayoum Governorate over during the summer of 2011. A total of 278 American cockroach (Periplaneta americana), and 508 house flies Musca domestica var. vicina were collected. The insects were collected indoors and outdoors. Flies were abundant in defecation areas and around houses. The recovered zoonotic parasites identified were cysts of Entamoeba histolytica Cryptosporidium parvum and Balantidium coli, and eggs of Ascaris lumbricoides, Anchylostoma deodunale, Enterobius vermicularis, and Trichuris trichura as well as larvae of Strongyloides stercoralis. PMID:22662605

  15. [Prevalence of intestinal parasitoses in a sample of Italian and immigrant workers employed in the food sector of Turin].

    PubMed

    Rosso, S; Miotti, T

    1991-06-01

    We studied the prevalence of intestinal parasites in a random sample of immigrants and Italians employed in the food industry. Prevalence ranged from 20.8% among immigrants to 5.6% among Italians. Where helminths are widespread among Indochinese, protozoa are more common among africans. Three cases of Entamoeba histolytica in asymptomatic subjects were identified and successfully treated. We observed high levels of Giardia intestinalis both in Italians (2.2%) and immigrants (3.9%). Prevalence of intestinal protozoa was significantly associated with year of immigration, showing higher levels for new immigrants. The public health implications of parasite screening and treatment among recent immigrants were also discussed. PMID:1838507

  16. Amebic infections in asymptomatic homosexual men, lack of evidence of invasive disease.

    PubMed Central

    Sorvillo, F J; Strassburg, M A; Seidel, J; Visvesvara, G S; Mori, K; Todd, A; Portigal, L; Finn, M; Agee, B A

    1986-01-01

    A survey for enteric infections in 140 asymptomatic homosexual men who attended a community clinic revealed a high prevalence of infection with Entamoeba histolytica (27.1 per cent) and Giardia lamblia (15.7 per cent). In contrast, the prevalence of elevated indirect hemagglutination (IHA) titers (greater than or equal to 1:128), which indicate invasive amebiasis, was low (5.7 per cent). Our findings suggest that only a limited amount of invasive amebic disease is occurring in this group of homosexual men. PMID:2874747

  17. Surveillance for parasites in unaccompanied minor refugees migrating to Germany in 2015.

    PubMed

    Heudorf, Ursel; Karathana, Maria; Krackhardt, Bernhard; Huber, Meike; Raupp, Peter; Zinn, Christian

    2016-01-01

    In 2015, most of the refugees arriving in Germany originated from countries with poor hygienic and sanitary conditions. Stool samples of 1,230 minor refugees unaccompanied by adults were investigated for possible parasites. Giardia lamblia was by far the most frequently detected parasite (n=165); all other parasites were considerably less frequent and encountered in the following order: Hymenolepis nana (n=23), Entamoeba histolytica (n=17), Trichuris trichiura (n=8), and Blastocystis hominis (n=1). Ascaris lumbricoides was not detected among any of the screened refugees. Considerable differences in prevalence rates in refugees originating from different countries could be observed. PMID:26958459

  18. Crosstalk at the initial encounter: Interplay between host defense and ameba survival strategies

    PubMed Central

    Guo, Xiaoti; Houpt, Eric; Petri, William A.

    2009-01-01

    The host-parasite relationship is based on a series of interplays between host defense mechanisms and parasite survival strategies. Progress has been made in understanding the role of host immune response in amebiasis. While host cells elaborate diverse mechanisms for pathogen expulsion, amebae have also developed complex strategies to modulate host immune response and facilitate their own survival. This paper will give an overview of current research on the mutual interactions between host and Entamoeba histolytica in human and experimental amebiasis. Understanding this crosstalk is crucial for the effective design and implementation of new vaccines and drugs for this leading parasitic disease. PMID:17702556

  19. Balantidium Coli liver abscess: first case report from India.

    PubMed

    Kapur, P; Das, A K; Kapur, P R; Dudeja, M

    2016-03-01

    Protozoal infections are common in the tropics. Amoebic colitis is the commonest of these infections and can lead to liver abscess as a complication. Balantidium coli is a rare free moving protozoal parasite which is known to infest human large intestine causing a type of colitis very similar to that caused by Entamoeba histolytica. However this pathogen is not known to cause liver invasion in humans. We report here a case of liver abscess caused by B. coli, which is probably the first such case reported in Indian literature. PMID:27065613

  20. Neglected waterborne parasitic protozoa and their detection in water.

    PubMed

    Plutzer, Judit; Karanis, Panagiotis

    2016-09-15

    Outbreak incidents raise the question of whether the less frequent aetiological agents of outbreaks are really less frequent in water. Alternatively, waterborne transmission could be relevant, but the lack of attention and rapid, sensitive methods to recover and detect the exogenous stages in water may keep them under-recognized. High quality information on the prevalence and detection of less frequent waterborne protozoa, such as Cyclospora cayetanensis, Toxoplasma gondii, Isospora belli, Balantidium coli, Blastocystis hominis, Entamoeba histolytica and other free-living amoebae (FLA), are not available. This present paper discusses the detection tools applied for the water surveillance of the neglected waterborne protozoa mentioned above and provides future perspectives. PMID:27281375

  1. Regioselective phosphorylation of myo-inositol with BINOL-derived phosphoramidites and its application for protozoan lysophosphatidylinositol.

    PubMed

    Aiba, Toshihiko; Sato, Masaki; Umegaki, Daichi; Iwasaki, Takanori; Kambe, Nobuaki; Fukase, Koichi; Fujimoto, Yukari

    2016-07-12

    A regioselective phosphorylation method for myo-inositol was developed by utilizing readily preparable BINOL-derived phosphoramidites. The method also facilitated the complete separation of the diastereomeric products by simple chromatography. Based on this phosphorylation and Ni-catalyzed alkyl-alkyl cross-coupling reaction for long fatty acids, we achieved the first synthesis of a lysophosphatidylinositol, EhPIa having long fatty acid C30:1, as a partial structure of glycosylphosphatidylinositol (GPI) anchor from the cell membrane of a protozoa, Entamoeba histolytica. PMID:27326923

  2. Sexually transmitted infections manifesting as proctitis.

    PubMed

    Lamb, Chris A; Lamb, Elizabeth Iris Mary; Mansfield, John C; Sankar, K Nathan

    2013-01-01

    There is a rising incidence of several sexually transmitted infections (STIs), many of which can present with proctitis. Causative organisms include Neisseria gonorrhoeae, Chlamydia trachomatis, herpes simplex virus, Treponema pallidum (syphilis), Giardia lamblia (giardiasis) and Entamoeba histolytica (amoebiasis). This paper outlines important clinical discriminators and key investigations to distinguish these organisms from non-infective pathology that include inflammatory bowel disease, solitary rectal ulcer syndrome and Behçet's syndrome. Management of these infections is described and suggestions are made for successful gastroenterology clinical consultation when an STI is suspected. PMID:23914292

  3. Surveillance for parasites in unaccompanied minor refugees migrating to Germany in 2015

    PubMed Central

    Heudorf, Ursel; Karathana, Maria; Krackhardt, Bernhard; Huber, Meike; Raupp, Peter; Zinn, Christian

    2016-01-01

    In 2015, most of the refugees arriving in Germany originated from countries with poor hygienic and sanitary conditions. Stool samples of 1,230 minor refugees unaccompanied by adults were investigated for possible parasites. Giardia lamblia was by far the most frequently detected parasite (n=165); all other parasites were considerably less frequent and encountered in the following order: Hymenolepis nana (n=23), Entamoeba histolytica (n=17), Trichuris trichiura (n=8), and Blastocystis hominis (n=1). Ascaris lumbricoides was not detected among any of the screened refugees. Considerable differences in prevalence rates in refugees originating from different countries could be observed. PMID:26958459

  4. Review: amebic liver abscess in children - the role of diagnostic imaging

    SciTech Connect

    Merten, D.F.; Kirks, D.R.

    1984-12-01

    Amebiasis, infection with the protozoan Entamoeba histolytica, affects at least 10% of the world's population, with an incidence exceeding 30% in tropical and subtropical regions. Amebic liver abscess (ALA) is the most common extraintestinal form of invasive amebiasis and is a serious life-threatening disease in children. Recent experience indicates the prognosis of ALA in childhood to be improved with early identification of abscesses and prompt institution of treatment. The authors incorporate experience with a case of ALA in infancy with a review of current literature (1974-1983) to delineate clinical and radiologic features of ALA in childhood and further define the role of hepatic imaging in the diagnosis and treatment.

  5. [Protozoological stool examinations from 1980 to 1985 in the East German district of Rostock].

    PubMed

    Petersen, K

    1988-04-01

    Between 1980 and 1985 a total of 17,290 faecal specimens were examined for intestinal Protozoa at the Institute of Hygiene, District of Rostock, GDR. The samples were taken from GDR citizens travelling to tropical or semitropical regions, from foreigners from these regions visiting Rostock District, and from the local population. Intestinal Protozoa were found in 5.78%, 20.80% and 6.43%, respectively. 9 different species were diagnosed: Entamoeba coli, E. hartmanni, E. histolytica, Endolimax nana, Jodamoeba buetschlii, Giardia lamblia, Dientamoeba fragilis, Chilomastix mesnili, Trichomonas hominis. PMID:3415039

  6. Antiprotozoal activity of the constituents of Teloxys graveolens.

    PubMed

    Calzada, Fernando; Velázquez, Claudia; Cedillo-Rivera, Roberto; Esquivel, Baldomero

    2003-08-01

    Antiprotozoal activity-guided fractionation of a methanol extract of the aerial parts of Teloxys graveolens led to the isolation of one coumarinic acid derivative, melilotoside, and five flavonoids, pinocembrine, pinostrobin, chrysin, narcissin and rutin. Among them, melilotoside exhibited the most potent activity toward Entamoeba histolytica and Giardia lamblia (IC(50) 12.5 and 16.8 micro g/mL, respectively). Narcissin showed selectivity against E. histolytlica (IC(50) 17.2 micro g/mL). The results support data for the traditional use of T. graveolens in some gastrointestinal diseases. PMID:12916068

  7. Antiprotozoal activity of the constituents of Rubus coriifolius.

    PubMed

    Alanís, Alma Delia; Calzada, Fernando; Cedillo-Rivera, Roberto; Meckes, Mariana

    2003-06-01

    Extraction of the aerial parts of Rubus coriifolius, a medicinal plant used by the Maya communities in Southern Mexico to treat bloody diarrhoea, resulted in the isolation of seven known compounds (-)-epicatechin, (+)-catechin, hyperin, nigaichigoside F1, beta-sitosterol 3-O-beta-d-glucopyranoside, gallic acid and ellagic acid. All compounds were tested for their antiprotozoal activity against Entamoeba histolytica and Giardia lambia. Epicatechin was the main responsible for the antiprotozoal activity of the extract against both protozoa, its activity was comparable to emetine, but no exceeded that of metronidazole. PMID:12820241

  8. Structure, absolute configuration, and antidiarrheal activity of a thymol derivative from Ageratina cylindrica.

    PubMed

    Bustos-Brito, Celia; Sánchez-Castellanos, Mariano; Esquivel, Baldomero; Calderón, José S; Calzada, Fernando; Yepez-Mulia, Lilian; Hernández-Barragán, Angelina; Joseph-Nathan, Pedro; Cuevas, Gabriel; Quijano, Leovigildo

    2014-02-28

    The leaves of Ageratina cylindrica afforded a thymol derivative that was characterized by physical and spectroscopical methods as (8S)-8,9-epoxy-6-hydroxy-l0-benzoyloxy-7-oxothymol isobutyrate (1). The absolute configuration of 1 was established as 8S by vibrational circular dichroism spectroscopy in combination with density functional theory calculations and by evaluation of the Flack and Hooft X-ray parameters. Compound 1 showed weak antiprotozoal activity against Entamoeba histolytica and Giardia lamblia trophozoites and a high inhibitory effect on hyperpropulsive movement of the small intestine in rats. PMID:24502360

  9. Prevalence of intestinal protozoa infection among school-aged children on Pemba Island, Tanzania, and effect of single-dose albendazole, nitazoxanide and albendazole-nitazoxanide

    PubMed Central

    2013-01-01

    Background Pathogenic intestinal protozoa infections are common in school-aged children in the developing world and they are frequently associated with malabsorption syndromes and gastrointestinal morbidity. Since diagnosis of these parasites is difficult, prevalence data on intestinal protozoa is scarce. Methods We collected two stool samples from school-aged children on Pemba Island, Tanzania, as part of a randomized controlled trial before and 3 weeks after treatment with (i) single-dose albendazole (400 mg); (ii) single-dose nitazoxanide (1,000 mg); (iii) nitazoxanide-albendazole combination (1,000 mg–400 mg), with each drug given separately on two consecutive days; and (iv) placebo. Formalin-fixed stool samples were examined for the presence of intestinal protozoa using an ether-concentration method to determine the prevalence and estimate cure rates (CRs). Results Almost half (48.7%) of the children were diagnosed with at least one of the (potentially) pathogenic protozoa Giardia intestinalis, Entamoeba histolytica/E. dispar and Blastocystis hominis. Observed CRs were high for all treatment arms, including placebo. Nitazoxanide showed a significant effect compared to placebo against the non-pathogenic protozoon Entamoeba coli. Conclusions Intestinal protozoa infections might be of substantial health relevance even in settings where they are not considered as a health problem. Examination of a single stool sample with the ether-concentration method lacks sensitivity for the diagnosis of intestinal protozoa, and hence, care is indicated when interpreting prevalence estimates and treatment effects. PMID:23289920

  10. Comparative Study of the Efficacy and Tolerability of Secnidazole Suspension (single dose) and Tinidazole Suspension (two days dosage) in the Treatment of Amebiasis in Children.

    PubMed

    Salles; Bechara; Tavares; Martins; Sobrinho; Dietrich-Neto; Costa

    1999-04-01

    Amebiasis caused by Entamoeba histolytica may be considered the most aggressive parasitic disease affecting human intestine, causing acute amoebic colitis and extra-intestinal diseases of high morbidity and mortality. 5-nitroimidazoles are the drugs of choice. In this multicenter, open and random clinical trial, the efficacy and tolerability of secnidazole suspension in a single oral dose of 1ml/kg was compared with 0.5ml/kg doses of tinidazole suspension given for 2 consecutive days to 303 Entamoeba histolytica-positive children aged 2 to 13. Patients with extra-intestinal complications were excluded from the study. Clinical and parasitological follow-up using the Faust and Kato-Katz method were carried out 7, 14, and 21 days after treatment. Clinical improvement/cure was observed in 93% of the patients in the secnidazole group and 91% in the tinidazole group. Parasitological success was reported for 77% and 63% of the secnidazole and tinidazole patients, respectively, showing a significant statistical difference between the two groups (p=0.007). Both drugs were well tolerated, and the adverse effects reported were mild, consisting mainly of digestive disturbances. This comparative study showed that a single oral dose of 1ml/kg of secnidazole produced a significantly higher parasitological cure rate than 2 doses of tinidazole. Secnidazole is a safe and effective drug for the treatment of uncomplicated intestinal amebiasis. PMID:11098194

  11. Pulmonary Thromboembolism Complicating Amebic Liver Abscess: First Reported Case in the United States—Case Report and Literature Review

    PubMed Central

    McKenzie, Devon; Gale, Michael; Patel, Sunny; Kaluta, Grazyna

    2015-01-01

    Even in the absence of Amebic colitis, Amebic liver abscess (ALA) is the most common extraintestinal complication of Entamoeba histolytica infection. In the USA, it is most prevalent in middle aged immigrant males from endemic countries such as Africa, Mexico, and India. One of the complications of ALA is inferior vena cava (IVC) thrombosis, which is believed to result from the mechanical compression of the IVC and the consequent thrombogenic nidus elicited from the resultant inflammatory response. There are very few reported cases and even fewer in which the thrombus became a harbinger to pulmonary thromboembolism. We present the case of a 43-year-old male from West Africa who presented with the chief complaint of right upper quadrant abdominal pain for one week associated with persistent nonproductive cough. He had a positive serum Entamoeba histolytica antibody with CT scan findings of a hepatic abscess with thrombosis of the hepatic vein and inferior vena cava and numerous bilateral pulmonary emboli. This amebic liver abscess was successfully treated with metronidazole and paromomycin, whereas the pulmonary thromboembolism was managed with medical anticoagulation. Based on current knowledge, this is the first reported case in the USA. PMID:26294987

  12. Effects of Piper longum fruit, Piper sarmentosum root and Quercus infectoria nut gall on caecal amoebiasis in mice.

    PubMed

    Sawangjaroen, Nongyao; Sawangjaroen, Kitja; Poonpanang, Pathana

    2004-04-01

    The anti-amoebic effects of crude methanol extracts of Piper longum fruit, Piper sarmentosum root and Quercus infectoria nut gall against Entamoeba histolytica infecting the caecum of mice were studied. Caecal amoebiasis in mice was induced by injection of Entamoeba histolytica trophozoites directly into the caecum. The mice were then treated orally with the extract, a standard drug (metronidazole), or vehicle p.o. for five consecutive days, beginning 24 h after the infection and were examined on the sixth day. At a dose of 1000 mg/kg per day, the extracts of Piper longum fruit, Piper sarmentosum root and Quercus infectoria nut gall had a curative rate of 100, 40 and 26%, respectively. At a concentration of 500 and 250 mg/kg/day, extract from Piper longum fruit was still effective in 93 and 46% of the cases, respectively, while extract from Piper sarmentosum root at a dose of less than 1000 mg/kg per day did not cure any mice from amoebiasis. Extract of Quercus infectoria nut gall at a concentration of 500 and of 250 mg/kg per day cured 26 and 13% of mice, respectively. Metronidazole at a concentration of 125 and of 62.5 mg/kg per day had a curative rate of 100 and 60%, respectively. The severity of caecal wall ulceration was reduced in mice which received the extract and metronidazole as compared to the control animals. PMID:15120461

  13. Progress towards development of a vaccine for amebiasis.

    PubMed Central

    Stanley, S L

    1997-01-01

    The application of molecular biologic techniques over the past decade has seen a tremendous growth in our knowledge of the biology of Entamoeba histolytica, the causative agent of amebic dysentery and amebic liver abscess. This approach has also led to the identification and structural characterization of three amebic antigens, the serine-rich Entamoeba histolytica protein (SREHP), the 170-kDa subunit of the Gal/GalNAc binding lectin, and the 29-kDa cysteine-rich protein, which all show promise as recombinant antigen-based vaccines to prevent amebiasis. In recent studies, an immunogenic dodecapeptide derived from the SREHP molecule has been genetically fused to the B subunit of cholera toxin, to create a recombinant protein capable of inducing both antiamebic and anti-cholera toxin antibodies when administered by the oral route. Continued progress in this area will bring us closer to the goal of a cost-effective oral combination "enteric pathogen" vaccine, capable of inducing protective mucosal immune responses to several clinically important enteric diseases, including amebiasis. PMID:9336666

  14. [Non-pathogenic intestinal amoebae: a clinical-analytical overview].

    PubMed

    Sard, Bárbara Gomila; Navarro, Rafael Toledo; Esteban Sanchis, J Guillermo

    2011-03-01

    Human beings can be parasitized by various species of intestinal amoebae. Entamoeba histolytica is the only intestinal amoeba recognized to be pathogenic, while other amoeba species, E. dispar, E. moshkovskii, E. hartmanni, E. coli, E. polecki, Endolimax nana and Iodamoeba buetschlii are considered to be non-pathogenic. The aim of this review is to synthesize the main morphological characteristics of the trophozoite and cyst stages of each amoeba as the basis for precise microscopical diagnosis. The difficulty of morphological differentiation among species included in the so-called "Entamoeba complex" entails the use of immunological and molecular diagnoses. In addition, a summary of basic epidemiological, therapeutic and prophylactic aspects of these non-pathogenic amoebae is provided. All of these aspects are crucial since these amoebae are usually found to be present in human coproparasitological analyses and must be differentiated from the pathogenic species E. histolytica. Furthermore, they can be used as suitable biological tags of the hygienic state of the environment and the health and hygiene measures of the population. PMID:21458707

  15. Distribution and abundance of organic thiols

    NASA Technical Reports Server (NTRS)

    Fahey, R.

