AIG1 affects in vitro and in vivo virulence in clinical isolates of Entamoeba histolytica.

The disease state of amebiasis, caused by Entamoeba histolytica, varies from asymptomatic to severe manifestations that include dysentery and extraintestinal abscesses. The virulence factors of the pathogen, and host defense mechanisms, contribute to the outcomes of infection; however, the underlying genetic factors, which affect clinical outcomes, remain to be fully elucidated. To identify these genetic factors in E. histolytica, we used Illumina next-generation sequencing to conduct a comparative genomic analysis of two clinical isolates obtained from diarrheal and asymptomatic patients (strains KU50 and KU27, respectively). By mapping KU50 and KU27 reads to the genome of a reference HM-1:IMSS strain, we identified two genes (EHI_089440 and EHI_176590) that were absent in strain KU27. In KU27, a single AIG1 (avrRpt2-induced gene 1) family gene (EHI_176590) was found to be deleted, from a tandem array of three AIG1 genes, by homologous recombination between the two flanking genes. Overexpression of the EHI_176590 gene, in strain HM-1:IMSS cl6, resulted in increased formation of cell-surface protrusions and enhanced adhesion to human erythrocytes. The EHI_176590 gene was detected by PCR in 56% of stool samples from symptomatic patients infected with E. histolytica, but only in 15% of stool samples from asymptomatic individuals. This suggests that the presence of the EHI_176590 gene is correlated with the outcomes of infection. Taken together, these data strongly indicate that the AIG1 family protein plays a pivotal role in E. histolytica virulence via regulation of host cell adhesion. Our in-vivo experiments, using a hamster liver abscess model, showed that overexpression or gene silencing of EHI_176590 reduced and increased liver abscess formation, respectively. This suggests that the AIG1 genes may have contrasting roles in virulence depending on the genetic background of the parasite and host environment.

EhRab21 associates with the Golgi apparatus in Entamoeba histolytica.

Rab proteins constitute the largest group of small GTPases and act as molecular switches in a wide variety of cellular processes, including proliferation, cytoskeleton assembly, and membrane trafficking in all eukaryotic cells. Rab21 has been reported in several eukaryotic cells, and our results suggest that in Entamoeba histolytica, Rab21 is involved in the vesicular traffic associated with the Golgi apparatus, where its function appears to be important to maintain the structure of this organelle. In addition, proteins such as Rab1A and Sec24, identified in this work associated with EhRab21, participate in the traffic of COPII vesicles from the endoplasmic reticulum to the Golgi apparatus and are necessary to maintain the latter's structure in human cells. In addition, EhRab21 probably affects the lysosome biogenesis, as indicated by an increase in the number of lysosomes as a result of the increase in EhRab21 activity. The participation of EhRab21 in the pathogenesis of amebiasis was verified on the amoebic liver abscess formation model using hamsters (Mesocricetus auratus), in which the overexpression of EhRab21Q64L (positive dominant mutant protein) decreased the number of liver abscesses formed.

Amoebic liver abscess: an unusual cause for a right iliac fossa mass: a case report.

BACKGROUND: Amoebic liver abscess is the most common extra intestinal manifestation of amoebiasis in tropical countries. It usually presents with right hypochondrial pain, fever and anorexia. Amoebic liver abscess has gained clinical significance due to the wide variety of clinical presentations which can cause diagnostic dilemmas and high mortality in untreated cases. CASE PRESENTATION: We report a case of a 63-year-old male with a history of anorexia for 3 weeks, fever for 4 days and examination findings of tender hepatomegaly with a liver span of 15 cm in the mid clavicular line and a firm irregular mass in the right iliac fossa. Ultrasound scan of the abdomen showed two large liver abscesses with one of them leaking into the peritoneal cavity causing a localized pus collection, which had been walled off in the right iliac fossa. He was treated with metronidazole and liver abscesses were drained percutaneously under ultrasound scan guidance. The diagnosis of Entamoeba histolytica infection was confirmed with the serology and subsequently by PCR from the aspirated material. He made an uneventful recovery with resolution of the symptoms and right iliac fossa mass. CONCLUSION: Recognition of variable presentation of amoebic liver abscess is vital, considering the curable nature of this disease and potentially fatal outcome of untreated abscess. An intra-abdominal mass in a patient with amoebic liver abscess should raise the suspicion of a localized collection of pus and impending generalized peritonitis. Early diagnosis and prompt intervention can prevent the dreaded complication of peritonitis and toxemia, and hence reduce the consequent morbidity and mortality.

