Eric Hernández

Human Amebiasis: Breaking the Paradigm?

For over 30 years it has been established that the Entamoeba histolytica protozoan included two biologically and genetically different species, one with a pathogenic phenotype called E. histolytica and the other with a non-pathogenic phenotype called Entamoeba dispar. Both of these amoebae species can infect humans. E. histolytica has been considered as a potential pathogen that can cause serious damage to the large intestine (colitis, dysentery) and other extraintestinal organs, mainly the liver (amebic liver abscess), whereas E. dispar is a species that interacts with humans in a commensal relationship, causing no symptoms or any tissue damage. This paradigm, however, should be reconsidered or re-evaluated. In the present work, we report the detection and genotyping of E. dispar sequences of DNA obtained from patients with amebic liver abscesses, including the genotyping of an isolate obtained from a Brazilian patient with a clinical diagnosis of intestinal amebiasis that was previously characterized as an E. dispar species. The genetic diversity and phylogenetic analysis performed by our group has shown the existence of several different genotypes of E. dispar that can be associated to, or be potentiality responsible for intestinal or liver tissue damage, similar to that observed with E. histolytica.

Novelties on Amoebiasis: A Neglected Tropical Disease

In accordance with the 1997 documents of the World Health Organization (WHO), amoebiasis is defined as the infection by the protozoan parasite Entamoeba histolytica with or without clinical manifestations. The only known natural host of E. histolytica is the human with the large intestine as major target organ. This parasite has a very simple life cycle in which the infective form is the cyst, considered a resistant form of parasite: The asymptomatic cyst passers and the intestinal amoebiasis patients are the transmitters; they excrete cysts in their feces, which can contaminate food and water sources. E. histolytica sensu stricto is the potentially pathogenic species and E. dispar is a commensal non-pathogenic Entamoeba. Both species are biochemical, immunological and genetically distinct. The knowledge of both species with different pathogenic phenotypes comes from a large scientific debate during the second half of the 20th century, which gave place to the rapid development of diagnostics technology based on molecular and immunological strategies. During the last ten years, knowledge of the new epidemiology of amoebiasis in different geographic endemic and non-endemic areas has been obtained by applying mostly molecular techniques. In the present work we highlight novelties on human infection and the disease that can help the general physician from both endemic and non-endemic countries in their medical practice, particularly, now that emigration is undoubtedly a global phenomenon that is modifying the previous geography of infectious diseases worldwide.

Entamoeba histolytica and Entamoeba dispar infection in Mexican school children: genotyping and phylogenetic relationship

BackgroundThis study aimed to determine the frequency of Entamoeba histolytica and Entamoeba dispar infection in school children in the community of Tlaltizapan, in order to understand the dynamics of infection within the school and family spheres of this population. Amoebiasis is an unsolved public health problem and an endemic disease in Mexico. The incidence rate varies depending on the state; the most affected states show the highest numbers of new cases of amoebiasis per year. Previously, we reported the molecular frequency of infection with E. histolytica and/or E. dispar in other rural communities of the state of Morelos.MethodsChildren from 3 schools were studied to estimate the frequency of intestinal parasites through microscopic examination of fresh stool samples. The number of studied individuals were 309 school children. The molecular characterization of E. histolytica or E. dispar was carried out by Polymerase Chain Reaction (PCR) using species-specific primers to amplify short tandem repeats (STR) in non-coding sequences associated with the tRNA gene; the amplified fragments were sequenced and analyzed.ResultsEight different genotypes were obtained from E. dispar isolates with the molecular marker NKD3-D5. None of the cases in which the species E. histolytica was detected developed symptoms attributable to an invasive process of disease. Moreover, the parasitized condition appeared to have no significant impact on the development or nutritional status of affected children. Genotype 1, which corresponds to the reference strain E. dispar SAW760, considered a non-pathogenic amoeba, was the most prevalent.ConclusionsThe comparison of the genotypes of Entamoeba species did not show a correlation between children and their relatives. In this community, the species Entamoeba dispar genotype 1 was the most widespread. Based on the indicators of growth, development and nutrition status, the studied community seems to be reasonably adapted to constant exposure to intestinal parasites, since there were no evidences of a serious impact of the parasitized condition on the children’s health.

