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

BACKGROUND: This 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. METHODS: Children 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. RESULTS: Eight 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. CONCLUSIONS: The 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.

Analysis of the Bacterial Diversity in Liver Abscess: Differences Between Pyogenic and Amebic Abscesses.

Several recent studies have demonstrated that virulence in Entamoeba histolytica is triggered in the presence of both pathogenic and nonpathogenic bacteria species using in vitro and in vivo experimental animal models. In this study, we examined samples aspirated from abscess material obtained from patients who were clinically diagnosed with amebic liver abscess (ALA) or pyogenic liver abscess (PLA). To determine the diversity of bacterial species in the abscesses, we performed partial 16S rRNA gene sequencing. In addition, the E. histolytica and Entamoeba dispar species were genotyped using tRNA-linked short tandem repeats as specific molecular markers. The association between clinical data and bacterial and parasite genotypes were examined through a correspondence analysis. The results showed the presence of numerous bacterial groups. These taxonomic groups constitute common members of the gut microbiota, although all of the detected bacterial species have a close phylogenetic relationship with bacterial pathogens. Furthermore, some patients clinically diagnosed with PLA and ALA were coinfected with E. dispar or E. histolytica, which suggests that the virulence of these parasites increased in the presence of bacteria. However, no specific bacterial groups were associated with this effect. Together, our results suggest a nonspecific mechanism of virulence modulation by bacteria in Entamoeba.

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.

Blastocystis Isolates from Patients with Irritable Bowel Syndrome and from Asymptomatic Carriers Exhibit Similar Parasitological Loads, but Significantly Different Generation Times and Genetic Variability across Multiple Subtypes.

Blastocystis spp is a common intestinal parasite of humans and animals that has been associated to the etiology of irritable bowel syndrome (IBS); however, some studies have not found this association. Furthermore, many biological features of Blastocystis are little known. The objective of present study was to assess the generation times of Blastocystis cultures, from IBS patients and from asymptomatic carriers. A total of 100 isolates were obtained from 50 IBS patients and from 50 asymptomatic carriers. Up to 50 mg of feces from each participant were cultured in Barret's and in Pavlova's media during 48 h. Initial and final parasitological load were measured by microscopy and by quantitative PCR. Amplicons were purified, sequenced and submitted to GenBank; sequences were analysed for genetic diversity and a Bayesian inference allowed identifying genetic subtypes (ST). Generation times for Blastocystis isolates in both media, based on microscopic measures and molecular assays, were calculated. The clinical symptoms of IBS patients and distribution of Blastocystis ST 1, 2 and 3 in both groups was comparable to previous reports. Interestingly, the group of cases showed scarce mean nucleotide diversity (π) as compared to the control group (0.011±0.016 and 0.118±0.177, respectively), whilst high gene flow and small genetic differentiation indexes between different ST were found. Besides, Tajima's D test showed negative values for ST1-ST3. No statistical differences regarding parasitological load between cases and controls in both media, as searched by microscopy and by qPCR, were detected except that parasites grew faster in Barret's than in Pavlova's medium. Interestingly, slow growth of isolates recovered from cases in comparison to those of controls was observed (p<0.05). We propose that generation times of Blastocystis might be easily affected by intestinal environmental changes due to IBS probably because virulent strains with slow growth may be selected, reducing their genetic variability.

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.

Worldwide genealogy of Entamoeba histolytica: an overview to understand haplotype distribution and infection outcome.

Although Entamoeba histolytica is one of the most prevalent intestinal parasites, how the different strains of this species are distributed all over the world and how different genotypes are associated with the infection outcome are yet to be fully understood. Recently, the use of a number of molecular markers has made the characterization of several genotypes in those regions with high incidence of amoebiasis possible. This work proposes the first genealogy of E. histolytica, with an haplotype network based on two tRNA gene-linked array of Short Tandem Repeats (STRs) reported until today, and 47 sequences from 39 new isolates of Mexican Amoebic Liver Abscesses (ALA) samples. One hundred and three sequences were obtained from D-A locus, their information about the geographic region of isolation as well as clinical diagnosis were also collected. One hundred and five sequences from N-K2 locus were also obtained as well as the region of isolation, but the information about clinical diagnosis was not available in all cases. The most abundant and widely distributed haplotype in the world is the one of E. histolytica HM1:IMSS strain. This was found in Mexico, Bangladesh, Japan, China and USA and is associated to symptomatic patients as well as asymptomatic cyst passers. Many other haplotypes were found only in a single country. Both genealogies suggest that there are no lineages within the networks that may be related to a particular geographic region or infection outcome. A concatenated analysis of the two molecular markers revealed 12 different combinations, which suggests the possibility of genetic recombination events. The present study is the first to propose a global genealogy of this species and suggests that there are still many genotypes to be discovered. The genotyping of new isolates will help to understand the great diversity and genetic structure of this parasite.

Case report: 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.

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 20(th) 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 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.

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.

Reassessment of the epidemiology of amebiasis: state of the art.

