Entamoeba dispar: Could it be pathogenic

Abstract

Amebiasis is a disease caused by the protozoan parasite Entamoeba histolytica. This ameba can colonize the human intestine and persist as a commensal parasite, similar to Entamoeba dispar, an ameba considered to be non-pathogenic. The similarities between E. histolytica and E. dispar make the latter an attractive model for studies aimed at clarifying the pathogenesis of amebiasis. However, in addition to being an interesting experimental model, this relative of E. histolytica remains poorly understood. In the 1990, it was believed that E. dispar was unable to produce significant experimental lesions. This scenario began to change in 1996, when E. dispar strains were isolated from symptomatic patients in Brazil. These strains were able to produce liver and intestinal lesions that were occasionally indistinguishable from those produced by E. histolytica. These and other findings, such as the detection of E. dispar DNA sequences in samples from patients with amebic liver abscess, have revived the possibility that this species can produce lesions in humans. The present paper presents a series of studies on E. dispar that begin to reveal a new facet of this protozoan.

INTRODUCTION

The protozoan Entamoeba histolytica is the causative agent of amebiasis. There are approximately 50 million cases of amebiasis per year worldwide, and it kills nearly 100 thousand people annually.[1] Amebiasis is the most severe disease caused by protozoans that afflict the human intestine and the second leading cause of death among parasitic diseases.[2] It is characterized by two major clinical syndromes, amebic colitis and amebic liver abscess.[3] The importance of amebiasis stems from its widespread geographical distribution, its high incidence, and its association, in some cases, with severe and potentially fatal pathologies. It therefore remains a public health problem in developing countries despite the existence of medications.[4]

E. histolytica was first described over 130 years ago in Russia by Lösch in 1875, as cited by Jackson (1998),[5] and was formally named in 1903 by Schaudinn, as cited by Clark (1998).[6] In an attempt to explain the high prevalence of asymptomatic infected individuals, the DualistTheory was proposed by Brumpt (1925), as cited by Walsh (1986); this theory proposed the existence of two amebas that were morphologically identical but biologically distinct.[7] It was believed, however, that only E. histolytica should be able to penetrate tissues and produce invasive amebiasis. Subsequently, E. histolytica was separated into two distinct groups through isoenzyme electrophoresis: Pathogenic and non-pathogenic (NP), according to Brumpt 1925, as cited by Sargeaunt et al. (1978).[8] The NP species would then explain the prevalence of asymptomatic disease in approximately 90% of infected individuals worldwide.[7] The definitive proof that these two groups represented distinct species was obtained by deoxyribonucleic acid (DNA) analysis,[9] and the new species was called Entamoeba dispar, by Brumpt 1925, as cited by Diamond and Clark (1993).[10] The isoenzyme profiles, the differences in gene sequences, and the use of monoclonal antibodies confirmed the existence of two distinct species that have since been officially recognized by the World Health Organization (WHO).[1] Currently, it is estimated that E. histolytica and E. dispar infect approximately 12% of the world population, with infections with the latter being substantially more common and with infections with E. histolytica possibly representing 1% of infections.[1]

To differentiate infection by E. histolytica from infection by E. dispar, parasite antigen detection in stool samples[11] and gene segment amplification in both stool and amebic liver abscess samples[12] have been used. Epidemiological surveys have shown that most asymptomatic individuals infected by one of these species are colonized by E. dispar. However, in some regions, there is a high prevalence of E. histolytica in asymptomatic individuals and in patients with diarrhea.[13,14,15] Studies on the genetic variation among ameba species provide important clues to explain the different clinical presentations of E. histolytica infection and provide data on the geographical distribution and mobility of epidemiologically important strains. Studies on the epidemiology of amebiasisin endemic areas have used molecular approaches to characterize the prevalent species of Entamoeba in human infection. These studies have attempted to correlate the different species and sub-populations with the diverse spectrum of clinical forms: (a) Symptomatic infections caused by E. histolytica (intestinal or extra-intestinal amebiasis); (b) asymptomatic E. dispar infections; (c) mixed asymptomatic infections caused by E. histolytica and E. dispar; and (d) asymptomatic E. histolytica infections.[16]

With the recognition of the species E. dispar in 1997,[1] several studies were conducted comparing this species with E. histolytica. E. dispar is morphologically similar to E. histolytica and has been considered as a distinct species based on isoenzyme patterns of amebas isolated from asymptomatic patients and those with invasive disease.[8] The two Entamoeba species have nearly identical sets of genes encoding the major virulence factors. However, E. histolytica secretes approximately 10 to 1,000 times more cysteine proteases than E. dispar.[17] The activity of E. dispar amebaporesis approximately one-third of that observed in E. histolytica.[18] Furthermore, the Gal/GalNac lectin of E. dispar shows homology with two members of the heavy-chain family and four of the light-chain family in comparison to the E. histolytica lectin, leading to reduced adhesion and cytotoxicity in vitro.[19]

