Entamoeba histolytica: an update William Stauffera and Jonathan I. Ravdinb Purpose of review Over the past decade, since it was formally recognized that Entamoeba histolytica and Entamoeba dispar were two distinct species, studies in this field have made dramatic in-roads into the understanding of E. histolytica and the pathogenesis of invasive amoebiasis. Over the same period it has also become clear that the true incidence of E. histolytica infection, particularly in vulnerable populations such as low socioeconomic children, is exceedingly high. Understanding the epidemiology, pathophysiology, and the molecular and genetic biology of the organism will not only lead to improved diagnostic and treatment options but, ultimately, to the development of a safe and efficacious vaccine. Recent findings The recent advances in the genetic and molecular sciences have increased our understanding of the mechanisms that make E. histolytica unique among enteric protozoa in causing invasive disease. In addition, host factors, which predispose individuals or populations to infection or disease, are beginning to be elucidated. New diagnostic tools specific to E. histolytica are being exploited by clinicians and researchers to identify and treat patients as well as to add to the knowledge of the epidemiology and natural history of this infection. The ultimate goal – eradication of disease – is theoretically feasible since humans and primates are the only reservoirs of E. histolytica. Many talented and dedicated individuals are pursuing the development of an effective and safe amoebiasis vaccine. Summary E. histolytica remains an important pathogen in many populations of the world and although there has been substantial progress into understanding the disease major challenges still exist. Keywords Entamoeba histolytica, amoebiasis, amoebic abscess, amoebic colitis Curr Opin Infect Dis 16:479–485. # 2003 Lippincott Williams & Wilkins. a Department of Internal Medicine, Division of Infectious Disease and International Medicine, University of Minnesota, Minneapolis and Regions Hospital/HealthPartner’s Center for International Health and International Travel Clinic, St Paul and b Department of Medicine, University of Minnesota, Minneapolis, Minnesota, USA Correspondence to Dr Jonathan Ravdin, MD, Nesbitt Professor and Chairman, Department of Medicine, University of Minnesota, 516 Delaware Street SE, MMC 194, Minneapolis, MN 55455, USA Tel: +1 612 625 3654; fax: +1 612 636 3055; e-mail: [email protected] Current Opinion in Infectious Diseases 2003, 16:479–485 Abbreviations ALA GalNAc PCR amoebic liver abscess N-acetylgalactosamine polymerase chain reaction # 2003 Lippincott Williams & Wilkins 0951-7375 DOI: 10.1097/01.qco.0000092821.42392.ca Introduction Entamoeba histolytica infects hundreds of millions of people per year. While most individuals remain asymptomatic, perpetuating the natural cycle of the organism through fecal excretion of infective cysts, an important minority will suffer the severe morbidity associated with invasive disease (approximately 50 million) with an estimated 100 000 succumbing to the protozoa annually [1 . .]. During the 1990s enough evidence had accumulated to support the formal separation of two morphologically identical species of amoeba: the non-pathogenic Entamoeba dispar from the potentially pathogenic E. histolytica [2–6]. Morbidity and mortality data (in absolute numbers) which existed prior to this time pertaining to cases of invasive disease were not greatly affected by this reclassiﬁcation because all invasive disease was known to be caused by E. histolytica. However, because most prevalence and incidence data previously collected pertained to asymptomatic individuals, and it was clear that a majority of asymptomatic individuals with cysts detected in their stool were infected with non-pathogenic E. dispar, the true prevalence and incidence of E. histolytica became a matter of speculation. In 1997 an expert committee was convened which issued several recommendations to be given ‘highest priority’ in future research involving E. histolytica. Top among these recommendations was a call to investigate the true incidence and prevalence of E. histolytica infection. The committee also felt it imperative for proper planning and control strategies to not only identify the prevalence of asymptomatic carriers but to elucidate the likelihood of manifesting invasive disease and the frequency of transmission to naı¨ve individuals. Finally the report called for further immunologic, biochemical, molecular and genetic research to assist in understanding the pathogenesis of this organism as well as helping to pioneer new treatment and prevention strategies, and ultimately, to the creation of a safe and effective vaccine . This paper will discuss the most recent scientiﬁc literature and assist the reader by placing these recent discoveries in the context of previous work conducted in the study of E. histolytica. Epidemiology Some of the original epidemiologic descriptions distinguishing E. histolytica from E. dispar infections originated from studies conducted in South Africa. A community cross sectional survey of asymptomatic individuals found through the use of culture and zymodeme analysis an overall prevalence of the E. histolytica–E. dispar complex of approximately 10%. On further analysis it was 479 480 Gastrointestinal infections revealed that 90% of individuals were infected with the non-pathogenic E. dispar and 10% asymptomatically harbored E. histolytica . In the same study it was noted that E. dispar occurred more frequently in females than in males while E. histolytica had an equal prevalence and incidence in males and females. Further, it was also interesting to note that although the prevalence of E. histolytica infection was equal in all age groups and between sexes, males and individuals over 16 years of age had a much greater likelihood of invasive disease. It was subsequently noted in this population that approximately 10% of asymptomatic carriers of E. histolytica would develop invasive disease while a majority of others spontaneously cleared their infection by 1 year [9 .,10 . .]