Delta State University
Department of Biological Sciences
BIO 447/547 Parasitology
Course Notes


Entamoeba histolytica and Other Intestinal Amebae of Medical/Veterinary Importance

    Amebae are unicellular eukaryotic organisms, most of which are free-living in aquatic environments.  Many amebae (but not all) are known to have the ability to transform from an active feeding/reproducing stage, known as the trophozoite, into an inactive, encapsulated form, the cyst, that is somewhat resistant to unfavorable environmental conditions.  Amebae are among the simplest of the eukaryotes; although they possess genetic material bounded by a nuclear membrane, they lack several organelles (such as mitochondria, rough endoplasmic reticulum, and Golgi apparatus) that are found in many typical eukaryotic cells.  Several amebae have been demonstrated to harbor bacteria in their cytoplasm, possibly in mutualistic relationships.

    Amebae can be placed into three general categories based on the cyst stage of their life cycles: 

    (1) Amebae that do not form cysts.  In humans, Entamoeba gingivalis, an ameba that lives in the oral cavity, is not known to transform into cysts.  Many of the free-living aquatic species of amebae, such as Amoeba proteus, do not form cysts.  Most parasitic/commensal amebae have the ability to form cysts.

    (2) Amebae that form uninucleate cysts.  Several nonpathogenic amebae of humans and animals have uninucleate cysts, including Iodamoeba butschlii in humans and several Entamoeba spp. of animals (such as E. bovis in cattle and possibly other ruminants).  Entamoeba polecki, an ameba that lives as a trophozoite in the large intestine of pigs and primates (occasionally humans), has a single nucleus in its cyst stage.  E. chattoni, an amoeba that is commonly found in macaques also has a uninucleate cyst stage; it may be possible that some human infections reported to be E. polecki are actually infections with E. chattoni.  Free-living amebae that have been implicated as etiologic agents of primary amebic meningoencephalitis (PAM) typically form uninucleate cysts.

    (3) Amebae that form multinucleate cysts.  Most amebae of medical/veterinary importance form cyst stages that each have more than one nucleus.  Nuclear division (karyokinesis) without simultaneous cytokinesis occurs in these amebic cysts.  Most amebae with multinucleate cysts are quadrinucleate, i.e., they have four nuclei in each cyst.  Endolimax nana and Entamoeba hartmanni are nonpathogenic amebae of humans that form quadrinucleate cysts, while the mature cysts of nonpathogenic Entamoeba coli in humans and other mammals have eight nuclei.  Entamoeba anatis and E. gallinarum, amebae found in avian hosts, contain four nuclei per cyst.  Entamoeba invadens, a species that is potentially pathogenic in some reptiles, and E. ranarum, a pathogenic ameba in amphibians, are both species which form quadrinucleate cysts.  Entamoeba histolytica, the intestinal ameba that is routinely capable of causing clinical illness in humans and other primates, forms quadrinucleate cysts in its life cycle.

    Various species of amebae have been described that live in close associations with animals, including humans.  Most of these relationships between amebae and animals are examples of commensalism, because the amebae do not appear either to benefit or to harm the animal in any measurable way.  Parasitic/commensal amebae typically inhabit the digestive tract of their host, with most amebae dwelling in the lumen of the large intestine.  Entamoeba gingivalis trophozoites live in the mouth, gums, and tonsils of mammals, including humans, dogs, and cats.  Some species of Entamoeba, such as Entamoeba bovis, inhabit the rumen of ruminant mammals; rarely, amebae that are morphologically identical to E. bovis have been reported to cause serious invasive illness in some ruminants other than cattle.  Entamoeba histolytica in mammals, as well as E. invadens in certain reptilian hosts, has the ability to spread from the large intestine to extraintestinal tissues.  Free-living amebae that may under certain circumstances cause opportunistic infection typically invade locations outside of the digestive tract, such as the CNS and the eye.