Before its identification as a separate species, E. dispar was described as NP E. histolytica.[10] Experimental studies involving NP E. histolytica failed to demonstrate its ability to produce significant lesions in laboratory animals.[20,21] Moreover, after the description of E. dispar, experimental research continued to show similar results. Espinosa-Cantellano et al., inoculated 5 × 10⁵ and 5 × 10⁶ E. dispar trophozoites in hamsters by the intra-hepatic route. Seven days after infection, the authors observed only focal inflammatory infiltrate with no formation of necrosis and granuloma.[22] These findings, coupled with in vitro observations that E. dispar secretes toxic products in lower amounts and with lower activity than E. histolytica, reinforced the idea that E. dispar was commensal and thus incapable of generating human and experimental lesions. In fact, the axenic E. dispar SAW 760 strain produced a milder cytopathic effect on MDCK epithelial cells compared to that produced by E. histolytica.[23]

The prevalence of E. dispar is not known. It is speculated that this species is responsible for most infections that were previously considered to be associated with E. histolytica. Studies worldwide show a higher prevalence of E. histolytica in developing countries; however, even in these countries, the prevalence of E. dispar is high.[15,24,25,26,27,28,29,30,31,32,33,34]

In Brazil, amebiasis is an important cause of morbidity.[35,36] E. histolytica is most prevalent in Manaus, where it infects 6.8% of the population,[37] in Fortaleza, where it infects 14.9% of the low-income population,[38] and in Belém, where it infects 29.5% of individuals living in the metropolitan area.[39] In the remaining areas of the country, studies show a higher prevalence of E. dispar. In Pernambuco, Belo Horizonte (Minas Gerais), and Salvador (Bahia), only E. dispar was detected, with the exception of Minas Gerais. Our group has found an infection rate of nearly 5% with E. histolytica/E. dispar in communities of great Belo Horizonte.[40] To date, 99% of these infections have been associated with E. dispar (unpublished data).

E. dispar is considered non-invasive and thus related to asymptomatic infections. In Brazil, non-dysenteric colitis represents a very frequent clinical presentation. The ameba isolates belonging to this group were analyzed by isoenzymes and PCR and identified as E. dispar. Our group was the first to identify E. dispar isolated from symptomatic cases.[41,42] The strains isolated in these cases were also able to infect experimental animals. As noted earlier, the first experimental studies involving NP E. histolytica (E. dispar) did not succeed in reproducing the amebic lesions. However, since 2000, studies published using xenic E. dispar strains isolated in Brazil began to show results different than those observed in previous reports. E. dispar strains isolated from patients living in the North and Southeast regions of Brazil were inoculated in the liver (5 × 10⁴ trophozoites) and cecum (1 × 10⁶ trophozoites) of hamsters and rats, respectively.[43] All animals showed hepatic and intestinal lesions similar to those caused by E. histolytica, as well as the presence of trophozoites. The same was reported by Costa et al., after inoculating the liver of hamsters with 2.5 × 10⁵ trophozoites of other E. dispar strains also isolated from symptomatic and asymptomatic individuals in Brazil.[44] Inoculation of the microbiota of each strain in control hamsters did not lead to liver lesions or produced only discrete purulent lesions, indicating that E. dispar trophozoites were responsible for the development of liquefactivenecrosis. Unlike the xenic strains, the use of axenic[22] and monoxenic[44,45] strains of E. dispar failed to produce amebic abscesses, demonstrating the importance of bacterial association to acquire pathogenicity through unknown mechanisms. Recently, two other studies described what has been observed since 2000. Similar to the MCR strain, the experimental amebic liver abscess was reproduced in hamsters inoculated with xenic E. dispar strains also isolated from Brazilian asymptomatic patients.[46,47]

Research on the genetic diversity of E. histolytica and E. dispar in individuals with asymptomatic infection or invasive disease has shown high polymorphism of both Entamoeba species.[16,48,49] Evidence for genetic diversity has been demonstrated through differences in the genotypes of E. histolytica isolated from stool and amebic liver abscess samples from the same patient.[50] Other authors also detected different genotypes of E. histolytica trophozoites isolated from two distinct amebic liver abscesses from the same patient.[51] As with certain E. histolytica strains, E. dispar strains may also exhibit varied biological behavior as a result of genetic diversity.[43]

The use of more precise methods for quantifying amebic lesions showed that the liver abscess produced by the E. dispar MCR strain is as important and significant as that produced by the E. histolytica EGG strain.[52] It is noteworthy that although the EGG strain was isolated from a symptomatic patient presenting dysenteric colitis and amebic abscess, the MCR strain was isolated from an asymptomatic patient. The kinetics of amebic liver abscess produced by a single E. dispar strain was studied over 8 days of infection, during which it was possible to describe changes such as the different phases of epithelioid granuloma, thrombosis and apoptosis.[53] Although polyclonal antibodies were used in these two studies, immunohistochemical identification of E. dispar trophozoites greatly aided the counting of parasites and the qualitative and quantitative assessment of lesions associated with parasitism.

