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PubMed2005

Genomic DNA microarrays for Entamoeba histolytica: applications for use in expression profiling and strain genotyping.

The parasite Entamoeba histolytica is a causative agent of dysentery and liver abscesses. Found predominantly in developing countries, this parasitic infection is responsible for significant morbidity and mortality. We have developed a genomic DNA microarray for E. histolytica. The array composed of 11,328 clones contains >2000 unique genes and was utilized for expression profiling and comparative genomic hybridizations of Entamoeba strains. We present a synopsis of our results to date and potential future applications of microarray technology for the study of Entamoeba biology.

Animals↗
PubMed2005

Entamoeba histolytica and Entamoeba dispar: prevalence infection in a rural Mexican community.

The frequency of Entamoeba histolytica and Entamoeba dispar infection was analyzed in a rural community in the state of Morelos, Mexico, through PCR technique by using specie specific primer. The E. histolytica specie was detected in 33 of 290 analyzed stool samples (11.4%), E. dispar specie was observed in 21 samples (7.2%) and both species of Entamoeba were detected in seven samples (2.4%). So a higher E. histolytica than E. dispar frequency infection was detected (13.8 versus 9.6%). Even though in our design we did not considered the follow-up of included individuals, the absence of invasive amebiasis cases in the studied population during our stay in town was unexpected.

Animals↗
PubMed2005

Entamoeba histolytica and/or Entamoeba dispar: infection frequency in HIV+/AIDS patients in Mexico city.

The objective of this work was to evaluate the frequency of Entamoeba histolytica/Entamoeba dispar intestinal infection in HIV+/AIDS subjects and their HIV- close relatives or sexual partners. Enteric parasites were investigated in stool samples by microscopic examination and E. histolytica and E. dispar were identified by PCR. We found by microscopic analysis in HIV+/AIDS group that the E. histolytica/E. dispar complex was present in 5.9% of the members, while in the HIV- group was 2.9%. With PCR we found that the E. histolytica prevalence was 25.3% in the HIV+/AIDS group and 18.5% in the HIV-group. The difference in the results obtained with the microscopic and PCR is due to the different sensibility of the procedures. Besides, we found patients who were infected with E. histolytica in both groups were asymptomatic cyst passers. Our results suggest that E. histolytica strains prevalent in the studied community appear to be of low pathogenic potential.

AIDS-Related Opportunistic Infections↗
PubMed2006

Molecular methods for diagnosis of Entamoeba histolytica in a clinical setting: an overview.

The range of clinical outcomes following Entamoeba histolytica infection is likely to be influenced by the different strains of the parasite already existing in our population. There is a need for developing faster, reliable and reproducible methods for identifying the different strains of E. histolytica. This would have a major impact on the subsequent course of treatment given to patients. In the post-genomic era, different loci of the Entamoeba genome have been targeted for developing suitable probes and genetic markers. This review highlights the development made in this direction and the possibility of using these methods for routine testing of this parasite in clinical samples.

Animals↗
PubMed2006

A Bayesian approach to acute infectious diarrhea in adults.

Acute infectious diarrhea is a yearly occurrence for most Americans, and is associated with 1 million hospitalizations and about 6000 deaths in the United States annually. Up to 80% of acute infectious diarrhea is caused by noroviruses, which produce a clinically mild illness with a predictable short course and good outcome that make laboratory testing and antimicrobial treatment unnecessary. Most diarrhea-causing bacteria and protozoa can cause a clinical illness "like norovirus"; when they do so in healthy adults neither specialized testing nor antimicrobials is required. The presence or absence of epidemiologic evidence (such as travel, hospitalization, antibiotic use, other exposures)and clinical evidence (such as diarrhea frequency and duration, severity of abdominal pain and fever, character of stool, presence of chronic illness or immune deficiency) can change the probability of "not norovirus" from as low as 8% to as high as 100%. Such probabilities guide the use of laboratory testing and antimicrobial therapy in patients who have acute infectious diarrhea.

Acquired Immunodeficiency Syndrome↗
PubMed2006

Diagnostic methods for differentiation of Entamoeba histolytica and Entamoeba dispar in carriers: performance and clinical implications in a non-endemic setting.