    1985-01-01

    The role of glutathione (GSH) in protecting against the toxicity of oxygen and oxygen by products is well established for all eukaryotes studied except Entamoeba histolytica which lacks mitochrondria, chloroplasts, and microtubules. The GSH is not universal among prokaryotes. Entamoeba histolytica does not produce GSH or key enzymes of GSH metabolism. A general method of thiol analysis based upon fluorescent labeling with monobromobimane and HPLC separation of the resulting thiol derivatives was developed to determine the occurrence of GSH and other low molecular weight thiols in bacteria. Glutathione is the major thiol in cyanobacteria and in most bacteria closely related to the purple photosynthetic bacteria, but GSH was not found in archaebacteria, green bacteria, or GRAM positive bacteria. It suggested that glutathione metabolism was incorporated into eukaryotes at the time that mitochondria and chloroplasts were acquired by endosymbiosis. In Gram positive aerobes, coenzyme A occurs at millimolar levels and CoA disulfide reductases are identified. The CoA, rather than glutathione, may function in the oxygen detoxification processes of these organisms.

  16. Strategies To Discover the Structural Components of Cyst and Oocyst Walls

    PubMed Central

    Bushkin, G. Guy; Chatterjee, Aparajita; Robbins, Phillips W.

    2013-01-01

    Cysts of Giardia lamblia and Entamoeba histolytica and oocysts of Toxoplasma gondii and Cryptosporidium parvum are the infectious and sometimes diagnostic forms of these parasites. To discover the structural components of cyst and oocyst walls, we have developed strategies based upon a few simple assumptions. Briefly, the most abundant wall proteins are identified by monoclonal antibodies or mass spectrometry. Structural components include a sugar polysaccharide (chitin for Entamoeba, β-1,3-linked glucose for Toxoplasma, and β-1,3-linked GalNAc for Giardia) and/or acid-fast lipids (Toxoplasma and Cryptosporidium). Because Entamoeba cysts and Toxoplasma oocysts are difficult to obtain, studies of walls of nonhuman pathogens (E. invadens and Eimeria, respectively) accelerate discovery. Biochemical methods to dissect fungal walls work well for cyst and oocyst walls, although the results are often unexpected. For example, echinocandins, which inhibit glucan synthases and kill fungi, arrest the development of oocyst walls and block their release into the intestinal lumen. Candida walls are coated with mannans, while Entamoeba cysts are coated in a dextran-like glucose polymer. Models for cyst and oocyst walls derive from their structural components and organization within the wall. Cyst walls are composed of chitin fibrils and lectins that bind chitin (Entamoeba) or fibrils of the β-1,3-GalNAc polymer and lectins that bind the polymer (Giardia). Oocyst walls of Toxoplasma have two distinct layers that resemble those of fungi (β-1,3-glucan in the inner layer) or mycobacteria (acid-fast lipids in the outer layer). Oocyst walls of Cryptosporidium have a rigid bilayer of acid-fast lipids and inner layer of oocyst wall proteins. PMID:24096907

  17. Family of receptor-linked protein tyrosine phosphatases in humans and Drosophila

    SciTech Connect

    Streuli, M.; Krueger, N.X.; Saito, H. ); Tsai, A.Y.M. )

    1989-11-01

    To understand the regulation of cell proliferation by tyrosine phosphorylation, characterization of protein tyrosine phosphatases is essential. The human genes LCA (leukocyte common antigen) and LAR encode putative receptor-linked PTPases. By using consensus sequence probes, two additional receptor-linked PTPase genes, DLAR and DPTP, were isolated from Drosophila melanogaster. The extra-cellular segments of both DLAR and DPTP are composed of multiple immunoglobulin-like domains and fibronectin type III-like domains. The cytoplasmic region of DLAR and DPTP, as well as human LCA and LAR, are composed of two tandemly repeated PTPase domains. PTPase activities of immunoprecipitated LCA and LAR were demonstrated by measuring the release of phosphate from a {sup 32}P-labeled (Tyr(P))peptide. Furthermore, the cytoplasmic domains of LCA, LAR, DLAR, and DPTP, expressed in Escherichia coli, have PTPase activity. Site-directed mutagenesis showed that a conserved cysteine residue is essential for PTPase activity.

  18. Parasitological diagnosis combining an internally controlled real-time PCR assay for the detection of four protozoa in stool samples with a testing algorithm for microscopy.

    PubMed

    Bruijnesteijn van Coppenraet, L E S; Wallinga, J A; Ruijs, G J H M; Bruins, M J; Verweij, J J

    2009-09-01

    Molecular detection of gastrointestinal protozoa is more sensitive and more specific than microscopy but, to date, has not routinely replaced time-consuming microscopic analysis. Two internally controlled real-time PCR assays for the combined detection of Entamoeba histolytica, Giardia lamblia, Cryptosporidium spp. and Dientamoeba fragilis in single faecal samples were compared with Triple Faeces Test (TFT) microscopy results from 397 patient samples. Additionally, an algorithm for complete parasitological diagnosis was created. Real-time PCR revealed 152 (38.3%) positive cases, 18 of which were double infections: one (0.3%) sample was positive for E. histolytica, 44 (11.1%) samples were positive for G. lamblia, 122 (30.7%) samples were positive for D. fragilis, and three (0.8%) samples were positive for Cryptosporidium. TFT microscopy yielded 96 (24.2%) positive cases, including five double infections: one sample was positive for E. histolytica/Entamoeba dispar, 29 (7.3%) samples were positive for G. lamblia, 69 (17.4%) samples were positive for D. fragilis, and two (0.5%) samples were positive for Cryptosporidium hominis/Cryptosporidium parvum. Retrospective analysis of the clinical patient information of 2887 TFT sets showed that eosinophilia, elevated IgE levels, adoption and travelling to (sub)tropical areas are predisposing factors for infection with non-protozoal gastrointestinal parasites. The proposed diagnostic algorithm includes application of real-time PCR to all samples, with the addition of microscopy on an unpreserved faecal sample in cases of a predisposing factor, or a repeat request for parasitological examination. Application of real-time PCR improved the diagnostic yield by 18%. A single stool sample is sufficient for complete parasitological diagnosis when an algorithm based on clinical information is applied. PMID:19624500

  19. Intestinal parasitic infection among children and neonatus admitted to Ibn-Sina Hospital, Sirt, Libya.

    PubMed

    Kasssem, Hamed H; Zaed, Hana Abdalsalam; Sadaga, Gazala A

    2007-08-01

    A total of 350 stool samples from 196 males and 154 female children and neonatus admitted in Ibn-Sina hospital, Sirt, were examined from June 2001 to May 2002, to determine the prevalence of intestinal parasites. Intestinal parasitic infections were identified in 196 (56%) of children and neonates. No intestinal helminthic parasites were detected but 13 intestinal protozoan parasites were detected. The most prevalent protozoan was Entamoeba histolytica /E. dispar (36.57%); Blastocystis hominis (12.57%), Giardia lamblia (10.29%), Isospora belli (3.14%) and Balantidium coli (0.86%), the latter was detected in non-Libyan children. The non-pathogenic ones were Entamoeba coli (15.14%), Endolimax nana (13.71%), Entamoeba hartmanni (4.29%), Chilomastix mesnilli (4.29%), Retortamonas intestinalis (3.43%), Dientamoeba fragilis (2%), Iodamoeba butschlii (0.86%) and Trichomonas hominis (0.86%). The result showed a significant difference exists between the prevalence of pathogenic and non-pathogenic protozoan parasites (P < 0.05). High prevalence of E. histolytica/ E. dispar followed by E. coli, E. nana, B. hominis and G. lamblia in both sexes of children, while the prevalence of other intestinal parasites were low in both sexes, significantly different existed in the prevalence of intestinal parasites between males and females children (t = 24.68; P < 0.05). Age groups had no effect on the prevalence of intestinal parasites (F = 0.66; P < 0.05). Significant differences existed in the prevalence between single and multiple infections with pathogenic protozoa. The socio-economic status of children parents revealed that high prevalence in children from medium socio-economic status. The family size had no significant effect on the prevalence of the intestinal parasites. PMID:17985574

  20. Intestinal parasites of children and adults in a remote Aboriginal community of the Northern Territory, Australia, 1994–1996

    PubMed Central

    Aland, Kieran; Kearns, Thérèse; Gongdjalk, Glenda; Holt, Deborah; Currie, Bart; Prociv, Paul

    2015-01-01

    Introduction Parasitic infections can adversely impact health, nutritional status and educational attainment. This study investigated hookworm and other intestinal parasites in an Aboriginal community in Australia from 1994 to 1996. Methods Seven surveys for intestinal parasites were conducted by a quantitative formol-ether method on faecal samples. Serological testing was conducted for Strongyloides stercoralis and Toxocara canis IgG by enzyme-linked immunosorbent assays. Results Of the 314 participants, infections were as follows: Trichuris trichiura (86%); hookworm, predominantly Ancylostoma duodenale (36%); Entamoeba spp. (E. histolytica complex [E. histolytica, E. dispar and E. moskovski], E. coli and E. hartmanni) (25%); S. stercoralis (19%); Rodentolepis nana (16%); and Giardia duodenalis (10%). Serological diagnosis for 29 individuals showed that 28% were positive for S. stercoralis and 21% for T. canis. There was a decrease in the proportion positive for hookworm over the two-year period but not for the other parasite species. The presence of hookworm, T. trichiura and Entamoeba spp. was significantly greater in 5–14 year olds (n = 87) than in 0–4 year olds (n = 41), while the presence of S. stercoralis, R. nana, G. duodenalis and Entamoeba spp. in 5–14 year olds was significantly greater than 15–69 year olds (n = 91). Discussion Faecal testing indicated a very high prevalence of intestinal parasites, especially in schoolchildren. The decrease in percentage positive for hookworm over the two years was likely due to the albendazole deworming programme, and recent evidence indicates that the prevalence of hookworm is now low. However there was no sustained decrease in percentage positive for the other parasite species. PMID:25960921

  1. Household and personal factors are sources of heterogenity in intestinal parasite clearance among Mexican children 6-15 months of age supplemented with vitamin A and zinc.

    PubMed

    Srinivasan, Punitha; Lawa, Ha'i Raga; Rosado, Jorge L; Al Mamun, Abdullah; Khatun, Mohsina; Santos, José I; Utzinger, Jürg; Long, Kurt Z

    2016-04-01

    A randomised, double-blind, placebo-controlled trial was carried out among Mexico children aged 6-15 months to determine how household characteristics modify vitamin A and zinc supplementation efficacy on Ascaris lumbricoides, Giardia intestinalis and Entamoeba histolytica/E. dispar infection durations. Children assigned to receive vitamin A every 2 months, a daily zinc supplement, a combined vitamin A-zinc supplement or a placebo were followed for 1 year. Parametric hazard models were fit to infection durations stratified by personal and household factors. Children supplemented with vitamin A and zinc combined from households lacking piped water and children in all three treatment arms from households with dirt floors had longer G. intestinalis and A. lumbricoides infection durations than their counterparts, respectively. Shorter E. histolytica/E.dispar durations were found among zinc-supplemented children of mothers who had <6 years of education and no indoor bathrooms. Heterogeneity in supplementation efficacy among children may reflect differences in exposure risk and baseline immune responses. PMID:26772449

  2. Disinfection efficacy of sodium dichloroisocyanurate (NADCC) against common food-borne intestinal protozoa.

    PubMed

    El Zawawy, Lobna A; El-Said, Doaa; Ali, Safia M; Fathy, Fouad M

    2010-04-01

    The present study was conducted to investigate the efficacy of sodium dichloroisocyanurate (NaDCC) on the infective stages of common food-borne intestinal protozoa; Entamoeba histolytica (E. histolytica), Giardia lamblia (G. lamblia), Cryptosporidium, Cyclospora and Microsporidia; beside its effect on raw green vegetables and fruits. Parasites, isolated from stool of patients with diarrhea or dysentery, were exposed to NaDCC solution (1g/l) for one and two hours. Disinfection effect of NaDCC was assessed by in-vitro viability, using trypan blue stain, and infectivity bioassay in laboratory animals as indicated by fecal and intestinal parasitic counts. Raw vegetables and fruits were dipped in NaDCC solution in the same concentration and exposure time as used for treatment of the parasites. Results revealed statistically significant reductions in viability and infectivity of all examined parasites indicating their susceptibility to NaDCC. Relative variations in susceptibility were revealed; E. histolytica and G. lamblia were most susceptible (100% reduction) followed by Microsporidia then Cryptospridium and Cyclospora. NaDCC did not affect the consistency, color, taste or flavor of raw green vegetables and fruits. The proved efficacy of NaDCC, in cheap and convenient dry tablet form, makes it a promising tool in decontaminating raw vegetables and fruits from food-borne protozoan parasites at household and restaurant levels as well as in catering and fresh produce industry. It is also recommended for disinfection of food preparation surfaces and equipment. PMID:20503596

  3. Assessment of gastrointestinal parasites in wild chimpanzees (Pan troglodytes troglodytes) in southeast Cameroon

    PubMed Central

    Bertout, Sébastien; Locatelli, Sabrina; Butel, Christelle; Pion, Sébastien; Mpoudi-Ngole, Eitel; Delaporte, Eric; Peeters, Martine; Mallié, Michèle

    2014-01-01

    We tested 114 faecal samples from wild simian immunodeficiency virus (SIV)-positive (n=43) and SIV-negative (n=71) chimpanzees (Pan troglodytes troglodytes) in southeast Cameroon for the presence of gastrointestinal parasites by direct smear. We observed cysts from different protozoa (Entamoeba coli and Entamoeba histolytica/Entamoeba dispar, Endolimax nana, Iodamoeba butschlii, Chilomastix mesnili, Balantidium coli and Blastocystis cells) and trophozoites from Troglodytella abrassarti and Balantidium coli. Eggs from different helminths (strongylids, Ascaris lumbricoides, Abbreviata caucasica, Trichuris sp., Capillaria sp., Enterobius anthropopeci, Bertiella sp., Hymenolepis diminuta and an undetermined fluke) were also observed. Finally, we observed eggs that could not be properly identified and classified. We did not observe any differences between the SIV+ and SIV− samples except for the unidentified eggs. The studied chimpanzees were highly parasitised by strongylid (85.1 % of prevalence), Troglodytella (43.8 %) and Blastocystis (2.9 %), and the frequency of the other parasites ranged from 0.9 to 8.8 %. These high levels of parasite infections could represent an additional burden in a population where there is a high rate of the SIV virus in circulation. PMID:24781023

  4. Diversity and prevalence of gastrointestinal parasites in seven non-human primates of the Taï National Park, Côte d’Ivoire

    PubMed Central

    Kouassi, Roland Yao Wa; McGraw, Scott William; Yao, Patrick Kouassi; Abou-Bacar, Ahmed; Brunet, Julie; Pesson, Bernard; Bonfoh, Bassirou; N’goran, Eliezer Kouakou; Candolfi, Ermanno

    2015-01-01

    Parasites and infectious diseases are well-known threats to primate populations. The main objective of this study was to provide baseline data on fecal parasites in the cercopithecid monkeys inhabiting Côte d’Ivoire’s Taï National Park. Seven of eight cercopithecid species present in the park were sampled: Cercopithecus diana, Cercopithecus campbelli, Cercopithecus petaurista, Procolobus badius, Procolobus verus, Colobus polykomos, and Cercocebus atys. We collected 3142 monkey stool samples between November 2009 and December 2010. Stool samples were processed by direct wet mount examination, formalin-ethyl acetate concentration, and MIF (merthiolate, iodine, formalin) concentration methods. Slides were examined under microscope and parasite identification was based on the morphology of cysts, eggs, and adult worms. A total of 23 species of parasites was recovered including 9 protozoa (Entamoeba coli, Entamoeba histolytica/dispar, Entamoeba hartmanni, Endolimax nana, Iodamoeba butschlii, Chilomastix mesnili, Giardia sp., Balantidium coli, and Blastocystis sp.), 13 nematodes (Oesophagostomum sp., Ancylostoma sp., Anatrichosoma sp., Capillariidae Gen. sp. 1, Capillariidae Gen. sp. 2, Chitwoodspirura sp., Subulura sp., spirurids [cf Protospirura muricola], Ternidens sp., Strongyloides sp., Trichostrongylus sp., and Trichuris sp.), and 1 trematode (Dicrocoelium sp.). Diversity indices and parasite richness were high for all monkey taxa, but C. diana, C. petaurista, C. atys, and C. campbelli exhibited a greater diversity of parasite species and a more equitable distribution. The parasitological data reported are the first available for these cercopithecid species within Taï National Park. PMID:25619957

  5. Dramatic decrease in prevalence of soil-transmitted helminths and new insights into intestinal protozoa in children living in the Chaco region, Bolivia.

    PubMed

    Macchioni, Fabio; Segundo, Higinio; Gabrielli, Simona; Totino, Valentina; Gonzales, Patricia Rojas; Salazar, Esteban; Bozo, Ricardo; Bartoloni, Alessandro; Cancrini, Gabriella

    2015-04-01

    We assessed the prevalence of intestinal parasites among 268 2-12-year-old children living in rural areas, small villages, and semi-urban areas of the Chaco region, south-eastern Bolivia. The overall parasitism was 69%. Only protozoa, helminths, or co-infections were observed in 89.2%, 5.9%, or 4.9% of the positive children, respectively. A significant progressive increase in overall parasite prevalence was found when passing from rural areas to small villages and semi-urban areas. The most commonly found species were Entamoeba coli (38.4%), Giardia intestinalis (37.7%), and Blastocystis spp. (16%). Hymenolepis nana was the most prevalent helminth (5.6%), followed by Ascaris lumbricoides and hookworms (1.5% and 0.4%) evidenced only in rural areas and in villages. Molecular diagnostics identified Blastocystis subtypes 9 and 2, and 5 infections by Entamoeba histolytica and 4 by Entamoeba dispar. The dramatic decrease in prevalence of soil-transmitted helminths with respect to that observed about 20 years ago (> 40%) evidences the success of the preventive chemotherapy intervention implemented in 1986. Health education and improved sanitation should be intensified to control protozoan infections. PMID:25711609

  6. [The prevalence of intestinal parasites in children brought to the Kars Maternal and Children's Hospital with complaints of gastrointestinal symptoms].

    PubMed

    Arslan, Mükremin Ozkan; Sari, Bariş; Kulu, Bahar; Mor, Neriman

    2008-01-01

    This study was carried out to determine the prevalence of intestinal parasites in 2-6 year-old children who were brought to Kars Maternal and Children's Hospital with complaints of gastrointestinal symptoms during March-June 2007. Fecal samples were taken from children and brought to the parasitology laboratory of the Faculty of Veterinary Medicine to be examined for intestinal parasites. Fecal samples were examined by centrifugal formalin ether, zinc-sulphate floatation, and modified acid fast techniques. Lugol solution was used during microscopic examination and suspected samples were also examined by the Giemsa dye technique. The prevalence of intestinal parasites in children was found to be 36.2% (50/138). Protozoan and helminth parasites were found to be 34.1% (47/138) and 2.9% (4/138) in the fecal samples examined, respectively. Giardia intestinalis (10.9%), Entamoeba histolytica/dispar (10.1%), Entamoeba coli (8%), Blastocystis hominis (6.5%), Endolimax nana (4.3%), Chilomastix mesnili (1.4%), Ascaris lumbricoides (1.4%), Entamoeba hartmanni (0.7%), Cyclospora cayetanensis (0.7%), Enterobius vermicularis (0.7%) and Hymenolepis nana (0.7%) were identified from the feces of children of Kars and vicinity. No Cryptosporidium spp. was detected. PMID:18985583

  7. Diversity and prevalence of gastrointestinal parasites in seven non-human primates of the Taï National Park, Côte d'Ivoire.

