Patricia Morán

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.

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.

The effect of formalin fixation on the polymerase chain reaction characterization of Entamoeba histolytica

Formalin fixation is the most common storage, transportation and preservation method for stool samples. However, fixation dramatically reduces our ability to extract from stool samples DNA that is a suitable template for polymerase chain reaction (PCR)-based diagnostic tests. In this study we evaluated the effects of formalin concentration and of the time stored in fixative on the success of PCR amplification. We found that the deleterious effects of formalin are both time and concentration dependent and may result from fragmentation of fixed DNA during its purification.

Prevalence of Entamoeba histolytica and Entamoeba dispar in a Highly Endemic Rural Population

Amebiasis is caused by the protozoan Entamoeba histolytica. This disease, or the asymptomatic infection, is associated with poor socioeconomic conditions, malnutrition, and poor hygiene behavior, all common in developing countries. There is evidence of the existence of two species of quadrinucleated amebas in the human population ( E. histolytica and Entamoeba dispar ) with different pathogenic capabilities, making necessary the reevaluation of the actual frequency of infection and disease, because is a tendency to overestimate it in endemic areas where other intestinal infections causative of dysentery or bloody diarrhea are found. The opposite is frequent in nonendemic areas, where the presence of amebas is overlooked in stool microscopic examination. In the present work, we studied the prevalence of E. histolytica or E. dispar infection in the rural community of Coahuixtla, State of Morelos, Mexico. The selection of this community was made on the basis of the reported rate of new cases of intestinal amebiasis or amebic liver abscess in the state of Morelos (1). E. histolytica and E. dispar identification was performed through the molecular analysis of the cysts isolated directly from stool samples.

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.

Entamoeba histolytica: antibody response to recent and past invasive events

Sero-epidemiological data from endemic amoebiasis areas are difficult to evaluate because the serology of individuals affected by an active process of Entamoeba histolytica tissue invasion is, at present, almost impossible to distinguish from that of individuals who have had an invasive event in the past. The present study compares serum antigenic recognition frequencies among three groups of individuals with different infective conditions: amoebic liver abscess patients; asymptomatic cyst passers; and individuals who have had amoebic liver abscess from one to three years before the study. Control groups consisted of Mexican and Canadian healthy adults. Western blots of E. histolytica membrane extract antigen were reacted with sera from the studied individuals, recognition frequency values were calculated and immunoplots of frequency differences were constructed. The results obtained suggest that the identification and purification of antigenic fractions, which are frequently recognized by sera of amoebic liver abscess patients (136, 132, 93, 70 and 62 kDa), or preferentially associated with past invasive events (144, 140 and 49 kDa), or related to the E. histolytica cyst passer condition (62 and 136 kDa), are important improvements in the use of serology for diagnosis and epidemiological studies in endemic areas of amoebiasis.

Epidemiology of amoebic liver abscess in Mexico: the case of Sonora

The division of the taxon once known as Entamoeba histolytica into two species, the pathogenic E. histolytica and the nonpathogenic E. dispar, has led to a need to re-evaluate the epidemiology of amoebic infection and disease in humans. Although all human infection with E. histolytica is now sometimes called amoebiasis, that term is restricted here to invasive intestinal or hepatic infection with the parasite. Amoebiasis, which is endemic in many tropical and subtropical areas, is thought to be associated with millions of cases of dysentery and amoebic liver abscess every year (Walsh, 1986). Even in so-called ‘endemic countries’, however, there is a considerable lack of knowledge about the epidemiology of Entamoeba infection and the invasive disease that it may cause. In his now-classical review, Elsdon-Dew (1968) reported that Mexico was a major centre of invasive amoebiasis in the Americas, with other global ‘hot-spots’ in Natal and South– east Asia. According to more recent epidemiological surveys based on modern clinical and diagnostic tools (Abd-Alla et al., 2000; Jackson et al., 2000; Blessmann et al., 2002), these observations made by Elsdon-Dew almost four decades ago — which were based mainly on the numbers of cases of amoebic liver abscess (ALA) being reported — still appear valid. Some of the earliest data on the prevalence of amoebiasis in Mexico come from the autopsy-based studies of Dr Bernardo Sepúlveda. In these investigations (Sepúlveda, 1970), 195 cases of ALA were detected during 6126 autopsies performed, between 1954 and 1967, at the Hospital General de la Ciudad de México, and another 200 such cases were observed during 3000 autopsies performed, between 1963 and 1969, at the Hospital General del Centro Médico Nacional. Although clearly biased and limited in scope, these data indicate that, 30–40 years ago, 3%–7% of Mexicans had ALA at the time of their deaths. In 1992, the Mexican Institute of Social Security (IMSS), which provides healthcare to over half of the Mexican population, recorded the treatment of 369,440 new cases of invasive intestinal amoebiasis (i.e. either amoebic colitis or dysentery) and 1922 cases of ALA (Treviño GarciaManzo et al., 1994). Fortunately, although asymptomatic intestinal infections appear less responsive, symptomatic amoebiasis generally responds extremely well to treatment with metronidazole, and the extended availability of this drug in Mexico has markedly decreased national levels of Entamoeba-attributable mortality over the last 50 years. Before 1970, for example, 9%– 13% of Mexican cases of ALA were fatal (Lee-Ramos and González-Montesinos, 1970; Alvarez and de la Rosa, 1971) but the corresponding level of fatality (as recorded by the IMSS) had fallen to just 1.2% by 1994. This decrease has been attributed not only to effective chemotherapy but also to more general improvements in the health services of Mexico, which have led to signs of an epidemiological transition, away from parasitoses and towards chronic degenerative diseases (Frenk et al., 1989, 1991). The more recent official data on the incidence of ALA in Mexico (Ximénez, 2000) show a fall from 8.5 cases/100,000 in 1995 to 3.66 cases/100,000 in 2000 (albeit with a ‘blip’, of 9.04 cases/100,000, Annals of Tropical Medicine & Parasitology, Vol. 101, No. 6, 533–538 (2007)

Western blot of Entamoeba histolytica antigenic fractions: reactivity analysis with sera from intestinal amoebiasis patients.

The reactivity of sera from acute-phase intestinal amoebiasis patients (two weeks evolution) was studied to determine which of the Entamoeba histolytica antigens are most frequently immunogenic. Sera were examined by means of immunoelectrotransferase assay using crude extract of HM1:IMSS E. histolytica trophozoites. Three populations of clinically healthy adults from Mexico, Canada and Germany, with no evidence of parasites in faeces, were used as controls. The frequency of antigen recognition was analysed. In ailing individuals, the bands of 23, 24, 26 and 51 kDa were recognized most frequently (65 and 60%) followed by the 62 kDa band (56%). The combination of some of these bands, namely 3.4, 4.1 and 6.7, with molecular weights of 62, 51 and 24 kDa, increased the recognition frequency of patients to 91.4%. These results constitute a first but important step towards the design of more accurate methods for the successful immunodiagnosis and epidemiology of acute intestinal amoebiasis.

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.