TNFα-mediated liver destruction by Kupffer cells and Ly6Chi monocytes during Entamoeba histolytica infection.

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.

Vagotomy induces deregulation of the inflammatory response during the development of amoebic liver abscess in hamsters.

BACKGROUND: The parasympathetic nervous system modulates the immune response in the abdominal-pelvic gut through the vagus nerve, which releases acetylcholine. This endogenous ligand acts on α7 nicotinic receptors expressed on immune cells. OBJECTIVE: To study the mechanism of the production and regulation of cytokines in parasympathectomized and control hamsters during the development of amoebic liver abscesses (ALA) caused by Entamoeba histolytica. METHODOLOGY: Six- to 8-week-old male hamsters with and without vagotomy were used in a model of ALA. The animals were infected with trophozoites (350,000; HM1:IMSS strain) via the intrahepatic route and sacrificed at 6, 12, and 24 h and at 2, 4, and 7 days postinfection. Immune parameters were recorded at each time point using morphometric techniques including immunofluorescence and immunohistochemistry assays. These parameters included signal transducer and activator of transcription 3 (STAT3) levels, pro- and anti-inflammatory cytokine levels, and nuclear factor-κB (NFκB) activation in neutrophils and macrophages. RESULTS: Compared to the control groups, the vagotomized (VAG) hamsters showed a significant increase in NFκB activation in neutrophils and macrophages, and higher levels of interleukin (IL)-1ß, IL-6, interferon-γ, and tumor necrosis factor-α. VAG hamsters showed an increase in the expression of IL-8 and phosphorylated STAT3 during the first 24 h postinfection as well as slightly increased levels of transforming growth factor-ß on days 2-7 postinfection. No significant differences were demonstrated in the levels of IL-10. CONCLUSIONS: These results suggest that the vagus nerve plays an important role in the regulation of inflammation during ALA formation.

Efficacy of aspiration in amebic liver abscess.

BACKGROUND: Amebic liver abscess (ALA) is a common and serious problem in our country. There are only a few controlled trials on the efficacy and advantages of combination therapy with percutaneous needle aspiration and pharmacotherapy, over pharmacotherapy alone for amebic liver abscess. MATERIAL AND METHODS: This study was conducted to compare the efficacy of two different treatment modalities i.e. drug treatment alone vs. drug treatment and aspiration of abscess cavity in patients with small (up to 5 cm) and large (5 cm to 10 cm) size ALA. This is one of the largest single center, prospective, randomized studies comparing the efficacy of aspiration in ALA. RESULTS: (i) Mean body temperature, liver tenderness, total leukocyte count (TLC), serum alanine aminotransferase (ALT) and liver span were significantly decreased in the aspiration group on days 8 and 15 as compared to non-aspiration group especially in large abscess (5 cm to 10 cm). (ii) Abscess cavity maximum diameter decreased significantly in aspiration group on days 8 and 15, and 1 month & 3 months in large abscess (5cm to 10 cm). CONCLUSIONS: (i) Needle aspiration along with metronidazole hastens clinical improvement especially in large (5 cm up to 10 cm) cavities in patients with ALA. (ii) Aspiration is safe and no major complications occurred. (iii) Hence, combination therapy should be the first choice especially in large ALA (5 cm to 10 cm).

Experimental amoebic liver abscess in hamsters caused by trophozoites of a Brazilian strain of Entamoeba dispar.