    PubMed

    Kouassi, Roland Yao Wa; McGraw, Scott William; Yao, Patrick Kouassi; Abou-Bacar, Ahmed; Brunet, Julie; Pesson, Bernard; Bonfoh, Bassirou; N'goran, Eliezer Kouakou; Candolfi, Ermanno

    2015-01-01

    Parasites and infectious diseases are well-known threats to primate populations. The main objective of this study was to provide baseline data on fecal parasites in the cercopithecid monkeys inhabiting Côte d'Ivoire's Taï National Park. Seven of eight cercopithecid species present in the park were sampled: Cercopithecus diana, Cercopithecus campbelli, Cercopithecus petaurista, Procolobus badius, Procolobus verus, Colobus polykomos, and Cercocebus atys. We collected 3142 monkey stool samples between November 2009 and December 2010. Stool samples were processed by direct wet mount examination, formalin-ethyl acetate concentration, and MIF (merthiolate, iodine, formalin) concentration methods. Slides were examined under microscope and parasite identification was based on the morphology of cysts, eggs, and adult worms. A total of 23 species of parasites was recovered including 9 protozoa (Entamoeba coli, Entamoeba histolytica/dispar, Entamoeba hartmanni, Endolimax nana, Iodamoeba butschlii, Chilomastix mesnili, Giardia sp., Balantidium coli, and Blastocystis sp.), 13 nematodes (Oesophagostomum sp., Ancylostoma sp., Anatrichosoma sp., Capillariidae Gen. sp. 1, Capillariidae Gen. sp. 2, Chitwoodspirura sp., Subulura sp., spirurids [cf Protospirura muricola], Ternidens sp., Strongyloides sp., Trichostrongylus sp., and Trichuris sp.), and 1 trematode (Dicrocoelium sp.). Diversity indices and parasite richness were high for all monkey taxa, but C. diana, C. petaurista, C. atys, and C. campbelli exhibited a greater diversity of parasite species and a more equitable distribution. The parasitological data reported are the first available for these cercopithecid species within Taï National Park. PMID:25619957

  8. [Incidence of intestinal parasites in municipal sanitary workers in Malatya].

    PubMed

    Karaman, Ulkü; Atambay, Metin; Aycan, Ozlem; Yoloğlu, Saim; Daldal, Nilgün

    2006-01-01

    The incidence of intestinal parasites is closely related to such factors as the socio-economic level of the society, nutritional and hygienic habits, climate, environmental conditions, infrastructure and degree of literacy. In this study, the municipal sanitary workers who are regarded as a high risk group in Malatya were examined for intestinal parasites. Cellophane slides and fecal samples from 241 workers were examined and intestinal parasites were found in 93 (39.0%). The most common parasite was Entamoeba coli (34). Other parasites detected include Enterobius vermicularis (32), Giardia intestinalis (22), Blastocystis hominis (8), Iodamoeba butschlii (5), Entamoeba histolytica (2), Taenia sp. (2), Chilomastix mesnili (2), Dientamoeba fragilis (2), Entamoeba hartmanni (1), Trichomonas intestinalis (1) Hymenolepis nana (1), and Ascaris lumbricoides (1). A training seminary was conducted in order to inform all the workers about means of protection. The workers were given suitable treatment and were called for control after a month. The examinations revealed a significant decrease in the incidence rate of parasites (qui-square test in dependent samples P<0.05). It was concluded that offering training seminaries for certain occupational groups under risk is efficient in terms of protection. PMID:17160847

  9. Prevalence of enteric parasites in homosexual patients attending an outpatient clinic.

    PubMed

    Peters, C S; Sable, R; Janda, W M; Chittom, A L; Kocka, F E

    1986-10-01

    A total of 372 pooled stool specimens from 274 homosexual men with diarrhea were submitted for parasitologic examination over a 2.5-year period. Each two-vial pooled specimen set contained portions of stool from 3 consecutive days in Formalin and polyvinyl alcohol. Of the 274 patients, 133 (48.5%) harbored one or more intestinal protozoa, with 161 (43.3%) of the 372 specimens submitted being positive for one or more organisms. The parasites identified included Entamoeba histolytica (71 patients), Giardia lamblia (22 patients), Endolimax nana (106 patients), Entamoeba coli (39 patients), Entamoeba hartmanni (25 patients), Dientamoeba fragilis (3 patients), Iodamoeba bütschlii (2 patients), and Chilomastix mesnili (2 patients). Cryptosporidium sp. (2 patients) and Isospora belli (1 patient) were also detected. Results of this study support the experience of other workers regarding high rates of infection with intestinal parasites in the homosexual population and also indicate that symptomatic individuals belonging to this acquired immunodeficiency syndrome risk group be screened for both common and uncommon intestinal pathogens. PMID:2877006

  10. Human parasitoses of the Malili area, South Sulawesi (Celebes) province, Indonesia.

    PubMed

    Joseph, S W; Carney, W P; Van Peenen, P F; Russell, D; Saroso, J S

    1978-06-01

    A biomedical survey was conducted in 9 villages in the Malili area of South Sulawesi, Indonesia. Blood specimens were examined for malaria and microfilariae; stool specimens were examined for intestinal parasites. Malaria parasitemias were rare; Plasmodium falciparum was detected in 10 and P. vivax in 11 of 985 blood smears. Malayan filariasis was endemic to all villages surveyed. The overall prevalence of detectable microfilaremias was 15%, varying from 34% in Kawata to 1% in Nuha. Microfilarial densities, expressed as MfD50 averaged 8.0 and varied from 1.1 in Timampu to 16.0 in Karabbe. Intestinal parasites were common. Although Schistosoma japonicum was not found, 97% of the examined had one or more intestinal parasites as follows: Ascaris lumbricoides (74%), Trichuris trichiura (65%), hookworm (62%), Entamoeba coli (38%), Endolimax nana (10%), Entamoeba histolytica (6%), Iodamoeba bütschlii (4%), Entamoeba hartmanni (3%), Giardia lamblia (2%) Chilomastix mesnili (1%) and Enterobius vermicularis (1%). Strongyloides stercoralis larvae and Hymenolepis nana eggs were detected once each and heterophyid-like eggs were detected twice. PMID:364674

  11. [Contribution to the knowledge of intestinal protozoa infestation in the hospital population of Barcelona (author's transl)].

    PubMed

    Portús, M; Prats, G

    1981-03-10

    This report concerns the analysis of 650 fecal samples submitted for parasitic investigation to the Microbiology Service of the Hospital de la Santa Creu i Sant Pau of Barcelona. The samples were analyzed by the technique of fixation and direct observation of Sapero and Lawless (MIFD), and with the biphasic concentration method of Blagg et al (MIFC). Intestinal protozoa were detected in 183 samples (28.2%), with a total of 232 protozoosis uncovered. The specific analysis gave the following results: Giardia lamblia, 10.9%; Dientamoeba fragilis, 7.8%; Entamoeba coli, 4.6%; Endolimax nana, 4.5%; Entamoeba hartmanni, 4.0%; Iodamoeba buetschlii, 2.9%; Entamoeba histolytica, 0.7%; Chilomastix mesnili, 0.2%; Trichomonas hominis, 0.2%. The results obtained by the two methods have been compared, observing the highest number of positive results with MIFD technique. Especial mention is made of the higher incidence of Dientamoeba fragilis in samples positive for Oxyuris further studies being needed to establish the basis for the relationship between both species of parasites. PMID:7206888

  12. Dramatic Decrease in Prevalence of Soil-Transmitted Helminths and New Insights Into Intestinal Protozoa in Children Living in the Chaco Region, Bolivia

    PubMed Central

    Macchioni, Fabio; Segundo, Higinio; Gabrielli, Simona; Totino, Valentina; Gonzales, Patricia Rojas; Salazar, Esteban; Bozo, Ricardo; Bartoloni, Alessandro; Cancrini, Gabriella

    2015-01-01

    We assessed the prevalence of intestinal parasites among 268 2–12-year-old children living in rural areas, small villages, and semi-urban areas of the Chaco region, south-eastern Bolivia. The overall parasitism was 69%. Only protozoa, helminths, or co-infections were observed in 89.2%, 5.9%, or 4.9% of the positive children, respectively. A significant progressive increase in overall parasite prevalence was found when passing from rural areas to small villages and semi-urban areas. The most commonly found species were Entamoeba coli (38.4%), Giardia intestinalis (37.7%), and Blastocystis spp. (16%). Hymenolepis nana was the most prevalent helminth (5.6%), followed by Ascaris lumbricoides and hookworms (1.5% and 0.4%) evidenced only in rural areas and in villages. Molecular diagnostics identified Blastocystis subtypes 9 and 2, and 5 infections by Entamoeba histolytica and 4 by Entamoeba dispar. The dramatic decrease in prevalence of soil-transmitted helminths with respect to that observed about 20 years ago (> 40%) evidences the success of the preventive chemotherapy intervention implemented in 1986. Health education and improved sanitation should be intensified to control protozoan infections. PMID:25711609

  13. Discordances Between Serology and Culture for Strongyloides in an Ethiopian Adopted Child With Multiple Parasitic Infections

    PubMed Central

    Soriano-Arandes, Antoni; Sulleiro, Elena; Zarzuela, Francesc; Ruiz, Edurne; Clavería, Isabel; Espasa, Mateu

    2016-01-01

    Abstract Rationale: infectious diseases screening of international adoptees is complex because of the concurrence of different pathogens in a child at same time. We describe an international adopted child born at Ethiopia infected by 5 different pathogens (Hymenolepis nana, Giardia intestinalis, Entamoeba histolytica, Strongyloides stercoralis, and Trichuris trichiura), 2 of them S. stercoralis and E. histolytica with a capacity to develop severe clinical complications if not detected promptly with appropriate diagnosis tests. Concerns of the patient: according to the screening protocol a stool sample is always processed for culture addressed to find out protozoan and helminthic pathogens but not specifically for S. stercoralis. Only, when eosinophilia is detected 3 serial stool samples are collected to rule out intestinal parasitic infection including S. stercoralis. Interventions: in our case, S. stercoralis would not have been detected if we had followed the protocol because eosinophilia was absent and its specific serology was negative. Fortunately, the initial inclusion of the feces charcoal culture for S. stercoralis allowed us to detect this infection. Outcomes: discordances between direct methods such as culture and indirect as serology or antigen test forces us to be very cautious before ruling out S. stercoralis or E. histolytica infection, respectively. Also, if a child from tropical areas has persistent symptoms (such as diarrhea or fever) that have not been treated we have to rule out other infections that have not been detected yet. Main lessons: The introduction of different sequencing tests and the insistence to find out pathogens such as S. stercoralis or E. histolytica was determinant to be able to cure this symptomatic child and to prevent potential severe clinical forms in case of immunosuppression. PMID:26962825

  14. Water-soluble ruthenium complexes bearing activity against protozoan parasites.

    PubMed

    Sarniguet, Cynthia; Toloza, Jeannette; Cipriani, Micaella; Lapier, Michel; Vieites, Marisol; Toledano-Magaña, Yanis; García-Ramos, Juan Carlos; Ruiz-Azuara, Lena; Moreno, Virtudes; Maya, Juan Diego; Azar, Claudio Olea; Gambino, Dinorah; Otero, Lucía

    2014-06-01

    Parasitic illnesses are major causes of human disease and misery worldwide. Among them, both amebiasis and Chagas disease, caused by the protozoan parasites, Entamoeba histolytica and Trypanosoma cruzi, are responsible for thousands of annual deaths. The lack of safe and effective chemotherapy and/or the appearance of current drug resistance make the development of novel pharmacological tools for their treatment relevant. In this sense, within the framework of the medicinal inorganic chemistry, metal-based drugs appear to be a good alternative to find a pharmacological answer to parasitic diseases. In this work, novel ruthenium complexes [RuCl2(HL)(HPTA)2]Cl2 with HL=bioactive 5-nitrofuryl containing thiosemicarbazones and PTA=1,3,5-triaza-7-phosphaadamantane have been synthesized and fully characterized. PTA was included as co-ligand in order to modulate complexes aqueous solubility. In fact, obtained complexes were water soluble. Their activity against T. cruzi and E. histolytica was evaluated in vitro. [RuCl2(HL4)(HPTA)2]Cl2 complex, with HL4=N-phenyl-5-nitrofuryl-thiosemicarbazone, was the most active compound against both parasites. In particular, it showed an excellent activity against E. histolytica (half maximal inhibitory concentration (IC50)=5.2 μM), even higher than that of the reference drug metronidazole. In addition, this complex turns out to be selective for E. histolytica (selectivity index (SI)>38). The potential mechanism of antiparasitic action of the obtained ruthenium complexes could involve oxidative stress for both parasites. Additionally, complexes could interact with DNA as second potential target by an intercalative-like mode. Obtained results could be considered a contribution in the search for metal compounds that could be active against multiple parasites. PMID:24740394

  15. Hsp90 Inhibitors as New Leads To Target Parasitic Diarrheal Diseases

    PubMed Central

    Shahinas, Dea; Bryant, Clifford; Hirata, Ken; Miyamoto, Yukiko; Hwang, Grace; Gut, Jiri; Renslo, Adam R.; Pillai, Dylan R.; Eckmann, Lars; Reed, Sharon L.; McKerrow, James H.

    2014-01-01

    Entamoeba histolytica and Giardia lamblia are anaerobic protozoan parasites that cause amebiasis and giardiasis, two of the most common diarrheal diseases worldwide. Current therapy relies on metronidazole, but resistance has been reported and the drug has significant adverse effects. Therefore, it is critical to search for effective, better-tolerated antiamebic and antigiardial drugs. We synthesized several examples of a recently reported class of Hsp90 inhibitors and evaluated these compounds as potential leads for antiparasitic chemotherapy. Several of these inhibitors showed strong in vitro activity against both E. histolytica and G. lamblia trophozoites. The inhibitors were rescreened to discriminate between amebicidal and giardicidal activity and general cytotoxicity toward a mammalian cell line. No mammalian cytotoxicity was found at >100 μM for 48 h for any of the inhibitors. To understand the mechanism of action, a competitive binding assay was performed using the fluorescent ATP analogue bis-ANS (4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid dipotassium salt) and recombinant E. histolytica Hsp90 preincubated in both the presence and absence of Hsp90 inhibitors. There was significant reduction in fluorescence compared to the level in the control, suggesting that E. histolytica Hsp90 is a selective target. The in vivo efficacy and safety of one Hsp90 inhibitor in a mouse model of amebic colitis and giardiasis was demonstrated by significant inhibition of parasite growth at a single oral dose of 5 mg/kg of body weight/day for 7 days and 10 mg/kg/day for 3 days. Considering the results for in vitro activity and in vivo efficacy, Hsp90 inhibitors represent a promising therapeutic option for amebiasis and giardiasis. PMID:24820073

  16. [Intestinal parasitoses detected in primary schoolchildren in the Van province].

    PubMed

    Taş Cengiz, Zeynep; Akbayram, Sinan; Ciçek, Mutalip; Yilmaz, Hasan

    2009-01-01

    This study was carried out on school children in primary schools in the Van province in order to investigate the distribution of intestinal parasitoses. The study was conducted in 7-15 year-old primary schoolchildren during the years from 2003-2005. Stool specimens were taken from 2975 school children; of these, 1289 were female and 1686, male. The specimens were examined by native-Lugol, flotation and trichrome staining methods in the parasitology laboratory of the Health Research and Training Hospital, Yüzüncü Yil University. One or more parasite species were detected in 1916 (64.4%) stool specimens of 2975 children. Parasitosis was detected in 65.7% of females and in 63.4% of males. Blastocystis hominis, Giardia intestinalis, Entamoeba coli, Iodamoeba butschlii, Chilomastix mesnili, Endolimax nana, Entamoeba histolytica/Entamoeba dispar, Entamoeba hartmanni, Enteromonas hominis, Retortamonas intestinalis, Dientamoeba fragilis, Trichomonas hominis, Ascaris lumbricoides, Hymenolepis nana, Trichuris trichiura, Enterobius vermicularis and Fasciola hepatica were detected in 27.4%, 16.4%, 14.9%, 3.9%, 2.9%, 1.8%, 1.1%, 0.6%, 0.5%, 0.5%, 0.4%, 0.2%, 7.5%, 5.3%, 0.7%, 0.6% and 0.03% of the specimens, respectively. In conclusion, there are still high rates of intestinal parasitoses in school children in this region because care is not taken in hygiene and the socioeconomic status is low in the Van province. PMID:20101580

  17. [Blastocystis hominis in Canavese: a retrospective study of samples received for fecal parasitological examination at the Ivrea- Castellamonte Hospital over 42 months].

    PubMed

    Pistono, P G; Dusi, M P; Ronchetto, F; Cestonaro, G; Guasco, C

    1991-01-01

    In the last ten years the interest in the controversial Blastocystis hominis microorganism of the intestine has greatly increased. The authors conducted a retrospective study of feces for parasitology investigations at the Ivrea-Castellamonte hospital. These investigations lasted 42 months (1/1/1988-31/6/1991) and the authors focused their attention on samples with 5 or more Blastocystis hominis at the microscopically examination with a 40X phase objective. During the study 2,138 samples of feces (belonging to 1552 patients) arrived. 284 samples (13.3%), corresponding to 200 patients (12.9%), proved to be positive for one or more parasites. In 155 samples (7.2%) regarding 109 patients (7.0%)--53 males, 56 females--Blastocystis was present. These patients were between 2 and 78 years of age. In 19 (17.4%) of these patients other parasites were associated, parasites such as Giardia lamblia (in 6 cases), Entamoeba coli (in 4 cases), Entamoeba spp. (in 2 cases), Entamoeba histolytica (in one case), Entamoeba hartmanni (in one case), Iodamoeba butschlii (in one case), Hymenolepis nana (in one case); helminths (in 3 cases). The most effect proved to be those patients between 21 and 50 years of age (53 of 98 patients). Few infants were affected. The greatest incidence of Blastocystis hominis infection occurred during the months of January to June. The authors conclude by considering how the incidence data of such parasitosis in the Canavese region can be superimposed on as much as has been observed in territories which are very different regarding social-economic and geoclimatic conditions. The authors think it is necessary to start a prospective clinical-epidemiological investigation in the Canavese region with a view to defining the clinical approach to the parasite under discussion in a better way. PMID:1844921

  18. Comparison of nested-multiplex, Taqman & SYBR Green real-time PCR in diagnosis of amoebic liver abscess in a tertiary health care institute in India

    PubMed Central

    Dinoop, K.P.; Parija, Subhash Chandra; Mandal, Jharna; Swaminathan, R.P.; Narayanan, P.