. It has been recognized that disease expression varies geographically. For example, invasive disease in Egypt is predominantly amoebic colitis  whereas in South Africa there is an excessive rate of amoebic liver abscess (ALA). In fact, in Hue City, Vietnam, an overall estimated frequency of ALA was recently reported to be as high as 21 cases per 100 000 inhabitants [9 .]. As part of a study investigating innate and acquired resistance to amoebiasis, 230 Bangladeshi children (age 2–5 years) were enrolled in a 2-year observational study [10 . .]. In this impoverished population 55% of children acquired E. histolytica infection during the 2-year period. Of these 55% who acquired infection, 80% of children remained asymptomatic while 20% had an associated diarrhea with 4% of these meeting the deﬁnition of amoebic colitis. Further, 17% acquired additional E. histolytica infections during the 2-year study which were felt to be due to a second genetically distinct strain (determined by polymerase chain reaction (PCR) for the serine-rich E. histolytica protein) [10 . .]. A recent study  of low socioeconomic status school children in Ecuador shed additional light upon the epidemiology of E. histolytica infection. This crosssectional survey found asymptomatic E. histolytica infection present in only seven of 178 children. However, it was interesting to note that more than 64% of children showed high serologic titers implying current or recent infection with E. histolytica . This high seroprevalence corresponds to another recent sero-survey conducted in Mexico . Further, this study summarized some of the difﬁculties in conducting prevalence studies as it revealed very non-concordant results between some previously well validated tests: two antigen detection assays, the Prospect Entamoeba histolytica Microplate assay and the Entamoeba histolytica II assay; several methods of direct microscopic exam; in-vitro culture; starch gel isoenzyme electrophoresis; and serologic analysis by three separate methods. This paper revisited the question of what should be considered an adequate screening test (sensitive, speciﬁc, inexpensive), and what should be consid- ered the true ‘gold standard’ in investigating individuals for E. histolytica and E. dispar infection . A related question arises frequently in clinical practice in low prevalence areas which receive large numbers of immigrants and refugees from endemic areas. Most medical screening protocols in the United States for immigrants and refugees arriving from the developing world suggest that three ova and parasite examinations are routinely collected . This screening is conducted to enhance the individuals’ health by treating intestinal parasitic infections as well as to protect the public health of the community. Entamoeba speciﬁcally is known to spread within families, institutions (i.e. day care centers) and occasionally may cause epidemics [15,16]. Despite these recommendations it is known that stool ova and parasite examinations are relatively insensitive and they are unable to distinguish E. histolytica from E. dispar infection. Further reducing the speciﬁcity is new information indicating an inability of microscopy to reliably distinguish E. histolytica  from another close relative Entamoeba moshkovskii. Currently the standard at most centers is to treat all asymptomatic individuals with cysts in the stool with an anti-protozoal intestinal agent. It is the authors’ experience that there is poor compliance with this approach as it subjects asymptomatic patients to prolonged multiple daily dosing of medications that are difﬁcult to administer and carry substantial adverse effects. It is therefore suggested that positive stool samples be conﬁrmed with stool antigen or serum serology studies for E. histolytica prior to initiation of treatment [18,19]. It remains to be determined if in addition to the stool ova and parasite examination other diagnostic screening examinations (i.e. stool antigen testing) would be a more sensitive, speciﬁc and costeffective addition to the routine medical screening of immigrants and refugees. Pathogenicity and clinical disease When the host comes into contact with the protozoa the result, in a majority of cases, will be establishment of a non-invasive infection . However, some individuals will acquire invasive disease predominantly in the form of amoebic colitis or ALA. The parasite–host interactions which determine the course of disease, although largely still a mystery, are beginning to come to light. The three virulence factors that are best understood include a parasite surface protein (lectin), the amoebapore, and the cytoproteins (cysteine proteases). Generally, a cyst of E. histolytica is ingested by a human host from fecally contaminated food or water. Excystation takes place in the intestinal