Transcriptome Analysis of Entamoeba histolytica Trophozoites during in vivo Contact-independent Mediated Host-parasite Interaction: Putative Pathways Related to PCD

Aim: In the present study, we exploited DNA microarray-based transcriptome analysis and showed overall changes in gene expression in vivo of amoebic trophozoites that interact with animal soluble factors using an intraperitoneal dialysis bag model to elucidate putative molecular pathways and genes involved in this interaction. Study Design: We exploited DNA microarray-based transcriptome analysis. Results: An analysis from a network including the interactions of up-regulated genes and their neighbors revealed the presence of 11 functionally related modules. Six of the modules obtained were related to endoplasmic reticulum (ER) functions, such as degradation, stress, proteasome-ubiquitination, phosphorylation, lipid metabolism, and protein sorting. Furthermore, major transcriptional changes displayed by the parasite at the beginning of interaction were attributed to the response to the host defense. These data are consistent with the notion that the concerted expression of genes necessary for survival such as increment in protein synthesis, cytoskeleton rearrangement, vesicular traffic and genes involved in cell death including calcium imbalance and the ER signals associated with protein degradation (ERAD) is an overall landscape during the in vivo interaction between the amoebic trophozoites and animal soluble factors, and suggest that the ER stress is one of the main pathways leading to programmed cell death in E. histolytica. Conclusion: The present findings on the global transcriptional changes displayed by the parasite at the early stages of interaction with host environments in peritoneal implantation indicate that a substantial proportion of concerted changes in gene expression in amoebic trophozoites are attributable to the parasite’s response for cell death signals due to ER stress. A detailed knowledge of the underlying molecular mechanism might suggest the efficient elimination of this parasite by promoting their death pathways.

A Rare Case of Caecal Perforation with Ruptured Amoebic Liver Abscess.

Amoebic colitis is a common infection in all population groups across all geographical barriers and is an easily treatable disease in its routine manifestations. However, he has its set of complications that carry poor prognosis. Amoebic liver abscess is a common complication but its association with caecal perforation is rare, and carries worst prognosis. No survivors have been documented in the available literature and the result was pretty much the same in our study.

Gastrointestinal Fistulization in Amebic Liver Abscess.

Background: Liver abscess is a common deep seated abscess in children; amebic liver abscess is associated with more local complications. Case characteristics: We report two preschool children presenting with short history of pain, fever and right upper quadrant pain. The abscess communicated with gastro-intestinal tract (ascending colon in case 1 and duodenum in case 2), and diagnosis of amebic liver abscess was confirmed by DNA PCR. Outcome: Both children were successfully managed with intravenous antibiotics and catheter drainage. Message: Gastrointestinal fistulization may be rarely seen in amebic liver abscess. Conservative management with antibiotics, catheter drainage and supportive care may suffice.

Prevalence Of Parasitic Infestations In School Going Children.

Background and Aim: Over two billion people worldwide are affected by intestinal protozoans and helminths. Intestinal helminthic infestations are most common among school-age children and tend to be of high intensity in this age group. These diseases can affect child development, educational achievement, reproductive health, and social and economic development. Aim of the study was to find out the prevalence of parasitic infestation in school going children between 5 years to 12 years of age. Methodology: Macroscopic examination of stool samples was followed by direct saline (0.9%) preparation & for ova iodine preparation. Mounted wet saline (0.9%) and iodine preparations were examined under microscope using x10 and x40 objective lens. Concentration method with saturated salt solution was done in all the samples collected. Statistical analysis has been used. Results and Conclusion: Out of all 85 students enrolled in this study, 4 were observed with different parasitic structures in their stool. The common parasitic infection in this study was Cyst of Entamoebahistolytica and eggs of Ancylostomaduodenale.

Amebic liver abscess in rat: morphological evidence of innate immune modulation by the sympathetic nervous system / Absceso hepático amebiano en rata: evidencia morfológica de la modulación de la respuesta inmune innata por el sistema nervioso simpático

All organs of the immune system are innervated and almost all neurotransmitter receptors are present on immune cells. We studied the effects of sympathetic innervation in the development of amebic liver abscess (ALA) in rats. Our results showed that lack of sympathetic innervation promote a decrease in size of ALA. We found scarce amoebas, increased the number of neutrophils and a few collagen fibers surrounding the abscess, meanwhile in control group, we observed abscesses areas with typical necrosis including trophozoites and neutrophils. Macrophages were differentially distributed surrounding abscess area in control and vehicle groups, but equally located in and outside of the abscesses in sympathectomized rat. No significant differences were observed on NK cells in analysed groups. In cytokines quantification studies, we observed down-expression of IFN-g and TNF-a, moreover, we found overexpression of IL-10 in sympathectomized and ALA group. In conclusion, our results suggest that elimination of sympathetic nerve fibers in a model rat of amebic liver abscess induces reduction of the innate immune response and presence of amebas through the liver at seven days post-inoculation.