    Much of the interest in the biology of the amebae has focused on elucidating the mysteries of amebic pathogenicity and virulence.  Most of this research is directed at E. histolytica, because it is a human/primate pathogen, although E. invadens in reptiles and E. ranarum in amphibians have attracted the attention of some veterinary researchers working to solve problems in the captive rearing of terrestrial poikilotherms.  Molecular biologists have begun to offer fresh explanations for some of the classic problems in the biology of the amebae related to disease in humans and animals. 

Morphology of Entamoeba histolytica

    There are two distinct, persistent stages in the life cycle of Entamoeba histolytica that are visible in feces: (1) the trophozoite, the uninucleate, self-motile, feeding, and reproducing form of the parasite, and (2) the cyst, consisting of a quadrinucleate syncytium surrounded by a protective outermost hyaline layer.  Other transient, short-lived life cycle stages for E. histolytica, such as the metacyst (the syncytial mass that remains after excystment in the small intestine) and the so-called "precyst" (the rounded uninucleate-binucleate trophozoite initiating encystment), are mentioned in detailed accounts of the life cycle of this parasite.  A description of the physical appearances of trophozoites and cysts of E. histolytica needs to consider the two main physical components of both of these stages: (1) the nucleus, and (2) the cytoplasm.  The fragility of amebae, especially trophozoites, and the difficulty encountered in viewing internal structures in live amebae using routine light microscopy, has led to the use of several preservation/staining techniques in assisting the long-term study of the morphology of Entamoeba histolytica stages using the light microscope.

    The nucleus of the typical E. histolytica trophozoite is spherical in shape, and has a variable position in the cytoplasm.  The diameter of the trophozoite nucleus is approximately 4-7 µm in diameter, which is usually 1/3-1/5 the greatest width of the entire trophozoite.  In stained preparations, made from freshly-collected, properly processed fecal specimens, the nuclear heterochromatin (dense, inactive chromatin) is distributed evenly in minute granules along the inner surface of the nuclear membrane and in a finely pinpoint, centrally-located karyosome (= endosome).  Some of the variation that has been described in the appearance of the nuclear heterochromatin in Entamoeba histolytica trophozoites probably results from the reproductive status of individual trophozoites, as well as delays in specimen processing after passage of feces, fecal chemistry, improper specimen fixation, etc.  As the nucleus of a trophozoite prepares for cell division, it increases in size, varies in shape (less spheroid, more ovoid-ellipsoid), the peripheral heterochromatin becomes thicker, and the karyosome may appear as several coarse fragments.  The cysts of E. histolytica contain 4 nuclei when completely developed; forms that are intermediate between the trophozoite and the mature cyst have fewer nuclei.  The morphology of nuclei of cysts of E. histolytica is generally similar to that of trophozoite nuclei, although nuclei in cysts are frequently ellipsoid-ovoid in shape, due to pressure from large adjacent cytoplasmic inclusions (chromatoidal bodies, glycogen-containing vacuoles, ingested red blood cells, etc.).  Nuclei in mature cysts of E. histolytica are typically equal in size.  The karyosomes of cyst nuclei of E. histolytica are more frequently fragmented than are the karyosomes of trophozoites.  The parasitic fungus Nucleophaga invades the nuclei of amebae, including E. histolytica, and may affect the normal appearance of this structure.

    E. histolytica trophozoites typically have a cytoplasm that appears as two distinct regions: an outer clear ectoplasm and the larger inner, granular endoplasm.  The division between the ectoplasm and endoplasm is not as distinct in stained specimens as it is in fresh living material.  In fresh living material, clear, broad, finger-like ectoplasmic pseudopodia (known as lobopodia) may be quickly extended from the trophozoite; other filamentous extensions, known as filopodia, extend from the ectoplasm and may assist the trophozoite in making contact with other cells.  Trophozoites of E. histolytica have a uniform, finely granular endoplasm when viewed after staining with most routine procedures.  Occasionally starch grains are visible in vacuoles in the endoplasm of E. histolytica trophozoites, although large particles of ingested particulate matter are less common in E. histolytica trophozoites than those of other amebae.  Glycogen is distributed in granules throughout the endoplasm of trophozoites.  Variable numbers of ingested erythrocytes may be seen in the cytoplasm of E. histolytica trophozoites.  Parasitic fungi, such as Sphaerita, are often observed in the cytoplasm of amebic trophozoites, including E. histolytica.  Electron microscopy has revealed that ribosomes in the cytoplasm of E. histolytica trophozoites are arranged in a pattern of helical arrays and crystalline arrangements of these helical arrays.  Most authorities report that E. histolytica lacks mitochondria and the enzymes associated with mitochondrial metabolic reactions, yet recently genes coding for mitochondrial proteins have been identified in the E. histolytica genome and a mitochondrion/hydrogenosome-like structure (known as either a "crypton" or a "mitosome") has been found in the cytoplasm of E. histolytica trophozoites.  The crypton/mitosome cannot be viewed using routine light microscopic techniques and the exact function of this structure is not entirely known.