Unpreserved faecal samples, suspected to contain Entamoeba histolytica/Entamoeba dispar cysts or trophozoites on the basis of microscopic examination, and serum samples from 416 patients were collected in a prospective study to determine whether stool antigen assays and detection of antibodies in serum are reliable methods to distinguish between carriers of E. histolytica and E. dispar in comparison to the reference test: real-time PCR. In 283 patients (68%) DNA of E. histolytica or E. dispar was amplified by real-time PCR: 6 patients with amoebic colitis (2%), 19 carriers of E. histolytica (6.7%), and 258 carriers of E. dispar (91.2%). In 133 patients (31%) no DNA of E. histolytica or E. dispar could be amplified in the stool samples. This patient group was used as control for the evaluation of diagnostic tests. Using real-time PCR as a reference test, the sensitivity and specificity of (1) the Entamoeba test for the diagnosis of E. histolytica/E. dispar carrier were 59% and 98%, (2) E. histolytica II for the diagnosis of E. histolytica carrier was 71% and 100%, and (3) serology for the diagnosis of E. histolytica infection was 83.3% and 95.2%, respectively. Applied to carriers that did not originate from an endemic country the sensitivity of serology for E. histolytica infection was 90% and specificity was 98.8%. In comparison to real-time PCR the performances of Entamoeba test and E. histolytica II lacked sensitivity for a reliable diagnosis of E. histolytica/E. dispar infection in a non-endemic setting. In carriers of E. histolytica/E. dispar from non-endemic countries the high specificity of serology can be used to establish the diagnosis of E. histolytica infection if antibodies are present.

Adolescent↗
PubMed2005

Transcriptional profiling of Entamoeba histolytica trophozoites.

We have developed an Entamoeba histolytica genomic DNA microarray and used it to develop a transcriptional profile of 1,971 E. histolytica (HM-1:IMSS) genes. The arrays accurately detected message abundance and 31-47% of amebic genes were expressed under standard tissue culture conditions (levels detectable by Northern blot analysis or RT-PCR respectively). Genes expressed at high levels ( approximately 2% of total) included actin (8.m00351), and ribosomal genes (20.m00312). Moderately expressed genes ( approximately 14% of total) included cysteine proteinase (191.m00117), profilin (156.m00098), and an Argonaute family member (11.m00378). Genes with low-level expression ( approximately 15% of total) included Ariel1 (160.m00087). Genes with very low expression ( approximately 16% of total) and those not expressed ( approximately 52% of total) included encystation-specific genes such as Jacob cyst wall glycoprotein (33.m00261), chitin synthase (3.m00544), and chitinase (22.m00311). Transcriptional modulation could be detected using the arrays with 17% of genes upregulated at least two-fold in response to heat shock. These included heat shock proteins (119.m00119 and 279.m00091), cyst wall glycoprotein Jacob (33.m00261), and ubiquitin-associated proteins (16.m00343; 195.m00092). Using Caco-2 cells to model the host-parasite interaction, we verified that host cell killing was dependent on live ameba. However, surprisingly these events did not appear to induce major transcriptional changes in the parasites.

Animals↗
PubMed2006

Kikuchi Fujimoto disease secondary to Entamoeba histolytica: case report.

Kikuchi Fujimoto disease (KFD) is a rare histiocytic necrotizing lymphadenitis which has a benign self-limiting clinical course. Its origin is unknown, but an abnormal autoimmune reaction has been suggested and infection is often considered to be an inciting agent. A 50-year-old man presented with fever, malaise, fatigue and sweat of 7 days duration, and diarrhea for 2 days. Physical examination revealed five mobile and painless cervical adenopathies. Entamoeba histolytica trophozoites and cysts were detected by microscopy of feces. Parenteral ornidazole treatment was commenced. Thorax computerized tomography showed lymph node sizes congruent with infection in the mediastinum, right hilus and right axillary region. Axillary lymph node biopsy and immunohistochemical analyses were then performed, and the results were consistent with histiocytic necrotizing lymphadenitis. From day 4 of antibiotic treatment the patient's body temperature decreased and reached a normal level on day 10. After discharge the patient returned for follow-up twice and was asymptomatic; his lymph nodes were either unpalpable or were decreased in size. We could not find any previous study or case report about a probable role for E. histolytica. Amebiasis can be a triggering factor in KFD or alternatively it is possible that its occurrence is coincidental.

Animals↗
PubMed2005

Cytokine and chemokine responses in patients co-infected with Entamoeba histolytica/dispar, Necator americanus and Mansonella perstans and changes after anti-parasite treatment.