Todos los órganos del sistema inmune están inervados y casi todos los receptores para neurotransmisores están presentes en las células de la respuesta inmune. Nosotros estudiamos el efecto de la inervación simpática en el desarrollo del Absceso Hepático Amebiano (AHA) en ratas. Nuestros resultados muestran que la inervación simpática promueve una disminución en el tamaño del AHA. Nosotros encontramos áreas fibróticas bien definidas con algunas amibas, mayor número de neutrófilos y pocas fibras de colágena rodeando el área de daño, mientras que en el grupo control, nosotros observamos áreas con necrosis, trofozoítos y pocos neutrófilos en el área fibrótica. Los macrófagos se observaron distribuidos en el área fibrótica en los animales simpatectomizados, mientras que en los controles encontramos a los macrófagos distribuidos en la periferia del absceso. No se encontró diferencia significativa en la distribución y cantidad de células NK. En el estudio de citocinas nosotros observamos una disminución de IFN-g y TNF-a y un incremento de IL-10 en animales simpatectomizados. En conclusión, nuestros resultados sugieren que la eliminación de las fibras del sistema nervioso simpático en el modelo de AHA en rata, reduce la respuesta inmune innata y persisten amebas en el tejido dañados a los 7 días post-inoculación.

A clinical study on amoebic liver abscess.

Background: Amoebic liver abscess presents with severe pain and high grade fever and if not diagnosed and treated promptly, may lead to complications and mortality. Aim and objectives: The objective of the present study was to estimate the incidence, need for aspiration and prognosis. The diagnosis was based on clinical features, positive Elisa test, ultrasonography, aspiration of anchovy sauce from the liver lesion, isolation of E. Histolytica (cyst/trophozoite) from the stool of the patient. Result: We had 65 cases in the study. There were 52 males & 13 females with a ratio of 4:1. Solitary abscess was found in 48(73.8%) patients which are located as follows; right lobe(43), left lobe(2) and in both lobe(3). 9% were aspirated at presentation due to their size or position. Only 4 (2%) were aspirated at first follow-up on third day due to non resolution of pain or fever or increase in size. All the patients are responded to standard treatment of metronidazole. Amoebic liver abscess is a common diagnosis in our setup. Conclusion: Clinical background and sonogram give a reasonable suggestion about amoebic etiology. If initial aspiration is not indicated due to size larger than10 cm or proximity to surface, conservative treatment with oral or intravenous metronidazole is successful.

Prevalencia de enteroparásitos y anticuerpos IgG anti-Entamoeba histolytica en indígenas de la comunidad de Toromo, estado Zulia, Venezuela / Prevalence of enteroparasites and IgG antibodies anti-Entamoeba histolytica in the indigenous community of Toromo, Zulia State, Venezuela

Para determinar la seroprevalencia de anticuerpos contra Entamoeba histolytica y la prevalencia de los enteroparásitos en indígenas de la comunidad de Toromo, estado Zulia, Venezuela, se estudiaron 69 individuos de ambos sexos, de 1 a 60 años de edad, en el año 2012. Se recolectaron muestras de heces y suero; a todas las muestras fecales se les realizó examen microscópico con solución fisiológica y lugol, concentración con formol-éter y coloración de Kinyoun; para la detección de anticuerpos IgG anti-Entamoeba histolytica en suero, se empleó una técnica de ELISA comercial. El 98,6% de los individuos estudiados estaban parasitados, predominando el poliparasitismo (88,2%), detectándose asociaciones de hasta 10 especies parasitarias. Entre las especies encontradas están, Ascaris lumbricoides el principal helminto (65%), y Blastocystis sp. (59%) el protozoario más frecuente. Entamoeba histolytica/dispar/moshkovskii se detectó por microscopia en 25 individuos (36% de prevalencia), donde se incluyó un caso de infección por Entamoeba histolytica, por haberse encontrado trofozoítos hematófagos. Se observó una seroprevalencia de anticuerpos contra E. histolytica, del 83%. De los resultados obtenidos, se concluye que existe una elevada seroprevalencia de anticuerpos contra E. histolytica, así como una alta prevalencia de enteroparásitos en la población indígena estudiada.