    2016-01-01

    Background & objectives: Amoebiasis is a common parasitic infection caused by Entamoeba histolytica and amoebic liver abscess (ALA) is the most common extraintestinal manifestation of amoebiasis. The aim of this study was to standardise real-time PCR assays (Taqman and SYBR Green) to detect E. histolytica from liver abscess pus and stool samples and compare its results with nested-multiplex PCR. Methods: Liver abscess pus specimens were subjected to DNA extraction. The extracted DNA samples were subjected to amplification by nested-multiplex PCR, Taqman (18S rRNA) and SYBR Green real-time PCR (16S-like rRNA assays to detect E. histolytica/E. dispar/E. moshkovskii). The amplification products were further confirmed by DNA sequence analysis. Receiver operator characteristic (ROC) curve analysis was done for nested-multiplex and SYBR Green real-time PCR and the area under the curve was calculated for evaluating the accuracy of the tests to dignose ALA. Results: In all, 17, 19 and 25 liver abscess samples were positive for E. histolytica by nested-multiplex PCR, SYBR Green and Taqman real-time PCR assays, respectively. Significant differences in detection of E. histolytica were noted in the real-time PCR assays evaluated (P<0.0001). The nested-multiplex PCR, SYBR Green real-time PCR and Taqman real-time PCR evaluated showed a positivity rate of 34, 38 and 50 per cent, respectively. Based on ROC curve analysis (considering Taqman real-time PCR as the gold standard), it was observed that SYBR Green real-time PCR was better than conventional nested-multiplex PCR for the diagnosis of ALA. Interpretation & conclusions: Taqman real-time PCR targeting the 18S rRNA had the highest positivity rate evaluated in this study. Both nested multiplex and SYBR Green real-time PCR assays utilized were evaluated to give accurate results. Real-time PCR assays can be used as the gold standard in rapid and reliable diagnosis, and appropriate management of amoebiasis, replacing the

  19. Molecular Epidemiology of Amoebiasis: A Cross-Sectional Study among North East Indian Population

    PubMed Central

    Nath, Joyobrato; Ghosh, Sankar Kumar; Singha, Baby; Paul, Jaishree

    2015-01-01

    Background Epidemiological studies carried out using culture or microscopy in most of the amoebiasis endemic developing countries, yielded confusing results since none of these could differentiate the pathogenic Entamoeba histolytica from the non-pathogenic Entamoeba dispar and Entamoeba moshkovskii. The Northeastern part of India is a hot spot of infection since the climatic conditions are most conducive for the infection and so far no systemic study has been carried out in this region. Methodology/Principal Findings Following a cross-sectional study designed during the period 2011–2014, a total of 1260 fecal samples collected from the Northeast Indian population were subjected to microscopy, fecal culture and a sensitive and specific DNA dot blot screening assay developed in our laboratory targeting the Entamoeba spp. Further species discrimination using PCR assay performed in microscopy, culture and DNA dot blot screening positive samples showed E. histolytica an overall prevalence rate of 11.1%, 8.0% and 13.7% respectively. In addition, infection rates of nonpathogenic E. dispar and E. moshkovskii were 11.8% (95% CI = 10.2, 13.8) and 7.8% (95% CI = 6.4, 9.4) respectively. The spatial distributions of infection were 18.2% (107/588) of Assam, 11.7% (23/197) of Manipur, 10.2% (21/207) of Meghalaya, and 8.2% (22/268) of Tripura states. Association study of the disease with demographic features suggested poor living condition (OR = 3.21; 95% CI = 1.83, 5.63), previous history of infection in family member (OR = 3.18; 95% CI = 2.09, 4.82) and unhygienic toilet facility (OR = 1.79; 95% CI = 1.28, 2.49) as significant risk factors for amoebiasis. Children in age group <15 yr, participants having lower levels of education, and daily laborers exhibited a higher infection rate. Conclusions/Significance Despite the importance of molecular diagnosis of amoebiasis, molecular epidemiological data based on a large sample size from endemic countries are rarely reported in the

  20. Gastrointestinal parasites of mountain gorillas (Gorilla gorilla beringei) in the Parc National des Volcans, Rwanda.

    PubMed

    Sleeman, J M; Meader, L L; Mudakikwa, A B; Foster, J W; Patton, S

    2000-09-01

    Ninety-eight fecal samples were collected from 74 free-living mountain gorillas (Gorilla gorilla beringei) from the Parc National des Volcans, Rwanda, between July 1995 and January 1997 and examined for parasites by Sheather's sugar and zinc sulfate flotation methods, trichrome staining, and larval cultures. All samples contained at least one parasite. Seventeen endoparasites were identified, including eight protozoa, seven nematodes, one cestode, and one trematode. Two species of arthropod mite were also recovered from the fecal samples. Parasites observed on fecal examinations included strongyle/trichostrongyle-type eggs (72/74) (representing Oesphagostomum sp., Trichostrongylus sp., Hyostrongylus spp., and possibly Murshidia sp.), Strongyloides sp. (1/74), Trichuris trichiura (2/74), Probstmayria sp. (7/74), Anoplocephala sp. (63/74), Entamoeba hartmanni cysts and trophozoites (19/70), Endolimax nana cysts (31/70), Iodamoeba buetschlii cysts (11/70), Endolimax nana or Iodamoeba buetschlii trophozoites (63/70). Entamoeba coli cysts and trophozoites (14/70), Entamoeba histolytica trophozoite (1/70), Chilomastix sp. cysts and trophozoites (31/70), and Giardia sp. cysts (2/70). In addition, one ascarid and one trematode egg were seen. There were no significant differences in the prevalence of parasites between males and females and between age groups: however, infants and juveniles appeared to have a lower prevalence of Anoplocephala gorillae, and the silverbacked males appeared to have a higher prevalence of Probstmayria sp. Parasite prevalence was consistent among the five social groups studied except Susa group had a significantly lower prevalence of Anoplocephala gorillae. Trichuris trichiura, Strongyloides sp., Chilomastix sp., and Endolimax nana were identified for the first time in this population, and it is possible that these parasites were of human origin. Although there were no obvious clinical effects due to the presence of these parasites, six parasites

  1. Naegleria fowleri: contact-dependent secretion of electrondense granules (EDG).

    PubMed

    Chávez-Munguía, Bibiana; Villatoro, Lizbeth Salazar; Omaña-Molina, Maritza; Rodríguez-Monroy, Marco Aurelio; Segovia-Gamboa, Norma; Martínez-Palomo, Adolfo

    2014-07-01

    The free living amoeba Naegleria fowleri is pathogenic to humans but also to other mammalians. These amoebae may invade the nasal mucosa and migrate into the brain causing cerebral hemorrhagic necrosis, a rapidly fatal infection. Knowledge of the cytolytic mechanism involved in the destruction of brain tissues by Naegleria trophozoites is limited. In other amoebic species, such as Entamoeba histolytica, we have previously reported the possible lytic role of small cytoplasmic components endowed with proteolytic activities, known as electrondense granules (EDG). Using transmission electron microscopy we now report that EDG, seldom found in long term cultured N. fowleri, are present in abundance in trophozoites recovered from experimental mice brain lesions. Numerous EDG were also observed in amoebae incubated with collagen substrates or cultured epithelial cells. SDS-PAGE assays of concentrated supernatants of these trophozoites, containing EDG, revealed proteolytic activities. These results suggest that EDG may have a clear role in the cytopathic mechanisms of this pathogenic amoeba. PMID:24721258

  2. Use of microdilution to assess in vitro antiamoebic activities of Brucea javanica fruits, Simarouba amara stem, and a number of quassinoids.

    PubMed Central

    Wright, C W; O'Neill, M J; Phillipson, J D; Warhurst, D C

    1988-01-01

    A microdilution technique for the assessment of in vitro activity against Entamoeba histolytica was devised and validated with metronidazole. The test was used to detect the antiamoebic activities of plant extracts prepared from the traditional remedies Brucea javanica fruits and Simarouba amara stems. The activity was associated with quassinoid-containing fractions. The 50% inhibitory concentrations for some quassinoids against amoebae were determined by using the microdilution method. These concentrations ranged from 0.019 micrograms.ml-1 for bruceantin, the most active quassinoid, to greater than 5 micrograms.ml-1 for glaucarubol, the least active compound tested. These results are discussed with reference to the known activities of these compounds against Plasmodium falciparum. Overall, the activities of the quassinoids against both protozoa are similar. The microdilution technique will be useful in the search for novel antiamoebic drugs. PMID:2908094

  3. Use of microdilution to assess in vitro antiamoebic activities of Brucea javanica fruits, Simarouba amara stem, and a number of quassinoids.

    PubMed

    Wright, C W; O'Neill, M J; Phillipson, J D; Warhurst, D C

    1988-11-01

    A microdilution technique for the assessment of in vitro activity against Entamoeba histolytica was devised and validated with metronidazole. The test was used to detect the antiamoebic activities of plant extracts prepared from the traditional remedies Brucea javanica fruits and Simarouba amara stems. The activity was associated with quassinoid-containing fractions. The 50% inhibitory concentrations for some quassinoids against amoebae were determined by using the microdilution method. These concentrations ranged from 0.019 micrograms.ml-1 for bruceantin, the most active quassinoid, to greater than 5 micrograms.ml-1 for glaucarubol, the least active compound tested. These results are discussed with reference to the known activities of these compounds against Plasmodium falciparum. Overall, the activities of the quassinoids against both protozoa are similar. The microdilution technique will be useful in the search for novel antiamoebic drugs. PMID:2908094

  4. Microwave-mediated enzyme-linked immunosorbent assay procedure.

    PubMed

    Nahar, Pradip; Bora, Utpal; Sharma, Gainda L; Kannoujia, Dileep Kumar

    2012-02-15

    Here we demonstrate a novel microwave-mediated enzyme-linked immunosorbent assay (MELISA) method that has dramatically reduced the enzyme-linked immunosorbent assay (ELISA) timing to less than 5 min with a result comparable to that obtained by 18-h conventional ELISA. Efficacy of the MELISA procedure is demonstrated by detecting human immunoglobulin G (IgG), rabbit IgG, human immunoglobulin E (IgE), human interleuken 1β (IL-1β), Entamoeba histolytica antibody, and Aspergillus fumigatus antibody. MELISA could be an excellent substitute for time-consuming conventional ELISA for rapid diagnosis of diseases in cases of medical urgency, outbreak of infectious diseases, and screening of samples in blood banks or emigration counters. PMID:22033289

  5. Detection of intestinal protozoa in the clinical laboratory.

    PubMed

    McHardy, Ian H; Wu, Max; Shimizu-Cohen, Robyn; Couturier, Marc Roger; Humphries, Romney M

    2014-03-01

    Despite recent advances in diagnostic technology, microscopic examination of stool specimens remains central to the diagnosis of most pathogenic intestinal protozoa. Microscopy is, however, labor-intensive and requires a skilled technologist. New, highly sensitive diagnostic methods have been developed for protozoa endemic to developed countries, including Giardia lamblia (syn. G. intestinalis/G. duodenalis) and Cryptosporidium spp., using technologies that, if expanded, could effectively complement or even replace microscopic approaches. To date, the scope of such novel technologies is limited and may not include common protozoa such as Dientamoeba fragilis, Entamoeba histolytica, or Cyclospora cayetanensis. This minireview describes canonical approaches for the detection of pathogenic intestinal protozoa, while highlighting recent developments and FDA-approved tools for clinical diagnosis of common intestinal protozoa. PMID:24197877

  6. Structure of methionine γ-lyase from Clostridium sporogenes.

    PubMed

    Revtovich, Svetlana; Anufrieva, Natalya; Morozova, Elena; Kulikova, Vitalia; Nikulin, Alexey; Demidkina, Tatyana

    2016-01-01

    Methionine γ-lyase (MGL) is a pyridoxal 5'-phosphate-dependent enzyme that catalyzes the γ-elimination reaction of L-methionine. The enzyme is a promising target for therapeutic intervention in some anaerobic pathogens and has attracted interest as a potential cancer treatment. The crystal structure of MGL from Clostridium sporogenes has been determined at 2.37 Å resolution. The fold of the protein is similar to those of homologous enzymes from Citrobacter freundii, Entamoeba histolytica, Pseudomonas putida and Trichomonas vaginalis. A comparison of these structures revealed differences in the conformation of two flexible regions of the N- and C-terminal domains involved in the active-site architecture. PMID:26750487

  7. Novel aryl carbamate derivatives of metronidazole as potential antiamoebic agents.

    PubMed

    Hayat, Faisal; Wahedi, Hussain Mustatab; Park, Seonghyeok; Tariq, Saba; Azam, Amir; Shin, Dongyun

    2016-01-01

    A series of novel aryl carbamate derivatives of metronidazole (MNZ) were designed, synthesized, and screened for antiamoebic activity. As compared to MNZ, most of the derivatives exhibited moderate to excellent activity against the HM1:IMSS strain of Entamoeba histolytica. Compounds 7, 14, 16, 19, and 21 exhibited the most promising antiamoebic activity with IC50 values of 0.24, 0.08, 0.26, 0.26, and 0.15 μM, respectively, compared to that of MNZ (1.78 μM). Moreover, from the toxicological studies of these compounds on human melanocytes, the melan-a cell line revealed that the potent compounds are nontoxic at concentrations ranging from 2.5 to 50 μM. PMID:26597858

  8. Identifying Etiological Agents Causing Diarrhea in Low Income Ecuadorian Communities

    PubMed Central

    Vasco, Gabriela; Trueba, Gabriel; Atherton, Richard; Calvopiña, Manuel; Cevallos, William; Andrade, Thamara; Eguiguren, Martha; Eisenberg, Joseph N. S.

    2014-01-01

    Continued success in decreasing diarrheal disease burden requires targeted interventions. To develop such interventions, it is crucial to understand which pathogens cause diarrhea. Using a case-control design we tested stool samples, collected in both rural and urban Ecuador, for 15 pathogenic microorganisms. Pathogens were present in 51% of case and 27% of control samples from the urban community, and 62% of case and 18% of control samples collected from the rural community. Rotavirus and Shigellae were associated with diarrhea in the urban community; co-infections were more pathogenic than single infection; Campylobacter and Entamoeba histolytica were found in large numbers in cases and controls; and non-typhi Salmonella and enteropathogenic Escherichia coli were not found in any samples. Consistent with the Global Enteric Multicenter Study, focused in south Asia and sub-Saharan Africa, we found that in Ecuador a small group of pathogens accounted for a significant amount of the diarrheal disease burden. PMID:25048373

  9. Infection Strategies of Intestinal Parasite Pathogens and Host Cell Responses.

    PubMed

    Di Genova, Bruno M; Tonelli, Renata R

    2016-01-01

    Giardia lamblia, Cryptosporidium sp., and Entamoeba histolytica are important pathogenic intestinal parasites and are amongst the leading causes worldwide of diarrheal illness in humans. Diseases caused by these organisms, giardiasis, cryptosporidiosis, and amoebiasis, respectively, are characterized by self-limited diarrhea but can evolve to long-term complications. The cellular and molecular mechanisms underlying the pathogenesis of diarrhea associated with these three pathogens are being unraveled, with knowledge of both the strategies explored by the parasites to establish infection and the methods evolved by hosts to avoid it. Special attention is being given to molecules participating in parasite-host interaction and in the mechanisms implicated in the diseases' pathophysiologic processes. This review focuses on cell mechanisms that are modulated during infection, including gene transcription, cytoskeleton rearrangements, signal transduction pathways, and cell death. PMID:26973630

  10. [Intestinal parasite infections in rural students of Chiloé archipelago, X Region, Chile].

    PubMed

    Mejías, G

    1993-01-01

    In order to contribute to the knowledge of intestinal parasite infection a survey was performed in 462 primary school children from eight rural localities sited in two islands of the Chiloe archipelago. Stool samples and perianal scotch tape scrapings for intestinal protozoa and helminths were microscopically examined. The prevalence rates of the main species found were: Ascaris lumbricoides 43.3%, Trichuris trichiura 26.8%, Enterobius vernicularis 20.1%, Entamoeba histolytica 5.6%, Giardia intestinalis 20.6% and Blastocystis hominis 63.2%. Poverty, deficiencies in hygienic practices and poor basic environmental sanitation are the contributing factors in the maintenance of the high prevalence of the intestinal parasitic infections found. A health education program, improvement of basic sanitary conditions and eventual treatment of infected people may contribute to control this undesirable situation. PMID:8110371

  11. Fulminant amebic colitis.

    PubMed

    Aristizábal, H; Acevedo, J; Botero, M

    1991-01-01

    Amebiasis is the acute and chronic disease produced by Entamoeba histolytica, an entity which occurs in endemic fashion in many of the tropical and subtropical areas of the world, capable of affecting diverse organs of the body, especially the colon. Amebiasis has different clinical forms of presentation, varying from the asymptomatic carrier state to severe, although not frequent, fulminant or necrotizing colitis, characteristically associated with high morbidity and mortality. We hereby report a series of 50 adult patients with fulminating amebic colitis managed at our institution between January, 1971 and July, 1989, with a global mortality of 60%. Early diagnosis, treatment with effective antiamebic agents--specifically metronidazole--and opportune aggressive surgical intervention have resulted in better survival rates. We had no survivors prior to 1970; our current survival rate is still a dismal 40%, indicating the very severe nature of the disease. PMID:2031357

  12. Infection Strategies of Intestinal Parasite Pathogens and Host Cell Responses

    PubMed Central

    Di Genova, Bruno M.; Tonelli, Renata R.

    2016-01-01

    Giardia lamblia, Cryptosporidium sp., and Entamoeba histolytica are important pathogenic intestinal parasites and are amongst the leading causes worldwide of diarrheal illness in humans. Diseases caused by these organisms, giardiasis, cryptosporidiosis, and amoebiasis, respectively, are characterized by self-limited diarrhea but can evolve to long-term complications. The cellular and molecular mechanisms underlying the pathogenesis of diarrhea associated with these three pathogens are being unraveled, with knowledge of both the strategies explored by the parasites to establish infection and the methods evolved by hosts to avoid it. Special attention is being given to molecules participating in parasite–host interaction and in the mechanisms implicated in the diseases’ pathophysiologic processes. This review focuses on cell mechanisms that are modulated during infection, including gene transcription, cytoskeleton rearrangements, signal transduction pathways, and cell death. PMID:26973630

  13. Microbiological and Pharmacological Evaluation of the Micropropagated Rubus liebmannii Medicinal Plant

    PubMed Central

    Jiménez-Arellanes, Adelina; Cornejo-Garrido, Jorge; Rojas-Bribiesca, Gabriela; Nicasio-Torres, María del Pilar; Said-Fernández, Salvador; Mata-Cárdenas, Benito David; Molina-Salinas, Gloria María; Tortoriello, Jaime; Meckes-Fischer, Mariana

    2012-01-01

    Rubus liebmannii is an endemic species from Mexico used in traditional medicine primarily to treat dysentery and cough. The in vitro activity against Giardia lamblia and Entamoeba histolytica that produces the ethanolic extract of the aerial parts of the plant led us to expand the pharmacological and phytochemical research of this species. Gastrointestinal disorders including amebiasis remain one of the health problems that need to be addressed and it is of interest to find alternatives that improve their treatment. Also, it is important to emphasize that R. liebmannii grows wild in the country and is not found in abundance; therefore, alternatives that avoid overexploitation of the natural resource are mandatory. Ongoing with the evaluation of the potentialities that R. liebmannii possesses for treating infectious gastrointestinal diseases, the aim of the present study was to evaluate the biological effects and the chemical composition of the micropropagated plant. PMID:22966243

  14. Intestinal and blood parasites in the North Lore District, Central Sulawesi, Indonesia.

    PubMed

    Carney, W P; Masri, S; Stafford, E E; Putrali, J

    1977-06-01

    Over 1,000 stool specimens from residents of the Napu and Besoa Valleys, Central Sulawesi, Indonesia were examined. Schistosoma japonicum was detected in 31% of Napu Valley residents while in only 2% of the Besoa Valley residents. Hookworm infections were the most frequently encountered helminth parasitisms in both valleys. Other helminth parasites encountered were: Ascaris lumbricoides, Trichuris trichiura, Enterobius vermicularis, Strongyloides stercoralis, Physaloptera sp., Diphyllobothrium sp., echinostome and heterophyid trematodes. Intestinal protozoa endemic to the area were: Entamoeba histolytica, E. coli, E. hartmanni, Iodamoebe bütschlii, Giardia lamblia, Chilomastix mesnili and Trichomonas hominis. Plasmodium falciparum was responsible for malaria parasitaemias in 5% of 1353 specimens examined and Brugia malayi microfilaraemias were detected in 10% of 972 specimens examined. PMID:335530

  15. Synthesis, characterization and antiamoebic activity of chalcones bearing N-substituted ethanamine tail.

    PubMed

    Leeza Zaidi, Saadia; Mittal, Sonam; Rajala, Maitreyi S; Avecilla, Fernando; Husain, Mohammad; Azam, Amir

    2015-06-15

    A series of chalcones (4-21) possessing N-substituted ethanamine were synthesized by the aldol condensation reaction of 1-(4-(2-substituted ethoxy)phenyl)ethanones with different aldehydes preceded by the reaction of 2-chloro N-substituted ethanamine hydrochloride and 4-hydroxy acetophenone. The structure of all the synthesized compounds was elucidated by various spectral and X-ray diffraction studies. The compounds were screened against HM1: IMSS strain of Entamoeba histolytica and cytotoxicity was performed on A549 (non-small cell lung cancer cell line) cells by MTT assay. Out of eighteen compounds twelve showed better activity then the standard drug metronidazole. The compound 9, 14 and 19 showed good cell viability, hence were least toxic. PMID:26021707