    There is no distinction between endoplasm and ectoplasm in the cytoplasm of E. histolytica cysts.  The cytoplasm of the E. histolytica cyst frequently contains dark staining, variably shaped (often rod-like) structures known as chromatoidal bodies which are typically not found in the trophozoite.  These dark-staining structures contain some glycogen and, when viewed using the electron microscope, are similar in appearance to the crystalline aggregates of ribosomes seen in trophozoites.  Other areas of glycogen accumulation in the cytoplasm may be visualized in cysts when they are treated with certain stains, such as iodine.  Cysts of E. histolytica possess a thin outermost protective hyaline membrane.

    Dimensions of the stages of E. histolytica vary considerably.  Trophozoites range between 12-60 µm across through their longest axis, with some trophozoites having been reported to be as large as 90 µm across and others as small as 8 µm across.  Most E. histolytica cysts are 10-20 µm in diameter.

    Morphologically, trophozoites and cysts of E. dispar cannot be readily distinguished from those of E. histolytica.  E. dispar and E. histolytica have differences in their nucleic acid sequences that permit specific identifications of these organisms to be made.  E. dispar is believed to be nonpathogenic in humans.  Other amebae that live in the intestine of humans differ in appearance from E. histolytica.

Distribution of Entamoeba histolytica

    The distribution of E. histolytica is dependent on the presence of susceptible hosts for the survival of trophozoites and environmental conditions that are favorable for the survival of cysts.  Humans are the most important definitive hosts for E. histolytica, although naturally-occurring infections in other animals, such as cattle, pigs, dogs, cats, and rats have been reported.  Nonhuman primates are commonly found infected with E. histolytica.  Several mammalian species, such as the hamster, jird, mouse, guinea pig, and rabbit have been found to be suitable experimental hosts for E. histolytica.  The hamster and the jird are especially useful as animal models for the study of amebiasis because the parasite is capable of spreading to the liver in these hosts.

The hyaline membrane that covers the cysts of E. histolytica is protective against some external environmental conditions, but their survivability is enhanced by certain situations.  Moisture, shade, and warm temperatures  favor the survival of E. histolytica cysts.  E. histolytica cysts are not able to withstand temperatures above 50^oC (122^oF), and they are not very resistant to desiccation.  In damp soil, cysts of E. histolytica survive for at least 8 days.  E. histolytica cysts in untreated water at room temperature may live from 9-30 days, while cysts kept in untreated water at a temperature of 4^oC may survive for approximately 3 months.  Survivability of E. histolytica cysts in water is decreased as the temperature of the water is increased and as the number of bacteria in the water is also increased.  The UV radiation in direct sunlight can be lethal to E. histolytica cysts depending on the duration of exposure.

E. histolytica has a cosmopolitan geographic distribution, because of the ubiquity of susceptible hosts and the ability of this organism to survive a wide range of naturally-occurring environmental conditions.  The environmental conditions that exist in the tropical subtropical regions of Earth are the most favorable for survival and transmission of E. histolytica, and most of the cases of amebiasis that occur in humans are acquired in these regions.