It has been claimed that amoebic molecules such as amoebapore, galactose/N-acetyl galactosamine inhibitable lectin, and cysteine proteases are responsible for host tissue destruction and are present in both pathogenic Entamoeba histolytica and non-pathogenic Entamoeba dispar. Some reports have provided evidence that after infection with E. dispar, pathological changes may occur in some humans. The aim of this study was to evaluate E. dispar pathogenicity by comparing it to the pathogenicity of E. histolytica through liver abscesses induced in hamsters. Syrian golden hamsters were challenged by intrahepatic inoculation with the 03C E. dispar strain or with two strains of E. histolytica (HM1:IMSS and EGG) to compare their virulence grades. As control groups, we used bacterial flora and Pavlova's modified medium. Lesions were verified at 1, 3 and 6 days after inoculation. Multiplex Polymerase Chain Reaction was performed to characterize each strain using EdP1/EdP2 and EhP1/EhP2 primers. The EGG and HM1:IMSS E. histolytica strains and 03C E. dispar were able to cause liver lesions. The EGG strain caused extensive hepatic abscesses, and trophozoites were found in the lesions throughout the three periods of study. The HM1:IMSS strain caused smaller abscesses when compared to EGG lesions; however, trophozoites were observed at 1 and 3 days after inoculation. The 03C E. dispar strain caused intermediate abscesses when compared to the others; trophozoites were observed in all periods analyzed. The EGG strain caused progressive evolution of the injury, which differed from the HM1:IMSS and 03C strains. These results strongly suggest that the 03C E. dispar strain is pathogenic in the experimental hamster model. Additional studies are necessary to identify potential factors that regulate the manifestation of virulence of this strain and others.

Amoebic liver abscess with negative serologic markers for Entamoeba histolytica: mind the gap!

A 38-year-old male German traveller returning from Asia presented with fever, night sweats and abdominal complaints. Abdominal ultrasonography revealed several fast-growing abscesses of the liver. Three blood cultures as well as serologic investigations for the detection of antibodies to Entamoeba histolytica, performed on day 3 and 7 after the onset of clinical symptoms, remained negative. Stool microscopy revealed the presence of amoeba cysts compatible with E. histolytica infection. Taking both the amoebic and bacterial etiology of the abscesses into consideration, the patient was treated with metronidazole and ciprofloxacin followed by paromomycin. Antibodies to E. histolytica tested positive shortly after anti-amoebic therapy was initiated. The patient fully recovered, and ultrasound follow-up showed complete resolution of the abscesses within 50 days. This case leads to the conclusion that amoebic liver abscess should be considered despite negative amoeba serology and that ultrasonography is an important diagnostic tool for the early diagnosis of extraintestinal amoebiasis.

Amoebic liver abscess production by Entamoeba dispar.

Although Entamoeba dispar displays a similar morphology to Entamoeba histolytica, cellular and molecular studies have revealed significant differences between these two amoebae, including the former being characterized as non-pathogenic and the later as pathogenic. However, recent in vivo and in vitro experiments have shown that E. dispar strains of different origin are capable of causing liver damage and destroying cell culture lines in the presence of common intestinal bacteria. These results suggested that E. dispar may present pathogenic behavior according to the specific E. dispar strain, culture and environmental conditions. To investigate this possibility, we carried out in vivo and in vitro studies using a xenic strain E. dispar (ICB-ADO) isolated from a symptomatic non-dysenteric Brazilian patient. This strain was able to induce liver necrosis in a hamster model that was more severe than that produced by E. histolytica. The ICB-ADO isolate also caused significantly more destruction of cultured MDCK cells and increased loss of transepithelial resistance than did the E. histolytica. Xenic E. dispar exhibited high proteolytic activity, which was partially inhibited by the addition of cysteine-protease inhibitors. Based on our biochemical and molecular characterization of E. dispar (ICB-ADO) xenic culture and its ability to produce liver abscesses, we conclude that this specific strain can indeed produce tissue damage, distinct from the frequently used non- pathogenic E. dispar SAW 760 strain.

