Entamoeba histolytica: Overexpression of the gal/galnac lectin, ehcp2 and ehcp5 genes in an in vivo model of amebiasis.

The parasite Entamoeba histolytica causes intestinal amebiasis and amebic liver abscess as its main extraintestinal manifestation. To study the in vivo events related to inflammation and the interactions between hosts and parasites during amebiasis, we designed a novel model of host-parasite interactions using cellulose membrane dialysis bags containing E. histolytica trophozoites. A bag is placed into the hamster peritoneal cavity, as has been reported in previous studies of programmed cell death (PCD) in E. histolytica trophozoites. To determine if virulence factors such as cysteine proteinases (EhCP2 and EhCP5) and Gal/GalNAc lectin could be involved in the host-parasite interaction using this model, we examined the relative expression of the ehcp2 and ehcp5 genes and the carbohydrate recognition domain (crd) of Gal/GalNAc lectin using real-time quantitative PCR (qRT-PCR). All analyzed genes were over-expressed 0.5h after the initiation of the host-parasite interaction and were then progressively down-regulated. However, Gal/GalNAc lectin had the greatest increase in gene expression 1.5h after host-parasite interaction; Gal/GalNAc lectin had a 250-fold increase with respect to the axenically grown trophozoites, which over-express Gal/GalNAc lectin in in vivo models. These results support the important role of these molecules in the initiation of cell damage by E. histolytica.

Morphological Findings in Trophozoites during Amoebic Abscess Development in Misoprostol-Treated BALB/c Mice.

During amoebic liver abscess (ALA) formation in susceptible animals, immune response is regulated by prostaglandin E2 (PGE2) dependent mechanisms. The aim of this study was to analyze the effect of misoprostol (MPL), a PGE1 analogue, on ALA formation in BALB/c mice. Male mice from BALB/c strain were intrahepatically infected with 7.5 × 10(5) trophozoites of E. histolytica strain HM1:IMSS and treated with 10(-4) M of MPL daily until sacrifice at 2, 4, and 7 days postinfection (p.i.). ALA formation was evaluated at 2, 4, and 7 days postinfection; trophozoite morphology was analyzed using immunohistochemistry and image analysis. Results showed an increase in frequency of ALA formation in infected and MPL-treated mice only at 2 days p.i. (P = 0.03). A significant diminution in the size of trophozoites was detected in abscesses from mice independently of MPL treatment (from 5.8 ± 1.1 µm at 2 days p.i. to 2.7 ± 1.9 µm at 7 days p.i.) compared with trophozoites dimensions observed in susceptible hamsters (9.6 ± 2.7 µm) (P < 0.01). These results suggest that MPL treatment may modify the adequate control of inflammatory process to allow the persistence of trophozoites in the liver; however, natural resistance mechanisms cannot be discarded.

Efeito da ciclohexilamina sobre trofozoitos de Giardia Lamblia / Cyclohexylamine effect on Giardia lamblia trophozoites

A giardíase é uma doença causada pelo protozoário flagelado Giardia lamblia, e sua sintomatologia é caracterizada pela eliminação de fezes esteatorréicas, dores abdominais e náuseas. Segundo o CDC estima-se que há cerca 1,2 milhões de casos por ano de giardíase, acometendo principalmente crianças em idade escolar. Atualmente, o tratamento da giardíase é realizado principalmente pelo uso do fármaco da família dos 5-nitromidazóis, metronidazol (Flagyl®), secnidazol e tinidazol em particular. Estes são confrontados em casos de resistência clínica causada pelo frequente uso inadequado do medicamento e/ou abandono do tratamento. Além disso, o metronidazol pode apresentar efeito carcinogênico em longo prazo em humanos. Desta forma, novos estudos com análogos e/ou inibidores de poliaminas podem levar à elucidação dos mecanismos de ação envolvidos, favorecendo o estabelecimento de novos regimes terapêuticos mais seguros e eficazes. Em nosso trabalho, foram testadas as substâncias ciclohexilamina (CHA) e o metronidazol que são produtos sintéticos, com o objetivo de avaliar os seus efeitos na proliferação celular, caracterização dos moduladores do metabolismo de poliaminas, avaliação nas mudanças no potencial redox e elucidação de seus possíveis mecanismos de ação nos trofozoítos de Giardia lamblia. Foi realizada uma avaliação da proliferação celular na presença de CHA para trofozoítos de Giardia lamblia, onde observamos que a substância demonstrou ter ação siginficativa apresentando um efeito dosedependente. Observamos que os trofozoítos de G. lamblia apresentam uma inibição significativa do crescimento em presença de concentrações milimolares do CHA, cujo IC50 em 72 horas foi de 1,646 mM...