This study examined the impact of concurrent parasite infections (amoebiasis, filariasis, necatoriasis) and the effect of anti-parasite treatment on cytokine and chemokine responses in singly and poly-parasitized patients. Cellular reactivity and parasite-specific Th1- and Th2-type cytokine and chemokine profiles were investigated before and six weeks after treatment. In those patients infected with three parasite species, cellular secretion of interleukin 5 (IL-5) and IL-12p40 by PBMC was strongly diminished (p<0.005) but IL-10 was elevated in parasite-infected patients (p<0.0001) in response to protozoa- and helminth-specific as well as bacteria-specific antigens. Macrophage inflammatory chemokines (MIP-1alpha/CCL3 and MIP-1beta/CCL4), macrophage-derived chemokine (MDC/CCL22) and neutrophil activating chemokine (IL-8/CXCL8) were produced by PBMC in similar amounts in endemic controls and singly and poly-parasitized patients, but thymus and activation-regulated chemokine (TARC/CCL17) was produced the highest by PBMC from patients with triple parasite infections (p<0.0001). Following anti-parasite therapy, secretion of IL-12p40 and IL-5 augmented significantly in treated patients while IL-10, MDC, MIP-1alpha, TARC and IL-8 substantially diminished (all p<10(-5)) when their PBMC were activated with parasite- and bacteria-specific antigens. In summary, PBMC from poly-parasitized patients responded to protozoa- and helminth-specific antigens with a compromised IL-5 and IL-12p40 but high IL-10 and a substantial chemokine release. Chemokines may attract and activate effector cells in peri-parasitic tissues to limit parasite proliferation and dissemination, while depressed IL-5 and IL-12p40 but prominent IL-10 may prevent eosinophil and cytotoxic cell-mediated inflammatory processes and pathogenesis to the host. The changes in this profile following anti-parasite therapy disclosed the dynamics of an immune adaptation associated with parasite accumulation and also with clearance of parasite infections.

Adult↗
PubMed2005

Dehydroepiandrosterone decreases while cortisol increases in vitro growth and viability of Entamoeba histolytica.

In vitro exposure of Entamoeba histolytica trophozoites to the sex steroids 17beta-estradiol, progesterone, and dehydrotestosterone had little effect on parasite viability or proliferation. However, treatment with the adrenal steroid dehydroepiandrosterone (DHEA) markedly inhibited parasite proliferation, adherence and motility, and at a certain dose it induced trophozoite lysis. The opposite effect on proliferation was found when the trophozoites were exposed to cortisol. Moreover, DHEA decreased while cortisol increased the parasite's DNA synthesis determined by 3H-thymidine incorporation. Trophozoite lysis by DHEA appeared to be caused by a necrotic rather than an apoptotic process, as observed in propidium iodide and terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling assays. A possible mechanisms of action was derived from experiments demonstrating that the activity of a putative 3-hydroxy-3-methyl glutaryl CoA reductase detected in trophozoite extracts was inhibited in the presence of DHEA. Contrary to its in vitro inhibitory effect, in vivo administration of DHEA to infected hamsters resulted in exacerbation of the amebic liver abscesses. These results demonstrated that androgen steroids act directly upon E. histolytica growth and viability, and may shed new light on some age and gender differences in disease progression, as well as finding application in the drug treatment of human amebiasis.

Animals↗
PubMed2005

E. dispar strain: analysis of polymorphism as a tool for study of geographic distribution.

The intra-species polymorphism of E. histolytica and E. dispar species in endemic area is an important tool for geographic distribution and spread mechanism studies. Since E. dispar and E. histolytica shears ecological niche, cell cycle, and transmission mechanism for human host, we studied the intra-specie variation and distribution of E. dispar strains obtained from cyst passers, in two neighbor rural communities in Morelos Mexico. We analyzed the polymorphic region of the quitinase protein gene in isolates of E. dispar. In 45 isolates from one community we identified 12 different CHI patterns while in 15 isolates from the other community we identified 5 different patterns. However both communities share 4 patterns. This finding suggests the presence of strains with different geographic mobility.

Animals↗
PubMed2005

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

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.

Animals↗
PubMed2004

In vitro and in vivo interaction of Entamoeba histolytica Gal/GalNAc lectin with various target cells: an immunocytochemical analysis.

The Gal/GalNAc lectin of Entamoeba histolytica trophozoites plays an important role in adhesion. The distribution and final destiny of the lectin during the interaction with host cells are poorly understood. Using monoclonal and polyclonal antibodies against the lectin we studied by immunocytochemistry the in vitro and in vivo interaction of E. histolytica trophozoites with human and hamster hepatocytes. We also analyzed the presence and distribution of the lectin in a mouse model of intestinal amoebiasis. In all cases, trophozoites were highly labeled by anti-lectin antibodies. Cultured human and hamster hepatocytes in contact with, or localized at the vicinity of parasites were also labeled by anti-lectin antibodies. Most of the labeled hepatocytes showed variable degrees of cell damage. Hepatocytes distantly localized from the parasites were also stained with the anti-lectin antibodies. Immunolabeling of tissue sections from different stages of the development of experimental amoebic liver abscess in hamsters showed inflammatory foci containing lectin-labeled trophozoites, hepatocytes, and sinusoidal and inflammatory cells. Lectin-containing hepatocytes had vacuolated cytoplasm with some nuclei with a condensed appearance. Damaged intestinal epithelium also was labeled with anti-lectin antibodies in a mouse model of intestinal amoebiasis. Electron microscopy of axenically cultured trophozoites using gold-labeled monoclonal and polyclonal anti-lectin antibody showed that plasma membrane, vacuole membranes and areas of cell cytosol were labeled. Higher deposits of gold particles in plasma membrane suggestive of cell secretion were observed. Our results demonstrated that Gal/GalNAc lectin was bound and captured by different target cells, and that host cells containing the lectin showed signs of cell damage. The contribution of lectin transfer to host cells in adherence and cell injury remains to be determined.