lumen to produce trophozoites. The trophozoite surface protein (lectin) recognizes the sugars galactose and N-acetylgalactosamine (GalNAc) on the host cell surface [20,21]. The Entamoeba histolytica Stauffer and Ravdin 481 interface of the galactose/GalNAc lectin with the host mucins lining the intestine  is the deﬁning moment of the infection. If the parasite lectin attaches to the host mucin glycoproteins that line the intestinal lumen, a non-invasive gut infection ensues. The life-cycle then continues as the trophozoites reproduce by clonal expansion in the mucin layer. Subsequently, the galactose/GalNAc lectin, along with mucin glycoproteins or other gut bacteria, initiates the developmental pathway leading to encystment . On the other hand, if the trophozoite penetrates the mucin layer and the lectin attaches directly to a host cell surface N-acetyl-D-galactosamine, a cascade of events occurs ultimately leading to death of the host cell  and progression of invasive disease. Once the trophozoite is committed to invasion, replication and encystment will not occur and the life-cycle will not be successfully completed. Therefore, invasive disease, as determined by the lectin protein, must be considered a maladaptation of the organism as it is disadvantageous to the protozoa’s survival. A complete review of the lectin protein is beyond the scope of this paper but interested readers are referred to an excellent recent review of the structure and function of this protein [1 . .]. E. histolytica trophozoites kill (in vitro) a wide variety of tissue cell lines including neutrophils, T lymphocytes and macrophages [24–26]. As mentioned, the killing of host cells is contact-dependent, initiated via the galactose/GalNAc lectin [20,27]. It has been shown that when the amoeba engages the host cell the cytoskeleton undergoes changes that are vital to the ability of the trophozoite to kill [1 . .]. A pore-forming protein complex is inserted into the host cell by the trophozoite that is similar to the human complement system (amoebapores) and is likely involved in pathogenicity . Killing ability is also dependent on calcium and cytolysis. It has been shown that within seconds of contact with the trophozoite the host cell calcium will precipitously rise 20-fold with cell death occurring within 15 min . Cysteine proteases are known to be important to the pathogenesis of E. histolytica and function by degrading extracellular proteins and through the disruption of cell monolayers . Approximately 20 genes, a majority of which are not expressed, have been identiﬁed that code for the cysteine proteases. Interestingly it was also recently demonstrated that the differential expression of these 20 genes varies between E. histolytica and E. dispar, explaining at least one mechanism that allows E. histolytica to invade the host . In the ﬁnal stages, when the host cell dies, it undergoes apoptosis as the nuclear chromatin condenses, the membrane forms blebs and the internucleosomal DNA fragments [32,33]. Activation of caspase 3, a distal effector molecule in the apoptotic pathway, seems to be a crucial step in cell killing (in vitro) and in the formation of ALA (in vivo) [32,34]. It has further been suggested, based on a study of the ability of E. histolytica to kill and phagocytose host cells, that virulence may be directly proportional to the amoeba’s ability to cause apoptosis and then phagocitize the dead cell as this process seems to substantially limit the host inﬂammatory response [35 .]. Molecular biology and genetics Molecular biology and genomics research is initiating revolutionary advances in medicine. Amoebiasis research is no exception with extraordinary insights being made into the understanding of the organism ranging from its teleologic origins to discoveries of novel gene products and functions. Molecular phylogeny has revealed that Entamoeba species (histolytica and dispar) are close to Dictystelium discoideum on one of the lowest branches of the eukaryotic tree [36 . .]. Although amoebae were thought to lack organelles (mitochondria, endoplasmic reticulum and Golgi apparatus) evidence to the contrary is coming to light. In addition to recently described nuclear-encoded mitochondrial genes and a remnant mitochondrial organelle [37,38], a study  found that a 51 kDa protein of E. histolytica has a signiﬁcant similarity to the amino acid sequence of the calreticulin-like protein of spinach leaves (77% homology) and of the calreticulin precursor of D. discoideum (60% homology) indicating an equivalent system to the eukaryotic endoplasmic reticulum and the Golgi apparatus . The recognition that the similar but non-pathogenic E. dispar, a much more successfully commensual parasite, differs from the potentially invasive E. histolytica has led to great opportunities from a genomics standpoint to compare species and elucidate the speciﬁc genes and proteins that make E. histolytica pathogenic. A joint project between the US Institute of Genomic Research (Rockville, Maryland, USA) and the UK Pathogen Sequencing Unit based at the Sanger Institute (Cambridge, UK) is endeavoring to sequence the complete E. histolytica genome and for comparative purposes, to partially sequence four other closely related intestinal protozoa (E. dispar, E. moshkovskii, E. terrapinae, E. invadens). It has become clear through this effort that E. histolytica has a small genome (approximately 20 MB) that is very repetitive and contains densely packed coding sequences with closely