In order to determine the seroprevalence of antibodies against Entamoeba histolytica and intestinal parasites in the indigenous community of Toromo, Zulia state, Venezuela, 69 individuals of both sexes, from 1 to 60 years of age were studied during the year 2012. Stool and serum samples were collected. Microscopic study of fecal specimens was carried out by saline solution dilution, Lugol staining, formol-ether concentration and Kinyoun’s stain. For determination of anti-Entamoeba histolytica IgG antibodies, a commercial ELISA technique in serum was used; 98.6% of the population were parasitized, predominating the polyparasitism (88.24%) and finding up to 10 different parasite species were associated. The main parasitic species found were: Ascaris lumbricoides (65%), followed by Blastocystis sp. (59%). Entamoeba histolytica/dispar/moshkovskii was detected by microscopy in 25 individuals (36% prevalence), which included a case of infection by Entamoeba histolytica as hematophagous trophozoites were observed. A seroprevalence of 83% for IgG anti-Entamoeba histolytica antibodies in the individuals studied was found. According to the results it is concluded, that there is a high seroprevalence of antibodies against E. histolytica, as well as a high prevalence of intestinal parasites in the indigenous population studied.

Nested PCR reveals elevated over-diagnosis of E. histolytica in Barcelona, Venezuela / Nested PCR revela elevado sobrediagnóstico de E. histolytica en Barcelona, Venezuela

The aim of this study was to identify the presence of Entamoeba histolytica and E. dispar by nested PCR in children attending the “Dr. Luis Razetti” Hospital, Barcelona, Anzoátegui State. Of the 1,141 fecal samples coproparasitologically evaluated by conventional microscopy, 150 were diagnosed positive for E. histolytica in 0-10 year-old-children, of both sexes. The signs, symptoms and a full coproparasitological report were obtained from all of these and nested PCR was performed to identify E. histolytica and E. dispar. The conventional microscopy results showed a diagnostic frequency of E. histolytica in 13.2% of the cases, of which 79.3% were positive only for this pathogen. However, nested PCR showed that of these, only 28% (42/150) were actually infected by Entamoeba spp., revealing a high over-diagnosis of E. histolytica. We also identified 9.3% E. histolytica, 4% E. dispar and 4.7% mixed infections. Diarrhea was the most common symptom, followed by abdominal pain and fever. Bloody stools were statistically associated with E. histolytica, but were also found for E. dispar infections. This study demonstrates that molecular techniques complementary to conventional methods enable the correct identification of Entamoeba spp., thus contributing to an improved epidemiological assessment of these parasites and implementation of the appropriate treatment.

Esta investigación planteó detectar por nested PCR Entamoeba histolytica y E. dispar en niños del Hospital “Dr. Luis Razetti” de Barcelona, estado Anzoátegui y su asociación con síntomas clínicos. De 1.141 muestras fecales evaluadas parasitológicamente por microscopía convencional, 150 fueron positivas a E. histolytica en niños de 0-10 años y de ambos sexos. Se obtuvo información de signos, síntomas y reporte parasitólogico completo de cada uno de los pacientes y se realizó nested PCR para identificar E. histolytica y E. dispar. Los resultados de la microscopía convencional demostraron una frecuencia de diagnóstico de E. histolytica del 13,2%. En el 79,3% de estas positivas se reportó esta especie como único patógeno. Sin embargo, la nested PCR evidenció que sólo 28,0% (42/150) de las mismas presentaron infecciones por Entamoeba, evidenciándose un elevado sobrediagnóstico de E. histolytica. Además se identificaron 9,3% infecciones por E. histolytica, 4,0% E. dispar, y 4,7%infecciones mixtas. La diarrea fue el síntoma más común, seguido de dolor abdominal y fiebre. La presencia de sangre demostró asociación estadísticamente significativa con E. histolytica, pero también se reportó en infecciones por E. dispar. Este estudio demuestra que las técnicas moleculares complementarias a los métodos convencionales, permiten la identificación correcta de especies de Entamoeba, contribuyendo con una mejor evaluación epidemiológica de estos parásitos y la aplicación adecuada del tratamiento.

Abscesos cerebelosos como complicación infrecuente de absceso hepático amebiano: Reporte de un caso

El absceso cerebral es una infrecuente y fatal complicación extraintestinal de la infección por E. histolytica. Presentamos el caso de un paciente que falleció por múltiples abscesos cerebelosos asociados con absceso hepático amebiano. Caso clínico: Se trata de paciente masculino, 62 años, proveniente del área metropolitana. Consulta por presentar dolor abdominal en hipocondrio derecho, nauseas, vómitos, fiebre y evacuaciones líquidas. Se diagnosticó absceso hepático de 12 x 8 cm de diámetro, por ultrasonido abdominal, correlacionado con la clínica y hallazgos de laboratorio (leucocitosis, elevación de transaminasas y fosfatasas alcalinas). Adicionalmente, el ELISA indirecto para determinar IgG para E. histolytica resultó positivo. La biopsia guiada por ultrasonido sugiere absceso hepático. Recibió tratamiento con metronidazol y ciprofloxacina por 10 días presentando mejoría clínica y de laboratorio. Sin embargo, consulta nuevamente con reaparición de los síntomas, pero se agrega cefalea occipital de fuerte intensidad. Al examen físico de reingreso se encuentran cifras tensiónales de 157/122 mmHg, refractarias al tratamiento, así como hallazgos de déficit neurológico sugestivos de síndrome cerebeloso. Se realizó tomografía de cráneo donde se evidencian múltiples imágenes hipodensas en probable relación con abscesos. Se planteó drenaje quirúrgico, sin embargo el paciente, falleció a las pocas horas.