Animals↗
PubMed2004

An experimental model for amoebic abscess production in the cheek pouch of the Syrian golden hamster, Mesocricetus auratus.

A new experimental model was developed in hamsters for amoebic abscess caused by Entamoeba histolytica. E. histolytica trophozoites were cultured in a liquid axenic medium, and then injected intradermally into the cheek pouch of the Syrian golden hamster, Mesocricetus auratus. Inoculation consistently resulted in abscess formation at the site in 20 of 22 (91%) study animals. The amoebic nature of the abscesses was confirmed by light microscopy and histopathologic examination. Abscess formation was maximal at day 12 post-inoculation. Potential applications of this simple and reliable model include further elucidation of the pathogenesis of invasive amoebiasis, studies of the host response to amoebae, and in vivo evaluation of chemotherapeutic agents that show in vitro efficacy against E. histolytica.

Amebiasis↗
PubMed2005

Risk factors for infection by the Entamoeba histolytica/E. dispar complex: an epidemiological study conducted in outpatient clinics in the city of Manaus, Amazon Region, Brazil.

An epidemiological study was conducted on a population attending outpatient clinics in Manaus, Amazon, Brazil to determine the prevalences of infection by the Entamoeba histolytica/E. dispar complex and by E. histolytica alone, as well as to identify the risk factors involved in transmission. The study was conducted in two phases: survey and case-control. Face-to-face interviews were carried out and faecal samples collected from 1578 individuals. Faeces were examined by optical microscopy and tested for the pathogenic E. histolytica specific antigen. Positivity to E. histolytica/E. dispar was 21.5% (340 cases). Cases were compared with 340 control samples, negative for the E. histolytica/E. dispar complex based on examination by optical microscopy. The analysis was conducted by logistic regression. The risk factors identified were: place of residence, age, ingestion of raw vegetables, quality of water consumed, number of rooms and bedrooms per house, and having other protozoan infections. Specific antigen detection tests identified 22 participants infected by E. histolytica (6.8%) among those positive for E. histolytica/E. dispar. There was a higher proportion of males among participants infected by E. histolytica than among those with E. dispar infections. The study population was asymptomatic or presented non-specific symptoms that could be attributed to amoebiasis.

Adult↗
PubMed2005

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

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

Animals↗
PubMed2006

Entamoeba histolytica-associated diarrheal illness is negatively associated with the growth of preschool children: evidence from a prospective study.

The enteric protozoa, Cryptosporidium, Giardia and Entamoeba histolytica, cause diarrhea in children. We investigated the association of enteric protozoan-associated diarrheal illness with the nutritional status and growth of preschool children in Dhaka, Bangladesh. The subjects were 221 children aged 2-5 years who were followed prospectively for diarrheal illness for 3 years. The weight and height of the children were measured at entry and at 4-month intervals. Cryptosporidium and E. histolytica were diagnosed with commercially available stool antigen detection kits. Giardia was diagnosed by conventional microscopy. Cryptosporidium- and Giardia-associated diarrheal illness was not associated with the growth of the children. Children with E. histolytica-associated diarrheal illness had lower weight for age Z-score changes (-0.103+/-0.120 vs. 0.176+/-0.052, P=0.038). Similarly, the change in height for age Z-score was lower in children with E. histolytica-associated diarrheal illness (-0.348+/-0.186 vs. 0.142+/-0.08, P=0.018). Children with E. histolytica-associated diarrheal illness were 2.93 times (95% CI 1.01-8.52, P=0.047) more likely to be malnourished and 4.69 times (95% CI 1.55-14.18, P=0.006) more prone to be stunted. Entamoeba histolytica-associated diarrheal illness was negatively associated with the growth of preschool children.

Age Factors↗
PubMed2006

Entamoeba histolytica encephalitis diagnosed by PCR of cerebrospinal fluid.

Extra-intestinal amebiasis is secondary to invasive intestinal infections and usually results in an amebic liver abscess. Other organs, including lungs, brain, skin, spleen and kidneys, may be involved. Diagnosis of the cerebral infection is of the utmost importance and is most often made by detection of the organism at the time of brain biopsy or at autopsy. We report the first case of Entamoeba histolytica encephalitis diagnosed by PCR of the cerebrospinal fluid. The patient was treated successfully with metronidazole. PCR is an increasingly useful tool for the diagnosis of central nervous system infection and can provide rapid diagnosis.

Adult↗