spaced genes lacking introns [1 . .,40–42]. It appears the sequences encoded in the genome arrangement are proteins involved in highly conserved functions and cellular processes. However, there are also areas of the genome that express amazing genetic polymorphisms . It has been proposed that these polymorphic loci may assist in determining geographic origins of isolates and may even assist in ascertaining routes of transmission . Certain unique features that are proving to be a challenge in decoding 482 Gastrointestinal infections the genome include a high adenosime-thymidine content which makes it refractory to large insert cloning and uncertain ploidy across the genome. Host defense and the potential for a vaccine Although the human host responds to E. histolytica by activating both cell-mediated and humoral immunity mechanisms, the organism has developed multiple methods of rendering many components of the human immune system ineffectual. Further, as with many infectious diseases, it appears that certain host responses worsen the pathologic sequelae of disease. On the other hand, some components are effective at limiting the disease process and producing protection against future infection. These components, particularly mucosal IgA, are currently being exploited in the pursuit to develop an effective vaccine. Host defense Both cell-mediated and humoral immune responses to E. histolytica develop with either asymptomatic or symptomatic infection. E. histolytica has developed multiple immunomodulatory effects on the host response that allows it to be a successful pathogen. Further, it is clear, as with many infectious diseases, the host responses may not only provide defense for the host but also are a key ingredient in the disease process [45,46]. Initial resistance to E. histolytica is dependent on natural or innate mechanisms. Beyond the physical barriers of the mucin and structures of the colonic epithelium the organism must also overcome the complement system. It has been recognized that the complement pathway is of limited defense for the host, particularly the alternative pathway as the trophozoite has ‘acquired resistance’ to the C5b-9 complex. The organism has acquired this ‘resistance’ through expressing an epitope on the GalNAc lectin heavy subunit that has sequence similarity and antigenic cross-reactivity to the human CD59 (a leukocyte antigen that prevents the assembly of the C5b-9 complex), effectively making it invisible to the host alternative complement system . Amoebic cysteine proteases also degrade C3a and C5a complement components . In-vitro data have demonstrated a role for cell-mediated response, especially interferon-g, tumor necrosis factor-a and interleukin-4, in the activation of macrophages and neutrophils to kill E. histolytica trophozoites . Although neutrophils seem to be essential in regard to initial host defense, it is interesting to note that E. histolytica produces a chemoattractant for neutrophils  and will kill these cells on contact . In addition, it has also been demonstrated that amoebas will lyse inactivated monocytes/macrophages  and new evidence suggests that tumor necrosis factor-a may, at least in part, act as a chemoattractant to these cells . Perhaps most importantly, both non-invasive and invasive E. histolytica infection has been shown to elicit mucosal immunoglobulin A and serum immunoglobulin G response to the lectin protein. The importance of this ﬁnding lay in the fact that mucosal immunoglobulin A response directed at the carbohydrate-recognition domain of the galactose/GalNAc lectin protein has shown to be protective against infection [36 . .,52]. An interesting observation was made recently in a study of Bangladeshi children [10 . .], which conﬁrmed the protective effect of mucosal anti-lectin immunoglobulin A but found that a serum immunoglobulin G response to the same protein seemed to be associated with a higher likelihood of future invasive disease [10 . .]. In a prospective study of adult patients cured of ALA and a large number of controls (family members or neighbors), it was found that high levels of intestinal anti-lectin immunoglobulin A antibodies are present in ALA patients for over 18 months and that they are highly immune to new infections by E. dispar , which contains functional lectin molecules that are antigentically cross reactive with the E. histolytica lectin . By stool culture criteria, new E. dispar infections were ﬁvefold more common in the control population than E. histolytica infections, highlighting the immunity observed in the ALA patients. A cysteine-rich portion of the galactose-inhibitable lectin heavy sub-unit (a portion of LC3) appears to be one of the main epitopes on the lectin as it recently was shown to induce humoral (immunoglobulins A and G) immunity in asymptomatic infected patients as well as in ALA patients . In addition, epitope speciﬁc mouse monoclonal antibodies against the cysteine rich region of the galactose–lectin’s heavy sub-unit have been shown to inhibit adherence to target cells [52,54–56]. Hence, the cysteine rich portion of the heavy subunit of the lectin would appear to be a potential vaccine target. In considering this protein as a target it is imperative that the epitope does not vary with genetically distinct stains of E. histolytica. It is therefore of interest to note a study that demonstrated the galactose/GalNAc lectin heavy subunit isolates from three distinct areas of the world (Bangladesh, Georgia, and Mexico) retain remarkable sequence conservation [57 .]