Giardiasis is a disease caused by the flagellate protozoan Giardia lamblia, and its symptomatology is characterized by steatorrhea, abdominal pain and nausea. According to the CDC, an estimate number of 1.2 million cases of giardiasis happen every year, affecting especially schoolchildren.Nowadays, giardiasis treatment is based on drugs from the 5-nitroimidazole family, particularly metronidazole (Flagyl), secnidazole and tinidazole. Those drugs are indiscriminately used by the population, and it's not uncommon to find them causing clinical resistance due to inappropriate utilization and/or tratment abandon. Besides that, metronidazole can present longterm carcinogenic effect in humans. Thus, new studies with analogs and/or polyamines inhibitors can lead to the clarification of the drugs action mechanis, favouring the establishment of new, safer and more efficient therapeutic regimens.Our work tested cyclohexylamine (CHA) and metronidazole, wich are synthetic products, in order to evaluate their effects on cell proliferation and on changes in redox potential, characterize polyamines metabolism modulator and describe their possible action mechanisms on Giardia lamblia trophozoites. We evaluated Giardia lamblia trophozoites cell proliferation in the presence of CHA; it was observe that the substance shows significant action, presenting dose-dependent effect. We also observed that G. lamblia trophozoites presented significant growth inhibition when exposed to millimolar concentrations of CHA - its IC50 in 72 hours was 1,646mM...

[Malaria infection and nutritional status: results from a cohort survey of children from 6-59 months old in the Kivu province, Democratic Republic of the Congo]. / Infection palustre et état nutritionnel: résultats d'une cohorte d'enfants âgés de 6 à 59 mois au Kivu en République démocratique du Congo.

BACKGROUND: Despite a reduction in the magnitude of endemic malaria reported in recent years, malaria and protein-energy malnutrition (PEM) still remain major causes of morbidity and mortality in sub-Saharan Africa among children under five. The relationship between malaria and malnutrition remains a topic of controversy. We aimed to investigate malaria infection according to nutritional status in a community-based survey. METHODS: A cohort of 790 children aged 6 to 59 months and residing in eastern Democratic Republic of the Congo was followed-up from April 2009 to March 2010 with monthly visits. Data on nutritional status, morbidity between visits, use of insecticide-treated nets and malaria parasitemia were collected at each visit. The Z scores height for age, weight for age and weight for height were computed using the reference population defined by the WHO in 2006. Thresholds for Z scores were defined at -3 and -2. A binary logistic model of the generalized estimating equation (GEE) was used to quantify the association between PEM indicators and malaria parasitemia. Odds ratio (OR) and their 95% confidence interval (95% CI) were computed. RESULTS: After adjustment for season, children with severe stunting (height for age Z score<-3) were at lower risk of malaria parasitemia greater or equal to 5000 trophozoits/µL of blood as compared to those in with a better nutritional status (height for age Z score≥-2) (OR=0.48, 95% CI: 0.25-0.91). CONCLUSION: Severely stunted children are at a lower risk of high-level malaria parasitemia.

Various confocal scan features of cysts and trophozoites in cases with Acanthamoeba keratitis.

PURPOSE: To describe the various confocal scan features of cysts and trophozoites in patients with Acanthamoeba keratitis and to specify the associated findings. METHODS: In a retrospective study of cases between June 2005 and June 2010, we reviewed all the recorded confocal scan images of patients given a high index in regards to clinical suspicion of Acanthamoeba keratitis, in order to specify the various morphometric and morphologic features of Acanthamoeba cysts and trophozoites and to characterize the associated findings in such cases. RESULTS: Confocal scan images of 170 eyes from 170 patients were reviewed. Bilayered, target-shaped, coffee-bean and rod-shaped appearances of the cysts were observed in 100%, 82.9%, 36.4%, and 17.5% of cases, respectively. Single file arrangement of the cysts was noticed in 22 cases. The mean size of the cysts was 18.9 µm (range 10-39.6). In all cases, trophozoites were observed as pear-shaped or irregularly wedge-shaped structures, some surrounded by a brilliant halo and some exhibiting fine pseudopodia-like extensions, with mean size of 30.2 µm (range 19.2-55.6). Keratoneuritis and the anterior stromal honeycomb pattern were seen in 28.2% and 5.9% of cases, respectively. CONCLUSIONS: To our knowledge, this is the largest case-series study on confocal scan features of Acanthamoeba cysts and trophozoites in cases with clinical diagnosis of Acanthamoeba keratitis specifying the morphologic and morphometric criteria of this infectious organism and the associated findings.