  16. Detection of Intestinal Protozoa in the Clinical Laboratory

    PubMed Central

    McHardy, Ian H.; Wu, Max; Shimizu-Cohen, Robyn; Couturier, Marc Roger

    2014-01-01

    Despite recent advances in diagnostic technology, microscopic examination of stool specimens remains central to the diagnosis of most pathogenic intestinal protozoa. Microscopy is, however, labor-intensive and requires a skilled technologist. New, highly sensitive diagnostic methods have been developed for protozoa endemic to developed countries, including Giardia lamblia (syn. G. intestinalis/G. duodenalis) and Cryptosporidium spp., using technologies that, if expanded, could effectively complement or even replace microscopic approaches. To date, the scope of such novel technologies is limited and may not include common protozoa such as Dientamoeba fragilis, Entamoeba histolytica, or Cyclospora cayetanensis. This minireview describes canonical approaches for the detection of pathogenic intestinal protozoa, while highlighting recent developments and FDA-approved tools for clinical diagnosis of common intestinal protozoa. PMID:24197877

  17. Seroepidemiologic study of giardiasis patients and high-risk groups in a midwestern city in the United States.

    PubMed

    Sullivan, R; Linneman, C C; Clark, C S; Walzer, P D

    1987-08-01

    Serum antibodies to Giardia lamblia were measured in giardiasis patients, in groups at high risk for intestinal parasite infection, and in controls by an indirect fluorescent antibody (IFA) technique. Symptomatic patients had the highest antibody titers, and antibodies remained present for up to 18 months in persons with chronic infection. Indochinese refugees and male homosexuals with the acquired immunodeficiency syndrome (AIDS) and pre-AIDS had higher mean antibody levels than did healthy controls, whereas sewer and highway maintenance workers had levels similar to those of controls. Serum antibodies to Entamoeba histolytica measured by an indirect hemagglutination antibody technique were detected in only a few Indochinese refugees. We conclude that serology is a promising tool in G. lamblia epidemiology and that further population studies would be of interest. PMID:2886069

  18. Development of a loop-mediated isothermal amplification assay for detection of Trichomonas vaginalis.

    PubMed

    Reyes, John Carlo B; Solon, Juan Antonio A; Rivera, Windell L

    2014-07-01

    A loop-mediated isothermal amplification (LAMP) assay targeting the 2-kbp repeated DNA species-specific sequence was developed for detection of Trichomonas vaginalis, the causative agent of trichomoniasis. The analytical sensitivity and specificity of the LAMP assay were evaluated using pooled genital swab and urine specimens, respectively, spiked with T. vaginalis trophozoites. Genital secretion and urine did not inhibit the detection of the parasite. The sensitivity of the LAMP was 10-1000 times higher than the PCR performed. The detection limit of LAMP was 1 trichomonad for both spiked genital swab and urine specimens. Also, LAMP did not exhibit cross-reactivity with closely-related trichomonads, Trichomonas tenax and Pentatrichomonas hominis, and other enteric and urogenital microorganisms, Entamoeba histolytica, Candida albicans, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. This is the first report of a LAMP assay for the detection of T. vaginalis and has prospective application for rapid diagnosis and control of trichomoniasis. PMID:24792836

  19. Recent advances in molecular biology of parasitic viruses.

    PubMed

    Banik, Gouri Rani; Stark, Damien; Rashid, Harunor; Ellis, John T

    2014-01-01

    The numerous protozoa that can inhabit the human gastro-intestinal tract are known, yet little is understood of the viruses which infect these protozoa. The discovery, morphologic details, purification methods of virus-like particles, genome and proteome of the parasitic viruses, Entamoeba histolytica, Giardia lamblia, Trichomonas vaginalis, and the Eimeria sp. are described in this review. The protozoan viruses share many common features: most of them are RNA or double-stranded RNA viruses, ranging between 5 and 8 kilobases, and are spherical or icosahedral in shape with an average diameter of 30-40 nm. These viruses may influence the function and pathogenicity of the protozoa which they infect, and may be important to investigate from a clinical perspective. The viruses may be used as specific genetic transfection vectors for the parasites and may represent a research tool. This review provides an overview on recent advances in the field of protozoan viruses. PMID:25019235

  20. Globalised world, globalised diseases: A case report on an amoebiasis-associated colon perforation.

    PubMed

    Redaèlli, Marcus; Mahmoohdzad, Jawad; Lang, Rahim; Schencking, Martin

    2013-05-16

    In 2010 the World Health Organisation estimated the number of infections with Entamoeba histolytica at about 50 million cases including 100000 fatal courses. In most cases this infection is a subclinical event with few or none symptoms noticeable for the patient. Courses of this disease and incidence of this parasite in industrialised nations are not yet fully investigated. Our case reports about a 68-year-old male patient from Turkey who lives for more than 30 years in Germany and had not been abroad during the past 2 years. Resistant asymptomatic amoebic dormant bodies caused an emergency-laparoscopy and revealed the seldom complication of a colon perforation. In the age of globalisation all providers in the health care systems are urged to acquaint themselves also with non-typical syndromes for the countries they work in order to reduce preventable morbidity respectively mortality rates. PMID:24303471

  1. Persistence of intestinal parasitic infections during the national de-worming campaign in schoolchildren of northwestern Mexico: a cross-sectional study

    PubMed Central

    Quihui-Cota, Luis; Morales-Figueroa, Gloria Guadalupe

    2012-01-01

    Background Intestinal parasitism remains a public health challenge in northwestern Mexico even when a twice yearly single dose of albendazole (400 mg) is administered to schoolchildren. We aimed to determine the current prevalence of intestinal parasitic infections in schoolchildren of northwestern Mexico. Methods The Faust and Kato Katz techniques were used to detect and identify the intestinal parasite species. One thousand two hundred and seventy eight children from 12 public schools were invited to participate in this study; 312 children participated in September 2003. Results Sixty eight percent of the subjects had intestinal parasites, 63% had protozoan infections, and 29%, 16% and 10% were infected with Giardia duodenalis, Hymenolepsis nana, and Entamoeba histolytica/dispar/moshkovskii respectively. Fifty children excreted eggs of Hymenolepsis nana. Conclusion Educational strategies should be considered to support the national de-worming campaign, because albendazole alone will not sufficiently improve the health conditions of vulnerable populations. PMID:24714136

  2. Metronidazole-triazole conjugates: Activity against Clostridium difficile and parasites

    PubMed Central

    Jarrad, Angie M.; Karoli, Tomislav; Debnath, Anjan; Tay, Chin Yen; Huang, Johnny X.; Kaeslin, Geraldine; Elliott, Alysha G.; Miyamoto, Yukiko; Ramu, Soumya; Kavanagh, Angela M.; Zuegg, Johannes; Eckmann, Lars; Blaskovich, Mark A.T.; Cooper, Matthew A.

    2015-01-01

    Metronidazole has been used clinically for over 50 years as an antiparasitic and broad-spectrum antibacterial agent effective against anaerobic bacteria. However resistance to metronidazole in parasites and bacteria has been reported, and improved second-generation metronidazole analogues are needed. The copper catalysed Huigsen azide-alkyne 1,3-dipolar cycloaddition offers a way to efficiently assemble new libraries of metronidazole analogues. Several new metronidazole-triazole conjugates (Mtz-triazoles) have been identified with excellent broad spectrum antimicrobial and antiparasitic activity targeting Clostridium difficile, Entamoeba histolytica and Giardia lamblia. Cross resistance to metronidazole was observed against stable metronidazole resistant C. difficile and G. lamblia strains. However for the most potent Mtz-triazoles, the activity remained in a therapeutically relevant window. PMID:26117821

  3. Prevalence of enteropathogens in stools of rural Maasai children under five years of age in the Maasailand region of the Kenyan Rift Valley.

    PubMed

    Joyce, T; McGuigan, K G; Elmore-Meegan, M; Conroy, R M

    1996-01-01

    Stool samples were collected during August 1994 from seventy rural Maasai children under the age of five years who were living in the Maasailand region of the Kenyan Rift Valley. Microbiological analysis was carried out on these samples to identify which intestinal pathogens were present among the infant population of the Maasai. Of the samples studied 54% were pathogen positive. The most common pathogen isolated was Giardia lamblia which was detected in 31% of the samples. Other pathogens that were detected include: Entamoeba histolytica (23%), Enteropathogenic Escherichia coli (13%), Strongyloides stercoralis (4%), Blastocystis hominis (3%) and Cryptosporidium sp (3%). Although all samples were screened for Campylobacter and rotavirus, neither pathogen was detected. Water samples were taken from all the water sources in the study area and analysed microbiologically. Results showed that all the sources were contaminated with the faecal E. coli whose populations ranged from 14 CFU/100 ml to greater than 1800 CFU/100ml. PMID:8625866

  4. Identifying etiological agents causing diarrhea in low income Ecuadorian communities.

    PubMed

    Vasco, Gabriela; Trueba, Gabriel; Atherton, Richard; Calvopiña, Manuel; Cevallos, William; Andrade, Thamara; Eguiguren, Martha; Eisenberg, Joseph N S

    2014-09-01

    Continued success in decreasing diarrheal disease burden requires targeted interventions. To develop such interventions, it is crucial to understand which pathogens cause diarrhea. Using a case-control design we tested stool samples, collected in both rural and urban Ecuador, for 15 pathogenic microorganisms. Pathogens were present in 51% of case and 27% of control samples from the urban community, and 62% of case and 18% of control samples collected from the rural community. Rotavirus and Shigellae were associated with diarrhea in the urban community; co-infections were more pathogenic than single infection; Campylobacter and Entamoeba histolytica were found in large numbers in cases and controls; and non-typhi Salmonella and enteropathogenic Escherichia coli were not found in any samples. Consistent with the Global Enteric Multicenter Study, focused in south Asia and sub-Saharan Africa, we found that in Ecuador a small group of pathogens accounted for a significant amount of the diarrheal disease burden. PMID:25048373

  5. Antiamoebic and spasmolytic activities of extracts from some antidiarrhoeal traditional preparations used in Kinshasa, Congo.

    PubMed

    Tona, L; Kambu, K; Ngimbi, N; Mesia, K; Penge, O; Lusakibanza, M; Cimanga, K; De Bruyne, T; Apers, S; Totte, J; Pieters, L; Vlietinck, A J

    2000-03-01

    Three major extracts from some traditional preparations, based on medicinal plants, used as antidiarrhoeal agents were investigated for their putative antiamoebic and spasmolytic activities in vitro. Results indicated that both biological activities are concentrated in the polyphenolic fraction, and not in the saponin or alkaloid containing fractions. The most active polyphenolic extracts were those from Euphorbia hirta whole plant, leaves of Alchornea cordifolia, Crossopteryx febrifuga, Nauclea latifolia, Psidium guajava, Tithonia diversifolia, stem bark of Harungana madagascariensis, Mangifera indica, Maprounea africana and Psidium guajava, inhibiting Entamoeba histolytica growth with MAC < 10 micrograms/ml. The same extracts, at a concentration of 80 micrograms/ml in an organ bath, also exhibited more than 70% inhibition of acetylcholine and/or KCl solution-induced contractions on isolated guinea-pig ileum. PMID:10782488

  6. In vitro antiplasmodial, antiamoebic, and cytotoxic activities of a series of bisbenzylisoquinoline alkaloids.

    PubMed Central

    Marshall, S J; Russell, P F; Wright, C W; Anderson, M M; Phillipson, J D; Kirby, G C; Warhurst, D C; Schiff, P L

    1994-01-01

    Twenty-four bisbenzylisoquinoline alkaloids were screened for antiplasmoidal, antiamoebic, and cytotoxic activities by use of in vitro microtests. Eight of the alkaloids had antiplasmodial activity, with a 50% inhibitory concentration (IC50) of less than 1 microM against a multidrug-resistant strain of Plasmodium falciparum (chloroquine had an IC50 of 0.2 microM). The three alkaloids most active against Entamoeba histolytica, aromoline, isotrilobine, and insularine, had IC50s of 5 to 11.1 microM (metronidazole had an IC50 of 1.87 microM). None of the 24 bisbenzylisoquinoline alkaloids exhibited significant cytotoxicity against the KB cell line, the most toxic being berbamine, with an IC50 of 17.8 microM (the IC50 of podophyllotoxin was 0.008 microM). Bisbenzylisoquinoline alkaloids merit further investigation as potential novel antimalarial agents. PMID:8141587

  7. Evaluation of Luminex xTAG Gastrointestinal Pathogen Analyte-Specific Reagents for High-Throughput, Simultaneous Detection of Bacteria, Viruses, and Parasites of Clinical and Public Health Importance

    PubMed Central

    Navidad, Jose F.; Griswold, David J.; Gradus, M. Stephen

    2013-01-01

    Acute diarrheal disease (ADD) can be caused by a range of pathogens, including bacteria, viruses, and parasites. Conventional diagnostic methods, such as culture, microscopy, biochemical assays, and enzyme-linked immunosorbent assays (ELISA), are laborious and time-consuming and lack sensitivity. Combined, the array of tests performed on a single specimen can increase the turnaround time (TAT) significantly. We validated a 19plex laboratory-developed gastrointestinal pathogen panel (GPP) using Luminex xTAG analyte-specific reagents (ASRs) to simultaneously screen directly in fecal specimens for diarrhea-causing pathogens, including bacteria (Campylobacter jejuni, Salmonella spp., Shigella spp., enterotoxigenic Escherichia coli [ETEC], Shiga toxin-producing E. coli [STEC], E. coli O157:H7, Vibrio cholerae, Yersinia enterocolitica, and toxigenic Clostridium difficile), parasites (Giardia lamblia, Cryptosporidium spp., and Entamoeba histolytica), and viruses (norovirus GI and GII, adenovirus 40/41, and rotavirus A). Performance characteristics of GPP ASRs were determined using 48 reference isolates and 254 clinical specimens. Stool specimens from individuals with diarrhea were tested for pathogens using conventional and molecular methods. Using the predictive methods as standards, the sensitivities of the GPP ASRs were 100% for adenovirus 40/41, norovirus, rotavirus A, Vibrio cholerae, Yersinia enterocolitica, Entamoeba histolytica, Cryptosporidium spp., and E. coli O157:H7; 95% for Giardia lamblia; 94% for ETEC and STEC; 93% for Shigella spp.; 92% for Salmonella spp.; 91% for C. difficile A/B toxins; and 90% for Campylobacter jejuni. The overall comparative performance of the GPP ASRs with conventional methods in clinical samples was 94.5% (range, 90% to 97%), with 99% (99.0% to 99.9%) specificity. Implementation of the GPP ASRs enables our public health laboratory to offer highly sensitive and specific screening and identification of the major ADD-causing pathogens

  8. Evaluation of Luminex xTAG gastrointestinal pathogen analyte-specific reagents for high-throughput, simultaneous detection of bacteria, viruses, and parasites of clinical and public health importance.

    PubMed

    Navidad, Jose F; Griswold, David J; Gradus, M Stephen; Bhattacharyya, Sanjib

    2013-09-01

    Acute diarrheal disease (ADD) can be caused by a range of pathogens, including bacteria, viruses, and parasites. Conventional diagnostic methods, such as culture, microscopy, biochemical assays, and enzyme-linked immunosorbent assays (ELISA), are laborious and time-consuming and lack sensitivity. Combined, the array of tests performed on a single specimen can increase the turnaround time (TAT) significantly. We validated a 19plex laboratory-developed gastrointestinal pathogen panel (GPP) using Luminex xTAG analyte-specific reagents (ASRs) to simultaneously screen directly in fecal specimens for diarrhea-causing pathogens, including bacteria (Campylobacter jejuni, Salmonella spp., Shigella spp., enterotoxigenic Escherichia coli [ETEC], Shiga toxin-producing E. coli [STEC], E. coli O157:H7, Vibrio cholerae, Yersinia enterocolitica, and toxigenic Clostridium difficile), parasites (Giardia lamblia, Cryptosporidium spp., and Entamoeba histolytica), and viruses (norovirus GI and GII, adenovirus 40/41, and rotavirus A). Performance characteristics of GPP ASRs were determined using 48 reference isolates and 254 clinical specimens. Stool specimens from individuals with diarrhea were tested for pathogens using conventional and molecular methods. Using the predictive methods as standards, the sensitivities of the GPP ASRs were 100% for adenovirus 40/41, norovirus, rotavirus A, Vibrio cholerae, Yersinia enterocolitica, Entamoeba histolytica, Cryptosporidium spp., and E. coli O157:H7; 95% for Giardia lamblia; 94% for ETEC and STEC; 93% for Shigella spp.; 92% for Salmonella spp.; 91% for C. difficile A/B toxins; and 90% for Campylobacter jejuni. The overall comparative performance of the GPP ASRs with conventional methods in clinical samples was 94.5% (range, 90% to 97%), with 99% (99.0% to 99.9%) specificity. Implementation of the GPP ASRs enables our public health laboratory to offer highly sensitive and specific screening and identification of the major ADD-causing pathogens

  9. Infection by Intestinal Parasites, Stunting and Anemia in School-Aged Children from Southern Angola

    PubMed Central

    Oliveira, Dinamene; Atouguia, Jorge; Fortes, Filomeno; Guerra, António

    2015-01-01

    Introduction Intestinal parasites are responsible for morbidity in children worldwide, especially in low income countries. In the present study we determine the prevalence of intestinal parasites and explore its association with anemia and stunting in school-aged children. Methods A cross-sectional study was conducted from September to October 2010 enrolling 328 children attending the primary school in Lubango, the second largest city after the capital Luanda. Stool samples were collected for parasite detection through microscopy and molecular identification of Entamoeba histolytica and Entamoeba dispar. Stunting was assessed using the z-scores of height for age and hemoglobin concentration was determined using a portable hemoglobin analyzing system. Results The global prevalence of pathogenic intestinal parasites was 44.2%, the most common being Ascaris lumbricoides (22.0%), Giardia lamblia (20.1%) and Hymenolepis nana (8.8%). Molecular detection revealed that 13.1% of the children carried E. dispar and 0.3% were infected with E. histolytica. The prevalence of stunting (mild to severe) was 41.5%. Stunting was more frequent in older children (p = 0.006, OR = 1.886), while anemia was more frequent in younger children (p = 0.005, OR = 2.210). The prevalence of anemia was 21.6%, and we found a significant association with infection by H. nana (p = 0.031, OR = 2.449). Conclusions This is one of the few published studies reporting intestinal parasites infection, nutritional status and anemia in children from Angola. Furthermore, the present work highlights the importance of regular intestinal parasites screening in children. PMID:26371758

  10. Structure-Based Design of Inhibitors of the Crucial Cysteine Biosynthetic Pathway Enzyme O-Acetyl Serine Sulfhydrylase.