Our group inoculated the E. dispar MCR strain into the liver and cecum of different hosts to evaluate their susceptibility. We found that the hamster is the fittest animal to develop an abscesss by E. dispar (100% of challenged animals) and that both the mouse and ther at presented amebiccolitis in approximately 70% of cases (unpublished data).

By studying the in vivo binding of antibodies and complement to trophozoites of the MCR strain, we observed that trophozoite binding and destruction was higher than in animals inoculated with the E. histolytica EGG strain.[54] Together with the larger amounts of E. dispar trophozoite debris observed, these findings confirmed the lower ability of this species to escape the action of the immune system. The joint study of MCR and EGG strains provided a better understanding of the relationship between the immune system and the development of amebic liver abscess. Regarding the virulence and pathogenicity inherent to each species, antibodies, complement, macrophages, and neutrophils seem to interfere in the selection of more resistant trophozoites and in the development of amebic liver abscesses.[54,55]

The explanation of the acquisition of pathogenicity of the E. dispar strains may lie in their interaction with bacteria. The observation that these lesions were obtained only after the association of bacteria with E. dispar suggest that the bacteria may be causing early injuries that favor the proliferation of and tissue invasion by trophozoites, by increasing the expression of virulence factors and/or by incorporating genes. In fact, the symbiosis between bacteria and E. histolytica or E. dispar may modulate phenotypic changes and virulence properties of ameba.[53,56,57]

A recent in vitro study showed that the increased virulence of the E. histolytica HM1: MSS strain was mediated by the presence of pathogenic entero bacteria. In this study, the researchers observed that the phagocytosis of Shigella dysenteriae and enteropathogenic Escherichia coli (EPEC) by E. histolytica trophozoites increased the cytopathic effect of trophozoites on epithelial cells as well as the cysteine protease activity and the expression of Gal/GalNAc-specific lectin. The authors also suggest that infection with enterobacteria may render the intestine epithelial cells more susceptible to the virulence mechanisms of E. histolytica.[58]

Although E. dispar is considered commensal for humans, evidence shows that additional in-depth studies are required to address this issue. E. dispar has been isolated from patients presenting symptoms of non-dysenteric colitis[41,42] and from a patient with dysenteric colitis.[59] Moreover, DNA sequences of E. dispar were detected and genotyped in samples from patients with amebic liver abscess, suggesting that E. dispar may also be involved in the development of lesions in the human intestine and liver.[16]

Given the evidence that E. dispar can produce significant experimental lesions in the presence of bacteria, along with the high frequency of intestinal co-infections that affect millions of people annually, we believe that the most important steps are further in-depth studies on the relationship between E. dispar and bacteria. Thus, we began a preliminary analysis of the possible interference of Salmonella typhimurium infection (intragastric intubation of 10⁸ colony-forming units (CFUs) at 14 hours before the amebic infection) with the MCR strain of E. dispar (intracecal inoculation of 5 × 10⁵ trophozoites) for approximately 7 days. In Wistar rats infected with E. dispar only, intestinal lesions were characterized by areas of mucosal destruction and submucosal inflammation (mixed inflammatory infiltrate, hyperemia, and edema), which sometimes reached the muscle and serosa. The presence of bacteria increased the intestinal necrosis area and the intensity of the inflammatory response. We scanned the necrotic areas through a JVC TK-1270/RGB micro-camera and measured these areas using the KS300 software in the Carl Zeiss image analyzer.[52] We morphometrically confirmed the development of a larger lesion area of the cecal mucosa in animals infected with both E. dispar and S. typhimurium (139,822 ± 30,089 μm² ) compared to the lesion area of the cecal mucosa in animals infected with E. dispar only (50,657 ± 5,131 μm² ) (P < 0.0001). Infection of animals with S. typhimurium alone did not lead to the destruction of the colonic mucosa as observed in E. dispar infection, and only the presence of areas of epithelial shedding was observed. The inflammation caused by S. typhimurium alone in the sub-mucosal layer was substantially reduced compared to that observed in animals co-infected with E. dispar and S. typhimurium. Future molecular biology studies will determine whether the bacteria favored the expression of amebic virulence factors and/or whether the epithelial lesions alone caused by bacteria favor trophozoite adhesion and invasion.