Evolutionary genomics of Entamoeba.

Entamoeba histolytica is a human pathogen that causes amoebic dysentery and leads to significant morbidity and mortality worldwide. Understanding the genome and evolution of the parasite will help explain how, when and why it causes disease. Here we review current knowledge about the evolutionary genomics of Entamoeba: how differences between the genomes of different species may help explain different phenotypes, and how variation among E. histolytica parasites reveals patterns of population structure. The imminent expansion of the amount genome data will greatly improve our knowledge of the genus and of pathogenic species within it.

Binding to complement factors and activation of the alternative pathway by Acanthamoeba.

Acanthamoeba can cause severe ocular and cerebral diseases in healthy and immunocompromised individuals, respectively. Activation of complement appears to play an important role in host defence against infection. The exact mechanism, however, is still unclear. The aim of the present study was to investigate the effect of normal human serum (NHS) and normal mouse serum (NMS) on Acanthamoeba trophozoites, the binding of different complement factors to Acanthamoeba and the activation of the complement system. Moreover, we aimed to work out any possible differences between different strains of Acanthamoeba. A virulent T4 strain, a non-virulent T4 strain and a virulent T6 strain were included in the study. It was shown that NHS, but not NMS clearly has amoebicidal properties. After 5min of incubation with NHS, amoebae showed plasma membrane disruption and extrusion of intracellular components. Cells were completely destroyed within 60min of incubation in NHS but stayed intact after incubation in heat-inactivated serum. The binding of human C3 and C9 to amoebae was established by immunoblotting. Although incubation with mouse serum did not result in lysis of Acanthamoeba trophozoites an immunofluorescence assay (IFA) demonstrated a strong deposition of mouse complement factor C3 activation products, moderate binding of C1q, but no binding of MBL-A and MBL-C. EDTA inhibited the binding of C3 to acanthamoebae. Binding of amoebae to C3b was observed with sera from C1qa-/- and MBL-A/C-/- mice, but not with serum from Bf/C2-/- mice demonstrating an activation of complement via the alternative pathway. There were no significant differences between the three Acanthamoeba strains investigated. Altogether, our results prove that NHS is amoebolytic and that Acanthamoeba binds to C3 and C9 and activates the complement system via the alternative pathway.

Perkinsus marinus in pleasure oyster Crassostrea corteziensis from Nayarit, Pacific coast of México.

Culture of the pleasure oyster Crassostrea corteziensis is emerging as an alternative to the Pacific oyster (Crassostrea gigas) for oyster producers, who face severe mortalities since 1997 in Northwest México. For determining the health status of this species, we conducted a histopathological analysis of cultured populations from two estuaries in the Pacific coast of México. Macroscopical analysis revealed animals with transparent and retracted mantle. Histopathological analysis of these specimens showed tissue alterations and parasitic forms consistent with Perkinsus sp. infection. Stages of the parasite identified included tomont and trophozoites with an eccentric vacuole characteristic of Perkinsus spp. Pieces of tissues of infected oysters were incubated in Fluid Thioglycollate Medium (FTM) resulting in blue-black hypnospores after incubation. The identity of the parasite was confirmed by species specific PCR-based assay in DNA samples from oysters, tissue fractions from FTM cultures, and deparaffined samples with Perkinsus-like parasite detected by histology. Sequencing of positive amplified fragments (307bp) showed a sequence similar to Perkinsus marinus strain TXsc NTS ribosomal RNA gene (100% coverage and 98% identity, GenBank Accession No. AF497479.1) and to P. marinus, Genomic DNA, (100% coverage and 97% identity, GenBank Accession No. S78416.1). The prevalence of P. marinus varied from 1 to 5% in Boca del Camichín and from 1 to 6% in Pozo Chino. In general, the intensity of infection was moderate. The infection was observed in oysters from 31 to 110mm of shell length. This is the first record of P. marinus in oysters from the North America Pacific coast and the first record in C. corteziensis. The origin of this parasite in the area is unknown, but it may be associated to introductions of Crassostrea virginica from the East coast of United States of America or Gulf of México.