    PubMed

    Mazumder, Mohit; Gourinath, Samudrala

    2016-01-01

    The cysteine biosynthetic pathway is of fundamental importance for the growth, survival, and pathogenicity of the many pathogens. This pathway is present in many species but is absent in mammals. The ability of pathogens to counteract the oxidative defences of a host is critical for the survival of these pathogens during their long latent phases, especially in anaerobic pathogens such as Entamoeba histolytica, Leishmania donovani, Trichomonas vaginalis, and Salmonella typhimurium. All of these organisms rely on the de novo cysteine biosynthetic pathway to assimilate sulphur and maintain a ready supply of cysteine. The de novo cysteine biosynthetic pathway, on account of its being important for the survival of pathogens and at the same time being absent in mammals, is an important drug target for diseases such as amoebiasis, trichomoniasis & tuberculosis. Cysteine biosynthesis is catalysed by two enzymes: serine acetyl transferase (SAT) followed by O-acetylserine sulfhydrylase (OASS). OASS is well studied, and with the availability of crystal structures of this enzyme in different conformations, it is a suitable template for structure-based inhibitor development. Moreover, OASS is highly conserved, both structurally and sequence-wise, among the above-mentioned organisms. There have been several reports of inhibitor screening and development against this enzyme from different organisms such as Salmonella typhimurium, Mycobacterium tuberculosis and Entamoeba histolytica. All of these inhibitors have been reported to display micromolar to nanomolar binding affinities for the open conformation of the enzyme. In this review, we highlight the structural similarities of this enzyme in different organisms and the attempts for inhibitor development so far. We also propose that the intermediate state of the enzyme may be the ideal target for the design of effective highaffinity inhibitors. PMID:26303427

  11. Human intestinal parasites in non-biting synanthropic flies in Ogun State, Nigeria.

    PubMed

    Adenusi, Adedotun Adesegun; Adewoga, Thomas O Sunday

    2013-01-01

    Filth-feeding and breeding, non-biting synanthropic flies have been incriminated in the dissemination of human enteropathogens in the environment. This study determined the species of non-biting synanthropic flies associated with four filthy sites in Ilishan, Ogun State, southwest Nigeria, and assessed their potentials for mechanical transmission of human intestinal parasites. 7190 flies identified as Musca domestica (33.94%), Chrysomya megacephala (26.01%), Musca sorbens (23.23%), Lucilia cuprina (8.76%), Calliphora vicina (4.59%), Sarcophaga sp. (2.78%) and Fannia scalaris (0.70%) were examined for human intestinal parasites by the formol-ether concentration and modified Ziehl-Neelsen techniques. Eggs of the following parasites: Ascaris lumbricoides (34.08%), Trichuris trichiura (25.87%), hookworms (20.45%), Taenia sp. (2.36%), Hymenolepis nana (1.11%), Enterobius vermicularis (0.56%), Strongyloides stercoralis (larvae; 3.89%) and cysts of Entamoeba histolytica/dispar (27.26%), Entamoeba coli (22.67%), Giardia lamblia (3.34%) and Cryptosporidium sp. (1.81%) were isolated from the body surfaces and or gut contents of 75.24% of 719 pooled fly batches. The helminths A. lumbricoides and T. trichiura and the protozoans, E. histolytica/dispar and E. coli were the dominant parasites detected, both on body surfaces and in the gut contents of flies. C. megacephala was the highest carrier of parasites (diversity and number). More parasites were isolated from the gut than from body surfaces (P < 0.05). Flies from soiled ground often carried more parasites than those from abattoir, garbage or open-air market. Synanthropic fly species identified in this study can be of potential epidemiological importance as mechanical transmitters of human intestinal parasites acquired naturally from filth and carried on their body surfaces and or in the gut, because of their vagility and feeding mechanisms. PMID:23290716

  12. Drug Targets and Mechanisms of Resistance in the Anaerobic Protozoa

    PubMed Central

    Upcroft, Peter; Upcroft, Jacqueline A.

    2001-01-01

    The anaerobic protozoa Giardia duodenalis, Trichomonas vaginalis, and Entamoeba histolytica infect up to a billion people each year. G. duodenalis and E. histolytica are primarily pathogens of the intestinal tract, although E. histolytica can form abscesses and invade other organs, where it can be fatal if left untreated. T. vaginalis infection is a sexually transmitted infection causing vaginitis and acute inflammatory disease of the genital mucosa. T. vaginalis has also been reported in the urinary tract, fallopian tubes, and pelvis and can cause pneumonia, bronchitis, and oral lesions. Respiratory infections can be acquired perinatally. T. vaginalis infections have been associated with preterm delivery, low birth weight, and increased mortality as well as predisposing to human immunodeficiency virus infection, AIDS, and cervical cancer. All three organisms lack mitochondria and are susceptible to the nitroimidazole metronidazole because of similar low-redox-potential anaerobic metabolic pathways. Resistance to metronidazole and other drugs has been observed clinically and in the laboratory. Laboratory studies have identified the enzyme that activates metronidazole, pyruvate:ferredoxin oxidoreductase, to its nitroso form and distinct mechanisms of decreasing drug susceptibility that are induced in each organism. Although the nitroimidazoles have been the drug family of choice for treating the anaerobic protozoa, G. duodenalis is less susceptible to other antiparasitic drugs, such as furazolidone, albendazole, and quinacrine. Resistance has been demonstrated for each agent, and the mechanism of resistance has been investigated. Metronidazole resistance in T. vaginalis is well documented, and the principal mechanisms have been defined. Bypass metabolism, such as alternative oxidoreductases, have been discovered in both organisms. Aerobic versus anaerobic resistance in T. vaginalis is discussed. Mechanisms of metronidazole resistance in E. histolytica have recently

  13. Sexual Dimorphism in the Control of Amebic Liver Abscess in a Mouse Model of Disease

    PubMed Central

    Lotter, Hannelore; Jacobs, Thomas; Gaworski, Iris; Tannich, Egbert

    2006-01-01

    Amebic liver abscess (ALA) is the most common extraintestinal manifestation of human infection by the enteric protozoan parasite Entamoeba histolytica. In contrast to intestinal infection, ALA greatly predominates in males but is rare in females. Since humans are the only relevant host for E. histolytica, experimental studies concerning this sexual dimorphism have been hampered by the lack of a suitable animal model. By serial liver passage of cultured E. histolytica trophozoites in gerbils and mice, we generated amebae which reproducibly induce ALA in C57BL/6 mice. Interestingly, all animals developed ALA, but the time courses of abscess formation differed significantly between the genders. Female mice were able to clear the infection within 3 days, whereas in male mice the parasite could be recovered for at least 14 days. Accordingly, male mice showed a prolonged time of recovery from ALA. Immunohistology of abscesses revealed that polymorphonuclear leukocytes and macrophages were the dominant infiltrates, but in addition, γ,δ-T cells, NK cells, and natural killer T (NKT) cells were also present at early times during abscess development, whereas conventional α,β-T cells appeared later, when female mice had already cleared the parasite. Interestingly, male and female mice differed in early cytokine production in response to ameba infection. Enzyme-linked immunospot assays performed with spleen cells of infected animals revealed significantly higher numbers of interleukin-4-producing cells in male mice but significantly higher numbers of gamma interferon (IFN-γ)-producing cells in female mice. Early IFN-γ production and the presence of functional NKT cells were found to be important for the control of hepatic amebiasis as application of an IFN-γ-neutralizing monoclonal antibody or the use of NKT knockout mice (Vα14iNKT, Jα 18−/−) dramatically increased the size of ALA in female mice. In addition, E. histolytica trophozoites could be reisolated from

  14. Sexual dimorphism in the control of amebic liver abscess in a mouse model of disease.

    PubMed

    Lotter, Hannelore; Jacobs, Thomas; Gaworski, Iris; Tannich, Egbert

    2006-01-01

    Amebic liver abscess (ALA) is the most common extraintestinal manifestation of human infection by the enteric protozoan parasite Entamoeba histolytica. In contrast to intestinal infection, ALA greatly predominates in males but is rare in females. Since humans are the only relevant host for E. histolytica, experimental studies concerning this sexual dimorphism have been hampered by the lack of a suitable animal model. By serial liver passage of cultured E. histolytica trophozoites in gerbils and mice, we generated amebae which reproducibly induce ALA in C57BL/6 mice. Interestingly, all animals developed ALA, but the time courses of abscess formation differed significantly between the genders. Female mice were able to clear the infection within 3 days, whereas in male mice the parasite could be recovered for at least 14 days. Accordingly, male mice showed a prolonged time of recovery from ALA. Immunohistology of abscesses revealed that polymorphonuclear leukocytes and macrophages were the dominant infiltrates, but in addition, gamma,delta-T cells, NK cells, and natural killer T (NKT) cells were also present at early times during abscess development, whereas conventional alpha,beta-T cells appeared later, when female mice had already cleared the parasite. Interestingly, male and female mice differed in early cytokine production in response to ameba infection. Enzyme-linked immunospot assays performed with spleen cells of infected animals revealed significantly higher numbers of interleukin-4-producing cells in male mice but significantly higher numbers of gamma interferon (IFN-gamma)-producing cells in female mice. Early IFN-gamma production and the presence of functional NKT cells were found to be important for the control of hepatic amebiasis as application of an IFN-gamma-neutralizing monoclonal antibody or the use of NKT knockout mice (Valpha14iNKT, Jalpha 18(-/-)) dramatically increased the size of ALA in female mice. In addition, E. histolytica trophozoites

  15. Infection Status of Hospitalized Diarrheal Patients with Gastrointestinal Protozoa, Bacteria, and Viruses in the Republic of Korea

    PubMed Central

    Cho, Shin-Hyeong; Lee, Jin-Hee; Lim, Yi-Young; Jeon, Ji-Hye; Yu, Jae-Ran; Kim, Tong-Soo; Lee, Won-Ja; Cho, Seung-Hak; Lee, Deog-Yong; Park, Mi-Seon; Jeong, Hye-Sook; Chen, Doo-Sung; Ji, Yeong-Mi; Kwon, Mi-Hwa

    2010-01-01

    To understand protozoan, viral, and bacterial infections in diarrheal patients, we analyzed positivity and mixed-infection status with 3 protozoans, 4 viruses, and 10 bacteria in hospitalized diarrheal patients during 2004-2006 in the Republic of Korea. A total of 76,652 stool samples were collected from 96 hospitals across the nation. The positivity for protozoa, viruses, and bacteria was 129, 1,759, and 1,797 per 10,000 persons, respectively. Especially, Cryptosporidium parvum was highly mixed-infected with rotavirus among pediatric diarrheal patients (29.5 per 100 C. parvum positive cases), and Entamoeba histolytica was mixed-infected with Clostridium perfringens (10.3 per 100 E. histolytica positive cases) in protozoan-diarrheal patients. Those infected with rotavirus and C. perfringens constituted relatively high proportions among mixed infection cases from January to April. The positivity for rotavirus among viral infection for those aged ≤ 5 years was significantly higher, while C. perfringens among bacterial infection was higher for ≥ 50 years. The information for association of viral and bacterial infections with enteropathogenic protozoa in diarrheal patients may contribute to improvement of care for diarrhea as well as development of control strategies for diarrheal diseases in Korea. PMID:20585526

  16. Gene discovery in the Acanthamoeba castellanii genome

    SciTech Connect

    Anderson, Iain J.; Watkins, Russell F.; Samuelson, John; Spencer,David F.; Majoros, William H.; Gray, Michael W.; Loftus, Brendan J.

    2005-08-01

    Acanthamoeba castellanii is a free-living amoeba found in soil, freshwater, and marine environments and an important predator of bacteria. Acanthamoeba castellanii is also an opportunistic pathogen of clinical interest, responsible for several distinct diseases in humans. In order to provide a genomic platform for the study of this ubiquitous and important protist, we generated a sequence survey of approximately 0.5 x coverage of the genome. The data predict that A. castellanii exhibits a greater biosynthetic capacity than the free-living Dictyostelium discoideum and the parasite Entamoeba histolytica, providing an explanation for the ability of A. castellanii to inhabit adversity of environments. Alginate lyase may provide access to bacteria within biofilms by breaking down the biofilm matrix, and polyhydroxybutyrate depolymerase may facilitate utilization of the bacterial storage compound polyhydroxybutyrate as a food source. Enzymes for the synthesis and breakdown of cellulose were identified, and they likely participate in encystation and excystation as in D. discoideum. Trehalose-6-phosphate synthase is present, suggesting that trehalose plays a role in stress adaptation. Detection and response to a number of stress conditions is likely accomplished with a large set of signal transduction histidine kinases and a set of putative receptorserine/threonine kinases similar to those found in E. histolytica. Serine, cysteine and metalloproteases were identified, some of which are likely involved in pathogenicity.

  17. [The intestinal parasites in the food handlers of Tunis area: study of 8502 stool samples (1998-2008)].

    PubMed

    Siala, E; Guidara, R; Ben Abdallah, R; Ben Ayed, S; Ben Alaya, N; Zallaga, N; Bouratbine, A; Aoun, K

    2011-01-01

    The food handlers control is one of the major measures of the National program against intestinal parasitoses in Tunisia. The collected data allow regular updates of the prevalence and profile of the involved parasitic species and note possible evolutions in time. From 1998 to 2008, 8502 stool samples of food handlers working in Tunis area were examined in the laboratory of Parasitology, Pasteur Institute of Tunis. The mean prevalence of intestinal parasites was 13.5% (1152 positive samples). The protozoa were the most detected parasites (93% of the species observed). Among the pathogenic or potentially pathogenic parasites, Dientamoeba fragilis (15.5%) and Giardia intestinalis (7.6%) were the most frequently encountered. Entamoeba (E.) histolytica/dispar was diagnosed in 41 subjects (a prevalence of 05%). Hymenolepis nana was the most frequent helminth (45% of the whole parasites). Therapeutic failures were noticed in 43.8% of infection by this cestode because of the lack of the appropriate antihelminthic. The identification using PCR of cysts and/or vegetative forms of E. histolytica/dispar revealed the exclusive presence of E. dispar. PMID:23461147

  18. Cytotoxicity of protein-carbon nanotubes on J774 macrophages is a functionalization grade-dependent effect.

    PubMed

    Montes-Fonseca, Silvia Lorena; Sánchez-Ramírez, Blanca; Luna-Velasco, Antonia; Arzate-Quintana, Carlos; Silva-Cazares, Macrina Beatriz; González Horta, Carmen; Orrantia-Borunda, Erasmo

    2015-01-01

    Carbon nanotubes (CNTs) are used as carriers in medicine due to their ability to be functionalized with chemical substances. However, cytotoxicity analysis is required prior to use for in vivo models. The aim of this study was to evaluate the cytotoxic effect of CNTs functionalized with a 46 kDa surface protein from Entamoeba histolytica (P46-CNTs) on J774A macrophages. With this purpose, CNTs were synthesized by spray pyrolysis and purified (P-CNTs) using sonication for 48 h. A 46 kDa protein, with a 4.6-5.4 pI range, was isolated from E. histolytica HM1:IMSS strain trophozoites using an OFFGEL system. The P-CNTs were functionalized with the purified 46 kDa protein, classified according to their degree of functionalization, and characterized by Raman and Infrared spectroscopy. In vitro cytotoxicity was evaluated by MTT, apoptosis, and morphological assays. The results demonstrated that P46-CNTs exhibited cytotoxicity dependent upon the functionalized grade. Contrary to what was expected, P46-CNTs with a high grade of functionalization were more toxic to J774 macrophages than P46-CNTs with a low grade of functionalization, than P-CNTs, and had a similar level of toxicity as UP-CNT. This suggests that the nature of the functionalized protein plays a key role in the cytotoxicity of these nanoparticles. PMID:26075262

  19. Cryptosporidium parvum Among Coprolites from La Cueva de los Muertos Chiquitos (600-800 CE), Rio Zape Valley, Durango, Mexico.

    PubMed

    Morrow, Johnica J; Reinhard, Karl J

    2016-08-01

    :  In the present study, 90 coprolites from La Cueva de los Muertos Chiquitos (CMC) were subjected to enzyme-linked immunosorbent assay (ELISA) tests for 3 diarrhea-inducing protozoan parasites, Entamoeba histolytica , Giardia duodenalis , and Cryptosporidium parvum , to determine whether these parasites were present among the people who utilized this cave 1,200-1,400 yr ago. These people, the Loma San Gabriel, developed as a culture out of the Archaic Los Caracoles population and lived throughout much of present-day Durango and Zacatecas in Mexico. The Loma San Gabriel persisted through a mixed subsistence strategy of hunting-gathering and agricultural production. The results of ELISA testing were negative for both E. histolytica and G. duodenalis across all coprolites. A total of 66/90 (∼73% prevalence) coprolites tested positive or likely positive for C. parvum . The high prevalence of C. parvum among CMC coprolites contributes to our growing knowledge of the pathoecology among the Loma San Gabriel who utilized CMC. Herein, we report the successful recovery of C. parvum coproantigens from prehistoric coprolites. The recovery of these coproantigens demonstrates the existence of C. parvum in Mesoamerica before European contact in the 1400s. PMID:27098916

  20. Cytotoxicity of Protein-Carbon Nanotubes on J774 Macrophages Is a Functionalization Grade-Dependent Effect

    PubMed Central

    Montes-Fonseca, Silvia Lorena; Sánchez-Ramírez, Blanca; Luna-Velasco, Antonia; Arzate-Quintana, Carlos; Silva-Cazares, Macrina Beatriz; González Horta, Carmen

    2015-01-01

    Carbon nanotubes (CNTs) are used as carriers in medicine due to their ability to be functionalized with chemical substances. However, cytotoxicity analysis is required prior to use for in vivo models. The aim of this study was to evaluate the cytotoxic effect of CNTs functionalized with a 46 kDa surface protein from Entamoeba histolytica (P46-CNTs) on J774A macrophages. With this purpose, CNTs were synthesized by spray pyrolysis and purified (P-CNTs) using sonication for 48 h. A 46 kDa protein, with a 4.6–5.4 pI range, was isolated from E. histolytica HM1:IMSS strain trophozoites using an OFFGEL system. The P-CNTs were functionalized with the purified 46 kDa protein, classified according to their degree of functionalization, and characterized by Raman and Infrared spectroscopy. In vitro cytotoxicity was evaluated by MTT, apoptosis, and morphological assays. The results demonstrated that P46-CNTs exhibited cytotoxicity dependent upon the functionalized grade. Contrary to what was expected, P46-CNTs with a high grade of functionalization were more toxic to J774 macrophages than P46-CNTs with a low grade of functionalization, than P-CNTs, and had a similar level of toxicity as UP-CNT. This suggests that the nature of the functionalized protein plays a key role in the cytotoxicity of these nanoparticles. PMID:26075262

  1. Antiamoebic and Antigiardial Activity of Clerodane Diterpenes from Mexican Salvia Species Used for the Treatment of Diarrhea.

    PubMed

    Calzada, Fernando; Bautista, Elihú; Yépez-Mulia, Lilian; García-Hernandez, Normand; Ortega, Alfredo

    2015-10-01

    Terpenoids from Salvia species have been identified to possess biological properties as antiprotozoal agents. Here, we evaluated the antiamoebic and antigiardial activities of 14 known clerodane and modified clerodane-type diterpenes isolated from five Mexican Salvia species against Entamoeba histolytica and Giardia lamblia, and analyzed the effects of the functionalities in decalin ring or in the whole clerodane framework to visualize the structural requirements necessary to produce an antiprotozoal activity. Among these, linearolactone was the most active clerodane diterpene against both protozoa with IC50 values of 22.9 μM for E. histolytica and of 28.2 μM in the case of G. lamblia. In this context it may be a lead compound for the development of novel therapeutic agent for the treatment of diarrhea and dysentery. The remaining diterpenes assayed showed moderate to weak activity against both protozoa. These findings give support to the use of Salvia species in the traditional medicine from México for the treatment of diarrhea. PMID:26190323

  2. Molecular Epidemiology of Human Intestinal Amoebas in Iran

    PubMed Central

    Hooshyar, H; Rostamkhani, P; Rezaian, M

    2012-01-01

    Many microscopic-based epidemiological surveys on the prevalence of human intestinal pathogenic and non-pathogenic protozoa including intestinal amoeba performed in Iran show a high prevalence of human intestinal amoeba in different parts of Iran. Such epidemiological studies on amoebiasis are confusing, mainly due to recently appreciated distinction between the Entamoeba histolytica, E. dispar and E. moshkovskii. Differential diagnosis can be done by some methods such as PCR-based methods, monoclonal antibodies and the analysis of isoenzyme typing, however the molecular study of these protozoa in Iran is low. Based on molecular studies, it seems that E. dispar is predominant species especially in the central and northern areas of Iran and amoebiasis due to E. histolytica is a rare infection in the country. It is suggested that infection with E. moshkovskii may be common among Iranians. Considering the importance of molecular epidemiology of amoeba in Iran and also the current data, the present study reviews the data currently available on the molecular distribution of intestinal human amoeba in Iran. PMID:23193500

  3. Detection of diarrhoea-causing protozoa in general practice patients in The Netherlands by multiplex real-time PCR.

    PubMed

    ten Hove, R; Schuurman, T; Kooistra, M; Möller, L; van Lieshout, L; Verweij, J J

    2007-10-01

    The diagnostic value of a multiplex real-time PCR for the detection of Entamoeba histolytica, Giardia lamblia and Cryptosporidium parvum/Cryptosporidium hominis was evaluated by comparing the PCR results obtained with those of routinely performed microscopy of faecal samples from patients consulting their general practitioner (GP) because of gastrointestinal complaints. Analysis of 722 faecal DNA samples revealed that the prevalence of G. lamblia was 9.3% according to PCR, as compared to 5.7% by microscopy. The number of infections detected was more than double in children of school age. Furthermore, G. lamblia infection was detected in 15 (6.6%) of 228 faecal samples submitted to the laboratory for bacterial culture only. C. parvum/C. hominis infections were not diagnosed by routine procedures, but DNA from these organisms was detected in 4.3% of 950 DNA samples. A strong association with age was noted, with Cryptosporidium being detected in 21.8% of 110 children aged <5 years. C. hominis was the most prevalent species. E. histolytica was not detected in this study population. Analysis of microscopy data revealed that the number of additional parasites missed by PCR was small. Overall, the study demonstrated that a multiplex real-time PCR approach is a feasible diagnostic alternative in the clinical laboratory for the detection of parasitic infections in patients consulting GPs because of gastrointestinal symptoms. PMID:17714523

  4. Longitudinal Evaluation of Enteric Protozoa in Haitian Children by Stool Exam and Multiplex Serologic Assay

    PubMed Central

    Moss, Delynn M.; Priest, Jeffrey W.; Hamlin, Kathy; Derado, Gordana; Herbein, Joel; Petri, William A.; Lammie, Patrick J.