Entamoeba histolytica calreticulin: an endoplasmic reticulum protein expressed by trophozoites into experimentally induced amoebic liver abscesses.

Entamoeba histolytica calreticulin (EhCRT) is remarkably immunogenic in humans (90-100% of invasive amoebiasis patients). Nevertheless, the study of calreticulin in this protozoan is still in its early stages. The exact location, biological functions, and its role in pathogenesis are yet to be fully understood. The aim of the present work is to determine the location of EhCRT in virulent trophozoites in vivo and the expression of the Ehcrt gene during the development of experimentally induced amoebic liver abscesses (ALA) in hamsters. Antibodies against recombinant EhCRT were used for the immunolocalization of EhCRT in trophozoites through confocal microscopy; immunohistochemical assays were also performed on tissue sections of ALAs at different times after intrahepatic inoculation. The expression of the Ehcrt gene during the development of ALA was estimated through both in situ RT-PCR and real-time RT-PCR. Confocal assays of virulent trophozoites showed a distribution of EhCRT in the cytoplasmic vesicles of different sizes. Apparently, EhCRT is not exported into the hepatic tissue. Real-time RT-PCR demonstrated an over-expression of the Ehcrt gene at 30 min after trophozoite inoculation, reaching a peak at 1-2 h; thereafter, the expression fell sharply to its original levels. These results demonstrate for the first time in an in vivo model of ALA, the expression of Ehcrt gene in E. histolytica trophozoites and add evidence that support CRT as a resident protein of the ER in E. histolytica species. The in vivo experiments suggest that CRT may play an important role during the early stages of the host-parasite relationship, when the parasite is adapting to a new environment, although the protein seems to be constitutively synthesized. Moreover, trophozoites apparently do not export EhCRT into the hepatic tissue in ALA.

Evaluation of the PEΔIII-LC3-KDEL3 Chimeric Protein of Entamoeba histolytica-Lectin as a Vaccine Candidate against Amebic Liver Abscess.

Entamoeba histolytica is an intestinal parasite that causes dysentery and amebic liver abscess. E. histolytica has the capability to invade host tissue by union of virulence factor Gal/GalNAc lectin; this molecule induces an adherence-inhibitory antibody response as well as to protect against amebic liver abscess (ALA). The present work showed the effect of the immunization with PEΔIII-LC3-KDEL3 recombinant protein. In vitro, this candidate vaccine inhibited adherence of E. histolytica trophozoites to HepG2 cell monolayer, avoiding the cytolysis, and in a hamster model, we observed a vaccine-induced protection against the damage to tissue liver and the inhibition of uncontrolled inflammation. PEΔIII-LC3-KDEL3 reduced the expression of TNF-α, IL-1ß, and NF-κB in all immunized groups at 4- and 7-day postinfection. The levels of IL-10, FOXP3, and IFN-γ were elevated at 7 days. The immunohistochemistry assay confirmed this result, revealing an elevated quantity of +IFN-γ cells in the liver tissue. ALA formation in hamsters immunized was minimal, and few trophozoites were identified. Hence, immunization with PEΔIII-LC3-KDEL3 herein prevented invasive amebiasis, avoided an acute proinflammatory response, and activated a protective response within a short time. Finally, this recombinant protein induced an increase of serum IgG.

Precision-cut hamster liver slices as an ex vivo model to study amoebic liver abscess.

Entamoeba histolytica is the etiological agent of amoebiasis, the second cause of global morbidity and mortality due to parasitic diseases in humans. In approximately 1% of the cases, amoebas penetrate the intestinal mucosa and spread to other organs, producing extra-intestinal lesions, among which amoebic liver abscess (ALA) is the most common. To study ALA, in vivo and in vitro models are used. However, animal models may pose ethical issues, and are time-consuming and costly; and cell cultures represent isolated cellular lineages. The present study reports the infection of precision-cut hamster liver slices with Entamoeba histolytica trophozoites. The infection time-course, including tissue damage, parallels findings previously reported in the animal model. At the same time amoebic virulence factors were detected in the infected slices. This new model to study ALA is simple and reproducible, and employs less than 1/3 of the hamsters required for in vivo analyses.