Brain abscess are a rare and fatal complication of infection with extraintestinal E. histolytica. We present a patient who died of multiple cerebellar abscesses associated with amebic liver abscess. Clinical case: male, 62 years old, from the metropolitan area. Who came in 4 weeks before his death due to abdominal pain in the right hypochondrium, nausea, vomiting, fever and diarrhea. Liver abscess were diagnosed in 12 x 8 cm diameter, by abdominal ultrasound, correlated with clinical and laboratory findings (leukocytosis, high transaminases and alkaline phosphatases). Additionally, the indirect ELISA to determine IgG to E. histolytica was positive. Biopsy guided by ultrasound is concluded as abscess. The patient received treatment with metronidazole and ciprofloxacin for 10 days showing improvement. However, checked again with recurrence of symptoms, but adds strong occipital headache intensity. Initial physical examination re-found the blood pressure 157/122 mmHg, refractory to treatment, and findings of neurological deficits suggestive of cerebellar syndrome. Cranial tomography was performed which showed multiple hypodense images in connection with probable abscess. Surgical drainage was raised; however the patient evolved torpidly and died within hours.

Effect of the monocyte locomotion inhibitory factor (MLIF) produced by E. histolityca on cytokines and chemokine receptors in T CD4+ lymphocytes

Entamoeba histolytica produces Monocyte Locomotion Inhibitory Factor (MLIF), which may contribute to the delayed inflammation observed in amoebic hepatic abscesses. Leukocytes are affected through the modulation of cytokine expression and/or production. We evaluated the effects of MLIF on the activation and production of intracellular cytokines in human CD4+ T lymphocytes by flow cytometry. Cells were stimulated for 24 h with PMA, MLIF, or PMA+MLIF. Cellular activation was measured using anti-CD69. Th1/Th2 production was studied by the expression of intracellular cytokines and cytokine/chemokine receptors. MLIF increased CD69 and induced the over-expression of the IL-l©¬, IFN-¥ã, IL-2, IL-4, and IL-10 intracellular cytokines; PMA+MLIF inhibited Th1 cytokine (IFN-¥ã) and increased Th2 cytokines (IL-4 and IL-10). The co-expression of the cytokine and chemokine receptors IFN-¥ã/CCR5 and IL-1©¬/CCR5 was inhibited by PMA+MLIF and Th2 co-expression was increased. MLIF effects varied depending on the conditions. MLIF alone activated the Th1 and Th2 cytokines and cytokine/receptor expression; however, PMA+MLIF increased the expression of Th2 but inhibited it in Th1.

Caracterización inmunológica de un anticuerpo monoclonal específico para E. Histolytica / Inmunological characterization of an specific monoclonal antibody for E. histolytica

En este reporte informamos sobre la caracterización de un anticuerpo monoclonal contra E. histolytica. El anticuerpo mostró ser específico para E. histolytica ya que no reaccionó por inmunofluorescencia ni por la prueba de ELISA como otras Entamoeba, específicamente con E. moshkovskii, E. invadens y de la cepa Laredo de tipo E. histolytica. Los estudios de inmunoelectrotransferencia de un antígeno de membrana de E. histolytica indican que el anticuerpo monoclonal reconoce tres polipéptidos de diferente peso molecular, que pueden ser el resultado de un precursor comín o debido a un artefacto causado por la degradación enzimática de uno de los componentes de la amiba

Traducción de ARN obtenido de Entamoeba histolytica y su caracterización con un anticuerpo monoclonal específico / Translation of RNA obtained of Entamoeba histolytica and its characterization with a specific monoclonal antibody

En este reporte describimos la obtención de productos de traducción de un ARN total de E. histolytica, en un sistema libre de células, y su reactividad con anticuerpos contra el parásito. Mientras los anticuerpos de pacientes con amibiasis invasora muestran una reactividad compleja con tales productos, el anticuerpo monoclonal específico para E. histolytica presenta una reactividad más definida, en la cual destacan tres componentes principales. La combinación de estas dos metodologías ofrece la posibilidad de obtener uno o varios mRNAs que puedan ser de utilidad, tanto en el diagnóstico como en la inmunoprofilaxis de esta enfermedad