Human Intestinal Microbiota: Interaction Between Parasites and the Host Immune Response

The human gut is a highly complex ecosystem with an extensive microbial community, and the influence of the intestinal microbiota reaches the entire host organism. For example, the microbiome regulates fat storage, stimulates or renews epithelial cells, and influences the development and maturation of the brain and the immune system. Intestinal microbes can protect against infection by pathogenic bacteria, viruses, fungi and parasites. Hence, the maintenance of homeostasis between the gut microbiota and the rest of the body is crucial for health, with dysbiosis affecting disease. This review focuses on intestinal protozoa, especially those still representing a public health problem in Mexico, and their interactions with the microbiome and the host. The decrease in prevalence of intestinal helminthes in humans left a vacant ecological niche that was quickly occupied by protozoa. Although the mechanisms governing the interaction between intestinal microbiota and protozoa are poorly understood, it is known that the composition of the intestinal bacterial populations modulates the progression of protozoan infection and the outcome of parasitic disease. Most reports on the complex interactions between intestinal bacteria, protozoa and the immune system emphasize the protective role of the microbiota against protozoan infection. Insights into such protection may facilitate the manipulation of microbiota components to prevent and treat intestinal protozoan infections. Here we discuss recent findings about the immunoregulatory effect of intestinal microbiota with regards to intestinal colonization by protozoa, focusing on infections by Entamoeba histolytica, Blastocystis spp, Giardia duodenalis, Toxoplasma gondii and Cryptosporidium parvum. The possible consequences of the microbiota on parasitic, allergic and autoimmune disorders are also considered.

Prevalent HLA Class II Alleles in Mexico City Appear to Confer Resistance to the Development of Amebic Liver Abscess

Amebiasis is an endemic disease and a public health problem throughout Mexico, although the incidence rates of amebic liver abscess (ALA) vary among the geographic regions of the country. Notably, incidence rates are high in the northwestern states (especially Sonora with a rate of 12.57/100,000 inhabitants) compared with the central region (Mexico City with a rate of 0.69/100,000 inhabitants). These data may be related to host genetic factors that are partially responsible for resistance or susceptibility. Therefore, we studied the association of the HLA-DRB1 and HLA-DQB1 alleles with resistance or susceptibility to ALA in two Mexican populations, one each from Mexico City and Sonora. Ninety ALA patients were clinically diagnosed by serology and sonography. Genomic DNA was extracted from peripheral blood mononuclear cells. To establish the genetic identity of both populations, 15 short tandem repeats (STRs) were analyzed with multiplexed PCR, and the allelic frequencies of HLA were studied by PCR-SSO using LUMINEX technology. The allele frequencies obtained were compared to an ethnically matched healthy control group (146 individuals). We observed that both affected populations differed genetically from the control group. We also found interesting trends in the population from Mexico City. HLA-DQB1*02 allele frequencies were higher in ALA patients compared to the control group (0.127 vs 0.047; p= 0.01; pc= NS; OR= 2.9, 95% CI= 1.09-8.3). The less frequent alleles in ALA patients were HLA-DRB1*08 (0.118 vs 0.238 in controls; p= 0.01; pc= NS; OR= 0.42, 95% CI= 0.19-0.87) and HLA-DQB1*04 (0.109 vs 0.214; p= 0.02; pc= NS; OR= 0.40, 95% CI= 0.20-0.94). The haplotype HLA-DRB1*08/-DQB1*04 also demonstrated a protective trend against the development of this disease (0.081 vs. 0.178; p=0.02; pc=NS; OR= 0.40, 95% CI= 0.16-0.93). These trends suggest that the prevalent alleles in the population of Mexico City may be associated with protection against the development of ALA.

Differential expression of pathogenic genes of Entamoeba histolytica vs E. dispar in a model of infection using human liver tissue explants