. Vaccine development The recognition of an extraordinarily high prevalence of E. histolytica infection in certain vulnerable populations of the world suggests that an effective and safe vaccine would not only prevent considerable mortality but that it would provide a valuable tool in decreasing morbidity in developing nations. This would be particularly true in children in developing nations as the epidemiology indicates that children in poverty are at an extremely high risk for developing infection. As humans are the only known reservoir for the parasite, other than Entamoeba histolytica Stauffer and Ravdin 483 primates such as baboons, a safe and effective vaccine could theoretically lead to eradication of the protozoa. Since it appears acquired immunity to E. histolytica is correlated with mucosal immunity, it follows that a vaccine that induces gut-associated lymphoid tissue would be a primary candidate. Several potential oral vaccine candidates have been reported in the literature [58 .]. A recent study of oral immunization of gerbils with a live-attenuated Salmonella vector expressing a portion of the galactose/GalNAc lectin  was shown to protect animals from ALA. Another innovative study  utilized Yersinia outer protein E to orally deliver heavy chain antigen (170CR2) through the Yersinia type III secretion pathway. This novel approach not only induced a signiﬁcant antibody response but was shown to be protective against ALA in the gerbil model . Finally, it was recently found through an in-vivo study conducted in mice  that an injectable codonoptimized DNA vaccine targeting a portion of the galactose-lectin heavy subunit (including the carbohydrate recognition domain), stimulated a Th1-type galactose-lectin speciﬁc cellular immune response as well as inducing development of serum antibodies that recognized a recombinant portion of the heavy subunit. Investigation is now underway to assess this vaccine via an oral delivery route. Diagnosis and treatment Traditionally E. histolytica is diagnosed by microscopy of the stool through identiﬁcation of either cysts or motile trophozoites. As previously mentioned, this technique is neither sensitive nor speciﬁc. The ‘gold standard’ has been considered amoebic culture and zymodeme determination although it is known to be far from 100% sensitive. Recent advances have made new diagnostic tools available to the clinician including serologic antibody tests and the commercial availability of stool E. histolytica-speciﬁc antigen testing . Antibody tests are generally used to conﬁrm positive stool ova and parasite examinations or antigen tests as the currently available serologic tests are unable to differentiate acute infection from past infection. In addition to the clinically available tools, other diagnostic tools have become available for research including PCR, sputum antigen testing, and serum antigen testing by enzyme-linked immunosorbent assay [63–65]. Most recently real-time PCR, able to distinguish E. histolytica from E. dispar, has been pioneered allowing large numbers of samples to be run with high sensitivity (0.1 parasite per gram of stool) and speciﬁcity [66,67]. available furazolidine . As E. dispar is the predominant intestinal organism in most settings, asymptomatic intestinal carriage of amoebae should only be treated if the organism has been conﬁrmed to be E. histolytica, or if this can be inferred from clinical events such as recent amoebic liver abscess. Invasive disease responds to nitroimidazoles such as metronidazole or tinidazole, which have no effect on intraluminal carriage [36 . .]. When these are necessary they should be given before paromomycin, because the diarrhea that is a common side effect of paromomycin may make it difﬁcult to assess the patient’s response to therapy [36 . .]. Occasionally therapeutic aspiration of an ALA or surgical intervention for complications of colonic disease may be necessary. Conclusion Amoebiasis is one of the most common and longest known parasitic infections of mankind. Over the last several years it has become clear that in vulnerable populations, such as in low socioeconomic children residing in the tropics, the incidence rates greatly exceed previous estimates. In fact, E. histolytica is likely one of the major causes of diarrhea in young children and has signiﬁcant negative health effects on this group. Further, it is now clear that invasive disease manifestations (colonic versus ALA) vary geographically being driven both by differences in the organism and through genetic susceptibility of the host. Signiﬁcant progress has also been made in the understanding of the mechanisms that make these intestinal protozoa unique in its ability to cause invasive disease in humans. The Gal/GalNAc lectin protein, which is principally responsible for invasion, has been further characterized using newly available molecular techniques. Also, the genetic mysteries of the organism are beginning to be revealed, helping to explain the pathogenic differences from closely related commensal organisms such as E. dispar. Several recent trials of vaccine candidates have been performed as well as veriﬁcation of new diagnostic techniques that may be used both clinically and in research. Although extensive work is still needed to understand this organism and create an effective vaccine, substantial progress has been made over the last several years. 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