    2014-01-01

    Haitian children were monitored longitudinally in a filariasis study. Included were stool samples examined for Giardia intestinalis and Entamoeba histolytica cysts, and serum specimens analyzed for immunoglobulin G (IgG) responses to eight recombinant antigens from G. intestinalis (variant-specific surface protein [VSP1–VSP5]), E. histolytica (lectin adhesion molecule [LecA]), and Cryptosporidium parvum (17- and 27-kDa) using a multiplex bead assay. The IgG responses to VSP antigens peaked at 2 years of age and then diminished and were significantly lower (P < 0.002) in children > 4.5 years than in children < 4.5 years. The IgG responses to Cryptosporidium tended to increase with age. The IgG responses to LecA and VSP antigens and the prevalence of stools positive for cysts were significantly higher (P < 0.037 and P < 0.035, respectively) in the rainy season than in the dry season. The multiplex bead assay provides a powerful tool for analyzing serologic responses to multiple pathogens. PMID:24591430

  5. A Retrospective Analysis of the Results of a Five-Year (2005–2009) Parasitological Examination for Common Intestinal Parasites from Bale-Robe Health Center, Robe Town, Southeastern Ethiopia

    PubMed Central

    Chala, Bayissa

    2013-01-01

    A cross-sectional retrospective survey using the past five years clinical records (2005–2009) was conducted. The study was aimed at assessing the status of common intestinal parasites from Bale-Robe Health Center, Southeastern Ethiopia, in 2009/2010. The survey involved collection of data recorded on intestinal parasite from the health center during 2005–2009. Precoded questionnaires and interviews were also supplemented for knowledge attitude practices survey (KAPs survey) to assess awareness level of treatment seekers. Analysis of the various associations and strength of significant variations among qualitative and quantitative variables were assessed. The results revealed that Entamoeba histolytica (36.1%) and Giardia lamblia (11.0%), both being protozoan parasites were found to be the most prevalent intestinal parasites encountered during 2005–2009. The least prevalent intestinal parasite recorded was Strongyloides stercoralis (1.1%). Most intestinal parasites were detected among age group of 15 years and above than 0–4 and 5–14 years as shown in Table 4. There was a significant correlation between intestinal parasites prevalence and the age of treatment seeking individuals (P < 0.05). A sharp increasing trend of E. histolytica and Ascaris lumbricoides infections was observed owing to low personal and environmental sanitation of the majority of the society. Initiation of health education at different levels could be recommended to mitigate infectious parasites in the area. PMID:27335857

  6. The Tropical Brown Alga Lobophora variegata: A Source of Antiprotozoal Compounds

    PubMed Central

    Cantillo-Ciau, Zulema; Moo-Puc, Rosa; Quijano, Leovigildo; Freile-Pelegrín, Yolanda

    2010-01-01

    Lobophora variegata, a brown alga collected from the coast of the Yucatan peninsula, Mexico, was studied for antiprotozoal activity against Giardia intestinalis, Entamoeba histolytica and Trichomonas vaginalis. The whole extract showed the highest activity against T. vaginalis, with an IC50 value of 3.2 μg/mL. For the fractions, the best antiprotozoal activity was found in non-polar fractions. The chloroform fraction of the extract contained a major sulfoquinovosyldiacylglycerol (SQDG), identified as 1-O-palmitoyl-2-O-myristoyl-3-O-(6‴-sulfo-α-d-quinovopyranosyl)-glycerol (1), together with small amounts of 1,2-di-O-palmitoyl-3-O-(6‴-sulfo-α-d-quinovopyranosyl)-glycerol (2) and a new compound identified as 1-O-palmitoyl-2-O-oleoyl-3-O-(6‴-sulfo-α-d-quinovopyranosyl)-glycerol (3). Their structures were elucidated on the basis of chemical and enzymatic hydrolysis and careful analysis of FAB-MS and NMR spectroscopic data. This is the first report on the isolation of SQDGs from L. variegata. The mixture of 1–3 showed good activity against E. histolytica and moderate activity against T. vaginalis with IC50s of 3.9 and 8.0 μg/mL, respectively, however, the activity of 1–3 is not as effective as metronidazole. These results afford ground information for the potential use of the whole extract and fractions of this species in protozoal infections. PMID:20479979

  7. Detection of selected intestinal helminths and protozoa at Hospital Universiti Sains Malaysia using multiplex real-time PCR.

    PubMed

    Basuni, M; Mohamed, Z; Ahmad, M; Zakaria, N Z; Noordin, R

    2012-09-01

    Intestinal parasites are the causative agents of a number of important human infections in developing countries. The objective of this study was to determine the prevalence of selected helminths and protozoan infections among patients admitted with gastrointestinal disorders at Hospital Universiti Sains Malaysia, Kelantan, Malaysia using multiplex real-time PCR. In addition microscopic examination was also performed following direct smear, zinc sulphate concentration and Kato-Katz thick smear techniques; and the presence of protozoan parasites was confirmed using trichrome and acid-fast stains. Of the 225 faecal samples analysed, 26.2% were positive for intestinal parasites by the multiplex real-time PCR, while 5.3% were positive by microscopy. As compared to microscopy, the multiplex real-time PCR detected 5.8 and 4.5 times more positives for the selected helminth and protozoan infections respectively. Among the selected helminths detected in this study, hookworm was the most prevalent by real-time PCR, while Ascaris lumbricoides was detected the most by microscopy. Meanwhile, among the selected protozoa detected in this study, Entamoeba histolytica was the most prevalent by real-time PCR, however microscopy detected equal number of cases with E. histolytica and Giardia lamblia. This study showed that real-time PCR can be used to obtain a more accurate prevalence data on intestinal helminths and protozoa. PMID:23018507

  8. Fulminant Amebic Colitis after Corticosteroid Therapy: A Systematic Review

    PubMed Central

    Shirley, Debbie-Ann; Moonah, Shannon

    2016-01-01

    Background Amebic colitis, caused by intestinal infection with the parasite, Entamoeba histolytica, is a common cause of diarrhea worldwide. Fulminant amebic colitis is the most devastating complication of this infection, associated with both high mortality and morbidity. We conducted a review of the English literature to describe cases of fulminant amebic colitis associated with exposure to corticosteroid medications in order to identify the risk factors for poor outcome and determine difficulties in diagnosis and treatment. Methodology and Principal Findings Articles reporting severe and fulminant forms of amebic colitis between 1991 and 2016 were collected. 525 records were screened to identify 24 cases for qualitative analysis associated with corticosteroid use. Cases arose from areas of high endemicity or travel to such areas. Most cases (14 of 24, 58%) were given corticosteroids for initially misdiagnosed colitis, mainly inflammatory bowel, resulting in rapid progression of disease. Nearly half of all cases underwent surgical intervention, and 25% of cases died, despite all patients eventually receiving treatment with metronidazole. The odds of death did not differ significantly by prior misdiagnosis, co-morbidities, bowel perforation or need for surgery. Conclusions and Significance Infection with E. histolytica should be considered prior to the administration of corticosteroids, in particular for patients residing in endemic areas or those with appropriate travel history, especially prior to the diagnosis of inflammatory bowel disease. The development of preventative and treatment interventions are needed to improve outcomes of fulminant disease. PMID:27467600

  9. Inactivation and unfolding of protein tyrosine phosphatase from Thermus thermophilus HB27 during urea and guanidine hydrochloride denaturation.

    PubMed

    Wang, Yejing; He, Huawei; Liu, Lina; Gao, Chunyan; Xu, Shui; Zhao, Ping; Xia, Qingyou

    2014-01-01

    The effects of urea and guanidine hydrochloride (GdnHCl) on the activity, conformation and unfolding process of protein tyrosine phosphatase (PTPase), a thermostable low molecular weight protein from Thermus thermophilus HB27, have been studied. Enzymatic activity assays showed both urea and GdnHCl resulted in the inactivation of PTPase in a concentration and time-dependent manner. Inactivation kinetics analysis suggested that the inactivation of PTPase induced by urea and GdnHCl were both monophasic and reversible processes, and the effects of urea and GdnHCl on PTPase were similar to that of mixed-type reversible inhibitors. Far-ultraviolet (UV) circular dichroism (CD), Tryptophan and 1-anilinonaphthalene -8-sulfonic acid (ANS) fluorescence spectral analyses indicated the existence of a partially active and an inactive molten globule-like intermediate during the unfolding processes induced by urea and GdnHCl, respectively. Based on the sequence alignment and the homolog Tt1001 protein structure, we discussed the possible conformational transitions of PTPase induced by urea and GdnHCl and compared the conformations of these unfolding intermediates with the transient states in bovine PTPase and its complex structures in detail. Our results may be able to provide some valuable clues to reveal the relationship between the structure and enzymatic activity, and the unfolding pathway and mechanism of PTPase. PMID:25255086

  10. Breast-Feeding Protects Infantile Diarrhea Caused by Intestinal Protozoan Infections

    PubMed Central

    Belal, Usama Salah; Abdellatif, Manal Zaki Mohamed; Naoi, Koji; Norose, Kazumi

    2013-01-01

    This study investigated the effect of breast-feeding in protection against protozoan infection in infants with persistent diarrhea. Infants were classified into 2 groups; 161 breast-fed infants and the same number of non-breast-fed infants. Microscopic examinations of stool were done for detection of parasites and measuring the intensity of infection. Moreover, serum levels of IgE and TNF-α were measured by ELISA. Cryptosporidium spp., Entamoeba histolytica/Entamoeba dispar, Giardia lamblia, and Blastocystis sp. were demonstrated in infants with persistent diarrhea. The percentage of protozoan infections was significantly lower in breast-fed infants than that in the non-breast-fed infants. The levels of IgE and TNF-α were significantly lower in the breast-fed group than in the non-breast-fed group. There were significant positive associations between the serum levels of IgE and TNF-α and the intensity of parasite infection in the breast-fed group. It is suggested that breast-feeding has an attenuating effect on the rate and intensity of parasite infection. PMID:24327776

  11. Comparative study of the prevalence of intestinal parasites in low socioeconomic areas from South chennai, India.

    PubMed

    Dhanabal, Jeevitha; Selvadoss, Pradeep Pushparaj; Muthuswamy, Kanchana

    2014-01-01

    Intestinal parasites cause one of the most important health problems through their effects in causing undernourishment morbidity and incapacitation due to their behavior particularly in children compared to adults. This study was intended to state the prevalence of intestinal parasites between the slum dwellers of different areas in south Chennai. Among the total of 256 samples collected between the ages of 0-50 yrs, 194 samples were positive. Standard laboratory techniques for parasitological diagnosis were carried out for each sample. Entamoeba coli (23%), Cyclospora sp. (22.2%), Entamoeba histolytica (21.8%), Giardia intestinalis (14.4%), Ascaris lumbricoides (6.2%), Trichuris trichiura (1.1%), and Hymenolepis nana (2.7%) were found in the dwellers of low socioeconomic areas. The data on the prevalence of parasites with respect to sex and age showed that the females harbored more numbers of parasites when compared to males. Further, with respect to age, children and teenagers had surplus parasites compared to old age groups. The percentage of educational status showed a reduction in the number of parasites in the higher education dwellers. These parasites could be prevented by possible grouping of better ecological design and hygiene. Conclusively, the examination of personal hygiene as well as routine medical examination and treatment is strongly recommended in the low socio-economic areas. PMID:24587897

  12. Parasite detection efficiencies of five stool concentration systems.

    PubMed Central

    Perry, J L; Matthews, J S; Miller, G R

    1990-01-01

    Fresh fecal material that was free of ova and parasites was pooled with 10% Formalin in a 1:4 ratio to prepare a standard specimen. Portions of 100 ml of this specimen were individually seeded with Cryptosporidium oocysts, Entamoeba coli, Entamoeba histolytica, and Giardia lamblia cysts; ova of Necator americanus; and Strongyloides larvae. Appropriate volumes of each parasite suspension were used to evaluate the Fecal Concentrator Kit (Remel, Lenexa, Kans.), Fecal Parasite Concentrator (Evergreen Scientific, Los Angeles, Calif.), Para-Pak Macro-Con (Meridian Diagnostics, Inc., Cincinnati, Ohio), and Trend FeKal CON-Trate (Trend Scientific, Inc., St. Paul, Minn.). A standardized gauze filtration method was used as the reference procedure. Tests were performed in triplicate with each individual parasite-concentrator combination, with three slides examined from each sediment. All of the systems effectively concentrated parasites compared with direct examination of unconcentrated fecal material. The Fecal Concentrator Kit provided the best overall performance. Clarity of sediment, lack of debris, and uniformity of background material were found to be important considerations for microscopic detection of parasites in concentrated specimens. PMID:2380347

  13. PREVALENCE OF INTESTINAL PARASITES AMONG FOOD HANDLERS OF SARI, NORTHERN IRAN

    PubMed Central

    SHARIF, Mehdi; DARYANI, Ahmad; KIA, Elham; REZAEI, Fateme; NASIRI, Mehrdad; NASROLAHEI, Mohtaram

    2015-01-01

    Parasitic infection is highly prevalent throughout the developing countries of the world. Food handlers are a potential source of infection for many intestinal parasites and other enteropathogenic infections as well. The aim of this study was to determine the prevalence of intestinal parasite carriers among food handlers attending the public health center laboratory in Sari, Northern Iran for annual check-up. The study was performed from August 2011 through February 2012. Stool samples were collected from 1041 male and female food handlers of different jobs aged between 18 to 63 years and were examined following standard procedures. Sociodemographic, environmental and behavioral data analysis of the food handlers were recorded in a separate questionnaire. Intestinal parasites were found in 161 (15.5%) of the studied samples. Seven species of protozoan or helminth infections were detected. Most of the participants were infected with Giardia lamblia (53.9%) followed by Blastocystis hominis (18%), Entamoeba coli (15.5%), Entamoeba histolytica/dispar (5.5%), Cryptosporidium sp. (3.1%), Iodamoeba butschlii (3.1%) and Hymenolepis nana (1.9%) as the only helminth infection. The findings emphasized that food handlers with different pathogenic organisms may predispose consumers to significant health risks. Routine screening and treatment of food handlers is a proper tool in preventing food-borne infections. PMID:25923893

  14. Intestinal parasitic infections among mentally handicapped individuals in Alexandria, Egypt.

    PubMed

    Shehata, Amany I; Hassanein, Faika

    2015-01-01

    This cross-sectional study was carried to determine the prevalence of intestinal parasitic infections among mentally handicapped individuals in Alexandria, Egypt, in the period from December 2012 till November 2013. The study was conducted on 200 institutionalized and non-institutionalized mentally handicapped individuals. Fresh stool samples were subjected to different stains including; trichrome for detecting intestinal protozoa, modified acid fast stain for intestinal coccidia and quick hot gram chromotrope stain for Microsporidia. Also they were processed by Kato-Katz and formol ethyl acetate techniques for intestinal helminths. Additionally, blood samples were collected for measuring hemoglobin levels. Out of 200 mentally handicapped individuals, 87 (43.5%) were infected. The infection rates were 44.6% and 42.6% for non-institutionalized and institutionalized people, respectively. Regarding gender, 46.7% and 38.5% were reported for the males and females respectively. The most common parasites detected were: Cryptosporidium sp. (23.5%), microsporidia (15%), Giardia lamblia (8.5%), Dientamoeba fragilis (8%), Cyclospora cyatanensis (7.5%), Blastocystis hominis (6.5%), Entamoeba histolytica (5.5%) and Entamoeba coli (2.5%). Rates for Isospora belli and Enterobius vermicularis were estimated to be 1.5% for each, while lower rate was reported for Iodamoeba butschlii (1.0%). Prevalence of infections among mentally handicapped individuals are indications for several risk factors, including improper sanitary hygiene and illiteracy about personal hygiene. Therefore, frequent investigations, health care and medical intervention are needed. PMID:26878626

  15. Prevalence of Intestinal Protozoans among Schoolchildren in Suburban Areas near Yangon, Myanmar.

    PubMed

    Kim, Min-Jae; Jung, Bong-Kwang; Cho, Jaeeun; Kim, Deok-Gyu; Song, Hyemi; Lee, Keon-Hoon; Cho, Seon; Htoon, Thi Thi; Tin, Htay Htay; Chai, Jong-Yil

    2016-06-01

    Although intestinal protozoans are common etiologies of diarrhea, few studies have been conducted in Myanmar. This study planned to investigate the prevalence of Giardia lamblia, Entamoeba coli, Entamoeba histolytica, and Endolimax nana among schoolchildren and their guardians in suburban areas near Yangon, Myanmar. We performed a cross-sectional survey among schoolchildren and their guardians from 7 primary schools in South Dagon and Hlaing Thar Yar districts, Yangon, Myanmar. Stool samples were observed with a microscope after concentration technique and iodine staining. Total 821 stool samples, including 556 from schoolchildren and 265 from guardians, were examined. The median age was 6 years old for schoolchildren and 36 years old for guardians. A 53.1% of the school children and 14.6 % of the guardians were males. The overall prevalence of each intestinal protozoan species was as follows: 3.4% (28/821) for G. lamblia; 3.5% (29/821) for E. coli; 1.2% (10/821) for E. histoytica, and 3.0% for E. nana. This study showed that intestinal protozoans are common in primary schoolchildren and their guardians in suburban areas near Yangon, Myanmar. Health interventions, such as hand washing education, improvement of sanitation, and establishment of water purification systems are urgently needed in this area. PMID:27417092

  16. Enteropathogens associated with diarrhea among military personnel during Operation Bright Star 96, in Alexandria, Egypt.