Gonadectomy inhibits development of experimental amoebic liver abscess in hamsters through downregulation of the inflammatory immune response.

Incidence of amoebic liver abscess (ALA) in human males is considerably higher than in females, suggesting a role for sex hormones in this parasite infection. We describe here the effect of hamster gonadectomization on the development of ALA. After monitoring the decrease of oestradiol in females and testosterone in males to undetectable levels by ELISA and Radio Immuno Assay (RIA) in serum, hamsters were intraportally infected with Entamoeba histolytica trophozoites and killed 7 days later. ALA was absent in 50% of male and 15% of female gonadectomized (Gdx) hamsters, in comparison with 100% infection in non-Gdx controls. This protection against ALA in Gdx hamsters was concomitant to a comparatively scarce inflammatory infiltrate and necrosis surrounding clusters of trophozoites in the liver tissue, as well as to a lack of response of spleen cells to Con A, evaluated in proliferation assays. As tissue damage in ALA has been associated with a local inflammatory Th1 response, we determined the profile of response in hamsters by immunohistochemistry on liver sections. In contrast to strong Th1 responses in non-Gdx animals, Gdx females and males exhibited Th2 and Th3 profiles of cytokines, respectively, suggesting that protection against ALA following gonadectomization, could be related to downregulation of liver Th1 response during amoebic infection.

Isolation and characterization of a potentially virulent species Entamoeba nuttalli from captive Japanese macaques.

We have recently proposed revival of the name Entamoeba nuttalli Castellani, 1908 for a virulent amoeba (P19-061405 strain) isolated from a rhesus monkey (Macaca mulatta) and located phylogenetically between E. histolytica and E. dispar. In this study, E. nuttalli was isolated from feces of captive Japanese macaques (M. fuscata) in an open-air corral in Japan. The sequence of the 18S rRNA gene in the isolates differed from the P19-061405 strain in 2 nucleotide positions, but was identical to the EHMfas1 strain isolated previously from a cynomolgus monkey (M. fascicularis). One of the E. nuttalli isolates from Japanese macaques, named the NASA6 strain, was axenized and cloned. In isoenzyme analysis, the mobilities of hexokinase and phosphate glucose isomerase in the NASA6 strain were identical to those in the P19-061405 and EHMfas1 strains, but the mobility of phosphoglucomutase was different. These results were supported by gene analyses of these enzymes. Inoculation of NASA6 strain trophozoites into the liver of hamsters led to formation of an amoebic liver abscess. The liver lesions were characterized by extensive necrosis associated with inflammatory reactions. These results demonstrate that the NASA6 strain is potentially virulent and that E. nuttalli should be recognized as a common parasite in macaques.

Entamoeba histolytica: cysteine proteinase activity and virulence. Focus on cysteine proteinase 5 expression levels.

Cysteine proteinase (CP) activity and CP5 mRNA levels were analyzed in eleven samples of Entamoeba histolytica isolated from patients presenting different clinical profiles. The virulence degree of the isolates, determined in hamster liver, correlated well with the clinical form of the patient and culture conditions. CP5 mRNA levels were also determined in sample freshly picked up directly from liver amoebic abscess. Differences were not observed in the levels of CP5 mRNA and CP specific activity among the cultured samples. However, different levels of CP5 mRNA were observed in trophozoite freshly isolated from hepatic amoebic lesions. These results reinforce the importance of CP5 for the virulence of amoebae and the need for studies with the parasite present in lesions to validate mechanisms involved in pathogenesis of amoebiasis.

Amebic liver abscesses: a new epidemiological trend in a non-endemic area?