The epidemiology of amebiasis has dramatically changed since the separation of Entamoeba histolytica and Entamoeba dispar species, and the worldwide prevalence of these species has not been estimated until recently. The most cited data regarding prevalence, morbidity, or mortality due to amebiasis is the 1986 Walsh report, in which 100,000 deaths are reported to occur worldwide each year due to medical complications of invasive amebiasis. However, the prevalence values of Entamoeba histolytica infection could be completely erroneous since the estimations were performed prior to the molecular characterization of E. histolytica and E. dispar species. Moreover, Entamoeba moshkovskii, another morphologically indistinguishable human parasitic Entamoeba, was not mentioned or considered as a contributor to the prevalence figures in endemic areas. However, recent available prevalence and morbidity data obtained through molecular techniques allow the construction of a more reliable map of endemic regions of amebiasis all over the world [the Asian subcontinent (India, Bangladesh), Africa, Asian Pacific Countries (Thailand, Japan), South and Central America (Mexico, Colombia)]. The epidemiology of infectious diseases focuses on identification of factors that determine disease distribution in time and space, transmission factors responsible for the disease, clinical manifestations, and progression in the host, with the goal being the design of realistic intervention and prevention strategies in a reasonable period of time. In the present review, we will describe how molecular tools have made actual knowledge regarding the epidemiology of amebiasis possible. We will also analyze the most relevant available data on prevalence, morbidity, geographic distribution, patterns of transmission, exposure, and risk factors for infection in the human host. Our intention is to emphasize the recent molecular typing methods applied in genotyping Entamoeba species and strains, and to assess their value and limitations. Finally, we will discuss those areas of the host-parasite relationship that are still not fully understood, and the scientific challenges to approach this important public health problem in the future.

Two different chitinase genotypes in a patient with an amebic liver abscess: a case report.

The present work deals with the identification of a patient with two liver abscesses containing two different strains of Entamoeba histolytica, as defined by chitinase gene plymorphisms.

Infection by human immunodeficiency virus-1 is not a risk factor for amebiasis.

The purpose of this study was to determine whether HIV-1 infected patients in our community were more susceptible to Entamoeba histolytica and Entamoeba dispar infection than non-HIV-infected individuals. The prevalence and frequency of invasive amebiasis was determined in 203 HIV+/AIDS subjects and 140 close relatives or sexual partners, all of whom were HIV-. Anti-E. histolytica antibodies (IgG, IgA) were assessed as indicators of E. histolytica invasive infection. Polymerase chain reaction (PCR) was used for the characterization of the Entamoeba species. The prevalence estimated with PCR data showed that E. histolytica infection was more common in the HIV+/AIDS group (25.32%), than in HIV- contacts (18.46%). E. histolytica + E. dispar infection was more frequent in HIV+/AIDS patients (13.3%), than in HIV- contacts (0.7%). E. histolytica and/or E. dispar infection was highly prevalent in HIV+/AIDS patients (34.1%) without evidence of recent or current invasive disease. Contacts of HIV+/AIDS patients who were infected with E. histolytica were asymptomatic cyst passers. Our results suggest that E. histolytica strains prevalent in the studied community appear to be of low pathogenic potential.

High prevalence rate of Entamoeba histolytica asymptomatic 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, using polymerase chain reaction (PCR). Sociodemographic variables as risk factors for the infection were assessed. Results highlighted the number of individuals with intestinal parasites (43.1%) in the community, indicating extensive fecalism. A high frequency of E. histolytica asymptomatic infection, higher than E. dispar infection (13.8% versus 9.6%), was detected by PCR. Anti-amebic antibody levels (IgG) in serum and saliva (IgA) samples were not associated with E. histolytica intestinal infection. These findings suggest a predominant distribution of E. histolytica strains of low invasive potential in this community.

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.

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.

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.

Defective production of reactive oxygen intermediates (ROI) in a patient with recurrent amebic liver abscess.

Previously, we reported the case of a man in the fourth decade of life afflicted with three independent episodes of amebic liver abscesses over a period of 4 years. Previous evidence has indicated that the cellular immune response is involved in protection against recurrent invasive amebic infection, and macrophage-mediated effector mechanisms appear important for host resistance to Entamoeba histolytica infection. The aim of the present work was to investigate locomotor activity and oxidative burst function of peripheral mononuclear cells of this individual after healing of the third amebic liver abscess. A locomotion assay using Boyden chemotaxis chambers and the respiratory burst evaluated by chemiluminescence were performed in both mononuclear phagocytes (MPs) and polymorphonuclear (PMN) leukocytes. Levels of salivary IgA and serum IgG anti-amebic antibodies were followed during 48 months after the second amebic liver abscess. Results obtained showed a deficiency in MP but not in PMN leukocyte respiratory burst. Respiratory burst is a major microbicidal mechanism in MP leukocytes; this also has been considered as a host resistance strategy against E. histolytica. It may be at least one risk factor in our patient that was responsible for recurrence of amebic liver abscess.

Calreticulin-like molecule in trophozoites of Entamoeba histolytica HM1:IMSS (Swissprot: accession P83003).

In this work, we report the partial sequence of a 51 kDa protein of Entamoeba histolytica that is highly immunogenic in humans. Partial sequencing of the N-terminal end showed that 18 of the first 20 amino acid residues of the protein were identified uniquely, indicating that the final product was a homogeneous protein preparation. The N-terminal sequence that was found was: KVYFEETFENGWKXIWSKW. Comparing the 19-amino acid sequence of the protein in automated databases shows significant similarity with amino acid sequences of the calreticulin-like protein of spinach leaves (77%) and of the calreticulin precursor of Dictyostelium discoideum (60%).