    PubMed

    Oyofo, B A; Peruski, L F; Ismail, T F; el-Etr, S H; Churilla, A M; Wasfy, M O; Petruccelli, B P; Gabriel, M E

    1997-06-01

    This study investigated the microbial causes of diarrheal disease among U.S. troops deployed near Alexandria, Egypt, during October 1995. Bacterial causes associated with 19 cases of diarrhea included: enterotoxigenic Escherichia coli (ETEC), 42% (21% heat-stable, 11% heat-labile, and 11% heat-stable/ heat-labile producers); enteropathogenic E. coli (5.3%); and enteroadherent E. coli (42%). Four cases of diarrhea were associated with enteroaggregative E. coli based on probe analysis for enteroaggregative heat-stable enterotoxin 1. Protozoan causes included; Entamoeba histolytica (11%), E. hartmanni (5%), E. nana (5%), Blastocystis hominis (5%), Chilomastix mesnili (11%), Dientamoeba fragilis (5%), Entamoeba coli (5%), and Cryptosporidium (5%). Shigella, Aeromonas, Plesiomonas, Vibrio, Campylobacter, and Salmonella were not detected. Of the eight ETEC cases, one was colonization factor antigen (CFA)/I only, one was both CFA/I and CFA/III, three were CFA/II, two were CFA/IV, and two were CFA-negative. Antibiograms of the ETEC and enteroadherent E. coli strains showed that all isolates were susceptible to norfloxacin, ciprofloxacin, and nalidixic acid but resistant to ampicillin, tetracycline, chloramphenicol, and sulfamethoxazole. PMID:9183160

  17. Amoebozoa possess lineage-specific globin gene repertoires gained by individual horizontal gene transfers.

    PubMed

    Dröge, Jasmin; Buczek, Dorota; Suzuki, Yutaka; Makałowski, Wojciech

    2014-01-01

    The Amoebozoa represent a clade of unicellular amoeboid organisms that display a wide variety of lifestyles, including free-living and parasitic species. For example, the social amoeba Dictyostelium discoideum has the ability to aggregate into a multicellular fruiting body upon starvation, while the pathogenic amoeba Entamoeba histolytica is a parasite of humans. Globins are small heme proteins that are present in almost all extant organisms. Although several genomes of amoebozoan species have been sequenced, little is known about the phyletic distribution of globin genes within this phylum. Only two flavohemoglobins (FHbs) of D. discoideum have been reported and characterized previously while the genomes of Entamoeba species are apparently devoid of globin genes. We investigated eleven amoebozoan species for the presence of globin genes by genomic and phylogenetic in silico analyses. Additional FHb genes were identified in the genomes of four social amoebas and the true slime mold Physarum polycephalum. Moreover, a single-domain globin (SDFgb) of Hartmannella vermiformis, as well as two truncated hemoglobins (trHbs) of Acanthamoeba castellanii were identified. Phylogenetic evidence suggests that these globin genes were independently acquired via horizontal gene transfer from some ancestral bacteria. Furthermore, the phylogenetic tree of amoebozoan FHbs indicates that they do not share a common ancestry and that a transfer of FHbs from bacteria to amoeba occurred multiple times. PMID:25013378

  18. Prevalence of intestinal parasites among food handlers of Sari, Northern Iran.

    PubMed

    Sharif, Mehdi; Daryani, Ahmad; Kia, Elham; Rezaei, Fateme; Nasiri, Mehrdad; Nasrolahei, Mohtaram

    2015-01-01

    Parasitic infection is highly prevalent throughout the developing countries of the world. Food handlers are a potential source of infection for many intestinal parasites and other enteropathogenic infections as well. The aim of this study was to determine the prevalence of intestinal parasite carriers among food handlers attending the public health center laboratory in Sari, Northern Iran for annual check-up. The study was performed from August 2011 through February 2012. Stool samples were collected from 1041 male and female food handlers of different jobs aged between 18 to 63 years and were examined following standard procedures. Sociodemographic, environmental and behavioral data analysis of the food handlers were recorded in a separate questionnaire. Intestinal parasites were found in 161 (15.5%) of the studied samples. Seven species of protozoan or helminth infections were detected. Most of the participants were infected with Giardia lamblia (53.9%) followed by Blastocystis hominis (18%), Entamoeba coli (15.5%), Entamoeba histolytica/dispar (5.5%), Cryptosporidium sp. (3.1%), Iodamoeba butschlii (3.1%) and Hymenolepis nana (1.9%) as the only helminth infection. The findings emphasized that food handlers with different pathogenic organisms may predispose consumers to significant health risks. Routine screening and treatment of food handlers is a proper tool in preventing food-borne infections. PMID:25923893

  19. [Water pollution and health risks at Yaoundé, Cameroon].

    PubMed

    Youmbi, Jean Ghislain Tabué; Feumba, Roger; Njitat, Valérie Tsama; de Marsily, Ghislain; Ekodeck, Georges Emmanuel

    2013-01-01

    The aim of this article consists of developing an ecosystemic approach to collect information about groundwater pollution in order to determine its origin and infer the health risks in Mingoa's watershed through the study of 12 spontaneous settlements. From an environmental point of view, family interviews and direct observations allowed us to inventory springs, wells and latrines, and to collect information. So, we have selected, on the basis of predefined criteria, 21 wells and springs for physico-chemical and bacteriological laboratory analyses. Two hundred children aged less than 5 years were subjected to KOAP examination. A number of 1224 latrines were inventoried, for an average use of 15 people per latrine, producing 913.3 kg/year of excreta. Most of 91% of people use these latrines. The superficial aquifers, connected to the wells and springs, present high values of nitrogen, phosphorous pollutant and fecal contamination indicators (coliforms and fecal streptococcus), which make water inappropriate to human consumption. PCA analysis establishes that water pollution comes mainly from traditional pit latrines. The overall prevalence of diarrhea and parasite infestation was important (=40%). The presence of cysts of Entamoeba histolytica, Ascaris limbricoides, and Entamoeba coli confirms this high prevalence and testify to very approximate hygiene and environment conditions. PMID:23916209

  20. INTESTINAL PARASITES IN PATIENTS WITH CHRONIC ABDOMINAL PAIN.

    PubMed

    Omran, Eman Kh; Mohammad, Asmaa N

    2015-08-01

    Information about intestinal parasites in Sohag (Upper Egypt) in patients with chronic abdominal pain is scarce. This study determined the intestinal parasites symptoms in 130 patients with chronic abdominal pain and cross-matched 20 healthy persons. Parasitic infection was confirmed by stool analysis.The most commonest clinical data with stool analysis was as following: 1-Entamoeba histolytica associated with nausea 20 (3 7.74%) followed by anorexia 19 (35.85%), 2-Entamoeba coli associated with diarrhea 3 (100%) followed by nausea 2 (66.67%) and vomiting 2 (66.67%), 3-Enetrobius vermicularis associated with nausea 2 (66.67%), diarrhea 2 (66.67%) followed by flatulence 1(33.33%), 4-Giardia lamblia associated with anorexia 3 (42.86%), vomiting 3 (42.86%) followed by diarrhea 2 (28.57%)., 6-Hymenolepis nana associated with anorexia 10 (40.00%) followed by flatulence 9 (36.00%), 7-Taenia saginata associated with dyspepsia 3 (60.00%) followed by flatulence 2 (40.00%), and 8-Ancylostoma duodenal associated with anorexia 2 (66.67%) and diarrhea 2 (66.67%). PMID:26485858

  1. Prevalence of Intestinal Protozoans among Schoolchildren in Suburban Areas near Yangon, Myanmar

    PubMed Central

    Kim, Min-Jae; Jung, Bong-Kwang; Cho, Jaeeun; Kim, Deok-Gyu; Song, Hyemi; Lee, Keon-Hoon; Cho, Seon; Htoon, Thi Thi; Tin, Htay Htay; Chai, Jong-Yil

    2016-01-01

    Although intestinal protozoans are common etiologies of diarrhea, few studies have been conducted in Myanmar. This study planned to investigate the prevalence of Giardia lamblia, Entamoeba coli, Entamoeba histolytica, and Endolimax nana among schoolchildren and their guardians in suburban areas near Yangon, Myanmar. We performed a cross-sectional survey among schoolchildren and their guardians from 7 primary schools in South Dagon and Hlaing Thar Yar districts, Yangon, Myanmar. Stool samples were observed with a microscope after concentration technique and iodine staining. Total 821 stool samples, including 556 from schoolchildren and 265 from guardians, were examined. The median age was 6 years old for schoolchildren and 36 years old for guardians. A 53.1% of the school children and 14.6 % of the guardians were males. The overall prevalence of each intestinal protozoan species was as follows: 3.4% (28/821) for G. lamblia; 3.5% (29/821) for E. coli; 1.2% (10/821) for E. histoytica, and 3.0% for E. nana. This study showed that intestinal protozoans are common in primary schoolchildren and their guardians in suburban areas near Yangon, Myanmar. Health interventions, such as hand washing education, improvement of sanitation, and establishment of water purification systems are urgently needed in this area. PMID:27417092

  2. [Distribution of intestinal parasites in patients presenting at the environmental-food and medicine analysis laboratory of Sivas municipality during the years 1993-2006].

    PubMed

    Ataş, Ahmet Duran; Alim, Ahmet; Ataş, Mehmet

    2008-01-01

    The purpose of the present study was to investigate retrospectively the distribution and prevalence of intestinal parasites in patients who presented at the environmental-food and medicine analysis laboratory of the Sivas municipality, Sivas, from January 1993-December 2006. A total of 732 stool specimens were examined for intestinal parasites using native-Lugol and formol ethyl ether methods. Also, 186 cellophane tape preparations were examined directly. Out of a total of 918 total specimens, intestinal parasites were found in 85 (9.3%) females and 178 (19.4%) males. Parasites were found in 188 (25.7%) of the stool specimens, coming from 54 (7.4%) females and 134 (18.3%) males. Of the 75 positive cellophane tape specimens, 31 (16.7%) were females and 44 (23.7%), male. The prevalence of intestinal parasites was evaluated according to years, sex and ages in which cases were seen. The distribution of intestinal parasites detected in stool specimens was as follows: 101 (13.8%) Entamoeba coli, 27 (3.7%) Entamoeba histolytica/dispar, 91 (12.4%) Giardia intestinalis, 30 (4.1%) Taenia saginata, 13 (1.8%) Hymenolepis nana, 7 (1%) Ascaris lumbricoides, 2 (0.3%) Trichuris trichiura and 1 (0.1%) Dicrocoelium dentriticum. Parasites detected in cellophane tape specimens included 64 (34.4%) Enterobius vermicularis and 11 (5.9%) Taenia saginata. PMID:18351554

  3. Amebiasis in HIV-1-infected Japanese men: clinical features and response to therapy.

    PubMed

    Watanabe, Koji; Gatanaga, Hiroyuki; Escueta-de Cadiz, Aleyla; Tanuma, Junko; Nozaki, Tomoyoshi; Oka, Shinichi

    2011-09-01

    Invasive amebic diseases caused by Entamoeba histolytica are increasing among men who have sex with men and co-infection of ameba and HIV-1 is an emerging problem in developed East Asian countries. To characterize the clinical and epidemiological features of invasive amebiasis in HIV-1 patients, the medical records of 170 co-infected cases were analyzed retrospectively, and E. histolytica genotype was assayed in 14 cases. In this series of HIV-1-infected patients, clinical presentation of invasive amebiasis was similar to that described in the normal host. High fever, leukocytosis and high CRP were associated with extraluminal amebic diseases. Two cases died from amebic colitis (resulting in intestinal perforation in one and gastrointestinal bleeding in one), and three cases died from causes unrelated to amebiasis. Treatment with metronidazole or tinidazole was successful in the other 165 cases. Luminal treatment was provided to 83 patients following metronidazole or tinidazole treatment. However, amebiasis recurred in 6 of these, a frequency similar to that seen in patients who did not receive luminal treatment. Recurrence was more frequent in HCV-antibody positive individuals and those who acquired syphilis during the follow-up period. Various genotypes of E. histolytica were identified in 14 patients but there was no correlation between genotype and clinical features. The outcome of metronidazole and tinidazole treatment of uncomplicated amebiasis was excellent even in HIV-1-infected individuals. Luminal treatment following metronidazole or tinidazole treatment does not reduce recurrence of amebiasis in high risk populations probably due to amebic re-infection. PMID:21931875

  4. Added value of multiplex Luminex Gastrointestinal Pathogen Panel (xTAG® GPP) testing in the diagnosis of infectious gastroenteritis.

    PubMed

    Wessels, E; Rusman, L G; van Bussel, M J A W M; Claas, E C J

    2014-03-01

    The Luminex Gastrointestinal Pathogen Panel (xTAG(®) GPP) detects in one assay the most common gastroenteritis-causing pathogens and toxins, namely adenovirus 40/41, norovirus genogroup (NG) I/II, rotavirus A, Clostridium difficile toxin A/B, Campylobacter sp., Escherichia coli O157, Enterotoxigenic E. coli heat-labile enterotoxin/heat-stable enterotoxin, Salmonella sp., Shiga-toxin producing E. coli, Shiga-like toxin (Stx)1/2, Shigella sp., Vibrio cholerae, Yersinia enterocolitica, Cryptosporidium sp., Entamoeba histolytica and Giardia sp. In this study, we compared the results that were obtained by testing 393 faecal samples, collected during November and December 2011 at our laboratory, using the xTAG(®) GPP assay with the results of the routine diagnostic procedure. This procedure includes culture for bacteria and real-time PCR for viruses and parasites, but only if the test was requested by the clinician. If the clinician did not request the test for an xTAG(®) GPP-positive target, real-time PCR assays were used to confirm xTAG(®) GPP positivity. Discrepant results were also tested with real-time PCR assays. A total of 83 targets were detected in 76 samples using xTAG(®) GPP. The xTAG(®) GPP assay detected 43 additional positives compared with the routine diagnostic procedure, of which 11 targets could not be confirmed by real-time PCR. The non-confirmed targets were Campylobacter (one sample), Salmonella (four samples), Shigella (one sample) and E. histolytica (five samples). The xTAG(®) GPP was shown to be a convenient and sensitive assay for detection of 15 major gastrointestinal pathogens in a single molecular test, but for detection of E. histolytica and Salmonella, a confirmatory assay is indicated. PMID:24131399

  5. Fascioliasis and Intestinal Parasitoses Affecting Schoolchildren in Atlixco, Puebla State, Mexico: Epidemiology and Treatment with Nitazoxanide

    PubMed Central

    Zumaquero-Ríos, José Lino; Sarracent-Pérez, Jorge; Rojas-García, Raúl; Rojas-Rivero, Lázara; Martínez-Tovilla, Yaneth; Valero, María Adela; Mas-Coma, Santiago

    2013-01-01

    Background The Atlixco municipality, Puebla State, at a mean altitude of 1840 m, was selected for a study of Fasciola hepatica infection in schoolchildren in Mexico. This area presents permanent water collections continuously receiving thaw water from Popocatepetl volcano (5426 m altitude) through the community supply channels, conforming an epidemiological scenario similar to those known in hyperendemic areas of Andean countries. Methodology and Findings A total of 865 6–14 year-old schoolchildren were analyzed with FasciDIG coproantigen test and Lumbreras rapid sedimentation technique, and quantitatively assessed with Kato-Katz. Fascioliasis prevalences ranged 2.94–13.33% according to localities (mean 5.78%). Intensities were however low (24–384 epg). The association between fascioliasis and the habit of eating raw vegetables was identified, including watercress and radish with pronouncedly higher relative risk than lettuce, corncob, spinach, alfalfa juice, and broccoli. Many F. hepatica-infected children were coinfected by other parasites. Entamoeba histolytica/dispar, Giardia intestinalis, Blastocystis hominis, Hymenolepis nana and Ascaris lumbricoides infection resulted in risk factors for F. hepatica infection. Nitazoxanide efficacy against fascioliasis was 94.0% and 100% after first and second treatment courses, respectively. The few children, for whom a second treatment course was needed, were concomitantly infected by moderate ascariasis burdens. Its efficacy was also very high in the treatment of E. histolytica/E. dispar, G. intestinalis, B. hominis, H. nana, A. lumbricoides, Trichuris trichiura, and Enterobius vermicularis. A second treatment course was needed for all children affected by ancylostomatids. Conclusions Fascioliasis prevalences indicate this area to be mesoendemic, with isolated hyperendemic foci. This is the first time that a human fascioliasis endemic area is described in North America. Nitazoxanide appears as an appropriate

  6. The Amoebicidal Effect of Ergosterol Peroxide Isolated from Pleurotus ostreatus.

    PubMed

    Meza-Menchaca, Thuluz; Suárez-Medellín, Jorge; Del Ángel-Piña, Christian; Trigos, Ángel

    2015-12-01

    Dysentery is an inflammation of the intestine caused by the protozoan parasite Entamoeba histolytica and is a recurrent health problem affecting millions of people worldwide. Because of the magnitude of this disease, finding novel strategies for treatment that does not affect human cells is necessary. Ergosterol peroxide is a sterol particularly known as a major cytotoxic agent with a wide spectrum of biological activities produced by edible and medicinal mushrooms. The aim of this report is to evaluate the amoebicidal activity of ergosterol peroxide (5α, 8α-epidioxy-22E-ergosta-6,22-dien-3β-ol isolated from 5α, 8α-epidioxy-22E-ergosta-6,22-dien-3β-ol) (Jacq.) P. Kumm. f. sp. Florida. Our results show that ergosterol peroxide produced a strong cytotoxic effect against amoebic growth. The inhibitory concentration IC50 of ergosterol peroxide was evaluated. The interaction between E. histolytica and ergosterol peroxide in vitro resulted in strong amoebicidal activity (IC50  = 4.23 nM) that may be due to the oxidatory effect on the parasitic membrane. We also tested selective toxicity of ergosterol peroxide using a cell line CCL-241, a human epithelial cell line isolated from normal human fetal intestinal tissue. To the best of our knowledge, this is the first report on the cytotoxicity of ergosterol peroxide against E. histolytica, which uncovers a new biological property of the lipidic compound isolated from Pleurotus ostreatus (Jacq.) P. Kumm. f. sp. Florida. PMID:26392373

  7. Study of the gastrointestinal parasitic fauna of captive non-human primates (Macaca fascicularis).

    PubMed

    Zanzani, Sergio Aurelio; Gazzonis, Alessia Libera; Epis, Sara; Manfredi, Maria Teresa

    2016-01-01

    The aim of this study was to examine helminths and protozoans in cynomolgus macaques (Macaca fascicularis) imported from registered breeding facilities in China and their relation to health risks for non-human primate handlers in biomedical research centers and in breeding facilities. Fresh fecal samples were collected from a total of 443 M. fascicularis and analyzed by copromicroscopical analysis, immunoenzymatic, or molecular assays. As to helminths, whose eggs were shed in 2.03% of the samples, Trichuris and Oesophagostomum were the only two taxa found, with low prevalence and low eggs per gram (EPG) values. Protozoans were more frequently detected (87.40%), with Entamoeba coli (85.19%) and Endolimax nana (79.26%) as the most prevalent species shed. Other parasites found by fecal smear examination were uninucleated-cyst-producing Entamoebas (78.52%), Iodamoeba bütschlii (42.96%), and Chilomastix mesnili (24.44%), while cysts of Balantidium coli (22.2%) were only observed by sedimentation. No coproantigens of Giardia duodenalis, Cryptosporidium spp., and Entamoeba histolytica complex were detected. Blastocystis sp. infection was noticed in 87.63% of macaques by PCR. These cynomolgus monkeys were infected with many subtypes (ST1, ST2, ST3, ST5, and ST7), where the predominant Blastocystis sp. subtypes were ST2 (77.5%), followed by ST1 (63.5%). Data collected confirmed the presence of potentially zoonotic parasites and a high parasite diversity, suggesting the need for appropriate and sensitive techniques to adequately control them and related health risks for handlers of non-human primates in biomedical research centers and in breeding facilities. PMID:26374536

  8. INTESTINAL PARASITES IN DIABETIC PATIENTS IN SOHAG UNIVERSITY HOSPITALS, EGYPT.

    PubMed

    Elnadi, Nada A; Hassanien, Hassan A; Ahmad, Amal M; Abd Ellah, Asmaa K

    2015-08-01

    Intestinal parasites usually create benign diseases, though they may induce complications with high morbidity and mortality to the immunocompromised, including diabetic patients. The study detected the prevalence of intestinal parasitic infections in diabetic patients, comparing to non-diabetic controls and other parameters. A total of 100 fecal samples were collected from diabetic patients at the outpatient clinic of Sohag University Hospitals and another 100 from cross matched controls. The samples were examined macroscopically and microscopically by direct smear and different concentration methods then stained by Modified Ziehl-Neelsen Acid fast stain. Glycated hemoglobin (Hb Alc) was measured to detect DM controlled patients. The data were organized, tabulated, and statistically analyzed. Intestinal parasites wer