We sought to establish an ex vivo model for examining the interaction of E. histolytica with human tissue, using precision-cut liver slices (PCLS) from donated organs. E. histolytica- or E. dispar-infected PCLS were analyzed at different post-infection times (0, 1, 3, 24 and 48 h) to evaluate the relation between tissue damage and the expression of genes associated with three factors: a) parasite survival (peroxiredoxin, superoxide dismutase and 70 kDa heat shock protein), b) parasite virulence (EhGal/GalNAc lectin, amoebapore, cysteine proteases and calreticulin), and c) the host inflammatory response (various cytokines). Unlike E. dispar (non-pathogenic), E. histolytica produced some damage to the structure of hepatic parenchyma. Overall, greater expression of virulence genes existed in E. histolytica-infected versus E. dispar-infected tissue. Accordingly, there was an increased expression of EhGal/GalNAc lectin, Ehap-a and Ehcp-5, Ehcp-2, ehcp-1 genes with E. histolytica, and a decreased or lack of expression of Ehcp-2, and Ehap-a genes with E. dispar. E. histolytica-infected tissue also exhibited an elevated expression of genes linked to survival, principally peroxiredoxin, superoxide dismutase and Ehhsp-70. Moreover, E. histolytica-infected tissue showed an overexpression of some genes encoding for pro-inflammatory interleukins (ILs), such as il-8, ifn-γ and tnf-α. Contrarily, E. dispar-infected tissue displayed higher levels of il-10, the gene for the corresponding anti-inflammatory cytokine. Additionally, other genes were investigated that are important in the host-parasite relationship, including those encoding for the 20 kDa heat shock protein (HSP-20), the AIG-1 protein, and immune dominant variable surface antigen, as well as for proteins apparently involved in mechanisms for the protection of the trophozoites in different environments (e.g., thioredoxin-reductase, oxido-reductase, and 9 hypothetical proteins). Some of the hypothetical proteins evidenced interesting overexpression rates, however we should wait to their characterization. This finding suggest that the present model could be advantageous for exploring the complex interaction between trophozoites and hepatocytes during the development of ALA, particularly in the initial stages of infection.

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

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.

Cutaneous Amebiasis The Importance of Molecular Diagnosis of an Emerging Parasitic Disease

Cutaneous amebiasis is the least common clinical form of human amebiasis in Mexico, sexual amebiasis was only occasionally observed before the late 1980s. However, in the last few decades, most of the documented cases of cutaneous amebiasis from around the world are sexually transmitted. We present two cases of sexually transmitted genital amebiasis. The molecular characterization of the Entamoeba species in the affected tissues underlines the importance of an etiological diagnosis using specific and sensitive techniques that avoid the rapid destruction of tissues and the irreversible sequelae to the anatomy and function of the affected organs. In addition, for those interested in the study of the human-amoebic disease relationship and its epidemiology, the detection of a new, mixed infection in an invasive case of amebiasis reveals new perspectives in the study of the extraordinarily complex host-parasite relationship in amebiasis.

Genetic Diversity and Distribution of Blastocystis Subtype 3 in Human Populations, with Special Reference to a Rural Population in Central Mexico

Blastocystis subtype 3 (ST3) is a parasitic protist found in the digestive tract of symptomatic and asymptomatic humans around the world. While this parasite exhibits a high prevalence in the human population, its true geographic distribution and global genetic diversity are still unknown. This gap in knowledge limits the understanding of the spread mechanisms, epidemiology, and impact that this parasite has on human populations. Herein, we provided new data on the geographical distribution and genetic diversity of Blastocystis ST3 from a rural human population in Mexico. To do so, we collected and targeted the SSU-rDNA region in fecal samples from this population and further compared its genetic diversity and structure with that previously observed in populations of Blastocystis ST3 from other regions of the planet. Our analyses reveled that diversity of Blastocystis ST3 showed a high haplotype diversity and genetic structure to the world level; however, they were low in the Morelos population. The haplotype network revealed a common widespread haplotype from which the others were generated recently. Finally, our results suggested a recent expansion of the diversity of Blastocystis ST3 worldwide.

Immune Response in Human Amebiasis: A Protective Response?

Infection with Entamoeba histolytica provokes human diseases that range from amebic colitis to the life-threatening amebic liver abscess; nonetheless, about 90 % of infected people do not develop any symptom. What circumstances or which events in the dynamics of the host–parasite relationship define the outcome of infection? What are the causes that allow the apparently spontaneous clearance of the infection? How protective is the immune response against E. histolytica? Why do some infected people remain asymptomatic and why do others develop clinical symptoms? What is the role of immunogenetic polymorphism in the final outcome of the infection? These questions are the major aims of many research groups around the world. In the present review, we analyze the potential participation of the immune system and the genetic variants of genes associated with immune response in the human host with its susceptibility or resistance to develop amebic invasive disease.