BACKGROUND: Amoebic liver abscess (ALA) is the most common extraintestinal complication of colonic amebiasis. In recent decades its incidence in developed European countries has significantly increased because of travel and immigration of individuals from highly endemic areas. We report our 29-year experience in echo-guided percutaneous needle/catheter drainage (EPND/EPCD) of ALA. PATIENTS AND METHODS: From May 1979 to November 2007, 68 ALA corresponding to 56 patients were diagnosed at our Department. All patients were treated with a metronidazole plus EPND/EPCD approach. RESULTS: The majority of the cases did not need more than two echo-guided punctures. Two patients, both male immigrants (HIV-negative), had unmodified lesions after two EPNDs: catheter drainage was performed. A quick worsening of their clinical conditions and onset of neurological symptoms occurred; in both patients, computed tomography (CT) revealed a brain abscess. Intravenous medical therapy was started, but both died 4 and 3 days, respectively, after the onset of neurological symptoms (overall mortality rate: 3.57%). CONCLUSION: The unfavorable outcome of two cases is a rare example of failure of percutaneous therapy of ALA. Mortality is a possible event even in a non-endemic area such as Italy. More observational data are needed to confirm the possibility of a new epidemiological trend.

Mechanisms of natural resistance of Balb/c mice to experimental liver amoebiasis.

Entamoeba histolytica is the parasite responsible for human amoebiasis. The analysis of the natural resistance mechanisms of some rodents to amoebic liver abscess (ALA) may reveal alternative pathogenicity mechanisms to those previously discovered in the experimental model of ALA in hamsters. In this work the natural resistance of BALB/c mice to ALA was explored by performing: (i) in vivo chemotaxis analysis with a specifically designed chamber; (ii) in vitro amoebic survival in fresh and decomplemented serum; (iii) histological temporal course analysis of ALA development in mice with different treatments (hypocomplementemic, hyperimmune and treated with iNOS and NADPH oxidase inhibitors) and (iv) mouse liver amoebic infection by both in situ implantation of ALA from hamsters and inoculation of parasites into the peritoneal cavity. The results show that E. histolytica clearance from the mouse liver is related to a low chemotactic activity of complement, which results in poor inflammatory response and parasite inability to cause tissue damage. Also, the absence of amoebic tropism for the mouse liver is correlated with resistance to experimental liver amoebiasis.

Curcumin Provides Hepatoprotection against Amoebic Liver Abscess Induced by Entamoeba histolytica in Hamster: Involvement of Nrf2/HO-1 and NF-κB/IL-1ß Signaling Pathways.

Amoebic liver abscess (ALA) is the most common extraintestinal amoebiasis caused by Entamoeba histolytica (E. histolytica). However, despite current knowledge and scientific advances about this infection, there are no effective treatments to prevent it. Herein, the antiamoebic capacity of curcumin in a hamster model was evaluated. Curcumin (150 mg/kg, p.o., daily during 10 days before infection) considerably prevents liver damage induced at 12 and 48 h post-intrahepatic inoculation of trophozoites and decreases ALT, ALP, and γ-GTP activities, and macroscopic and microscopic observations were consistent with these results. On the other hand, after one week of intraportal inoculation, liver damage was prevented by curcumin (150 mg/kg, p.o., daily, 20 days before amoebic inoculation and during the week of infection); liver/body weight ratios and tissue and histological stains showed normal appearance; in addition, the increases in ALT, ALP, and γ-GTP activities were prevented; the depletion of glycogen content induced by the amoebic damage was partially but significantly prevented, while NF-κB activity was inhibited and the expression of IL-1ß was reduced; Nrf2 production showed a tendency to increase it, and HO-1 protein was overexpressed. These results suggest for the first time that curcumin can be a compound with antiamoebic effect in the liver, suggesting that its daily use could help greatly decrease the incidence of this type of infection.

Primary cutaneous amebiasis with a fatal outcome.

We report a fatal case of disseminated amebiasis in a young African woman, which initially presented with an ulcerated cutaneous lesion on the left flank. The causative organism was confirmed by examination of a wet drop preparation from the ulcer discharge and by skin biopsy. The patient was not immunosuppressed and was treated unsuccessfully with metronidazole. Postmortem examination revealed the presence of intestinal amebiasis complicated by a liver abscess.