The Life Cycle of Entamoeba histolytica

    E. histolytica trophozoites typically live in the lumen and crypts of the large intestine, feeding on mucous secretions, bacteria, sloughed epithelial cells, and other intestinal contents.  These stages will reproduce asexually, via binary fission.  In the presence of unknown cues, the trophozoite will transform from an active, motile stage into a spherical uninucleate form, known as a "precyst", that usually contains a large glycogen mass in its endoplasm.  A clear hyaline membrane (a cyst wall) is secreted by the parasite around its outermost cellular surface.  The nucleus of the precyst undergoes karyokinesis, until four nuclei are present in the mature cyst.  The cyst exits the host via feces.  If the cyst is ingested and swallowed by a suitable definitive host, physiological cues provided by the pH of the gastric juice and the near-anaerobic environment of the stomach and intestine trigger the breakdown of the cyst wall by the parasite from the inside.  Excystation (the process by which the actively feeding/moving form of the parasite emerges from the cyst) is completed in the ileum, and the quadrinucleate syncytium (the metacyst) released from the cyst almost immediately undergoes division, yielding 4 so-called metacystic trophozoites.  The metacystic trophozoites are carried quickly to the large intestine, where they begin to feed and reproduce.  A new generation of cysts can usually be detected in the feces 2-5 days after the ingestion of viable E. histolytica cysts.

    In most human infections, E. histolytica trophozoites remain in the intestinal lumen, and do not cause any detectable illness.  Occasionally, trophozoites of E. histolytica enter the protective mucus layer covering the apical surfaces of mucosal epithelial cells in the large intestine; trophozoites that are able to penetrate the mucus layer may make direct contact with intestinal epithelial cells and this cytoadherence induces changes in the epithelium.  The changes in the mucosal epithelium make it possible for some trophozoites to enter into the mucosa, remaining between the epithelial cells and the basement membrane.  After entering the mucosa, these histozoic trophozoites are capable of feeding on host cells and will typically undergo asexual reproduction in the wall of the host's large intestine.

    After a population of E. histolytica trophozoites is established in the tissues of the wall of the large intestine, the organisms can move from this location to extraintestinal anatomic sites in the host.  E. histolytica trophozoites are able to spread from the original point of tissue invasion to other tissues via two primary routes: (1) direct spread from the infected tissue to adjacent uninfected tissue (extension), and (2) travel via the blood vessels/lymphatics to distant tissues (hematogenous spread).  In tissue sites, trophozoites of E. histolytica do not transform into cysts.  The most common organ invaded by E. histolytica, other than the large intestine, is the liver.

    Trophozoites that are ingested by suitable definitive hosts are not believed to be capable of producing infection, due to the effects of gastric juice on these unprotected stages.  Introduction of live trophozoites into the body per rectum, via unsterilized endoscopes/surgical equipment, contaminated enema apparatus, and multiple partner anal intercourse, can lead to human infection with E. histolytica.

The Effects of Entamoeba histolytica on the Definitive Host

    It is widely reported that most infections of humans with E. histolytica are asymptomatic.  This fact contradicts the reputation of E. histolytica as a pathogen.  The traditional description of E. histolytica as a biological species has been based on the physical appearance of the organism as seen with the light microscope (i.e., its phenotype).  In asexually-reproducing organisms with relatively simple structure, species descriptions based on phenotype alone may not sufficiently recognize the true biological diversity that exists within a population.  Progress in biology has yielded tools that permit more advanced phenotypic studies (such as biochemical composition), as well as genotypic investigation, of organisms that possess structural simplicity.  As use of these new methodologies has been directed at E. histolytica, awareness has increased concerning the biological variation that exists among populations of amebae that fit the morphologic definition of this species.  There are amebae that are morphologically consistent with the description of E. histolytica yet do not produce significant disease in humans.  One species of ameba that is indistinguishable from E. histolytica based on morphology alone is Entamoeba dispar.  E. dispar has been demonstrated to be genetically distinct from E. histolytica.  It is possible that comparative genetic analysis of other forms of E. histolytica may result in the recognition of additional species with varying abilities to cause disease in humans.  Prior to the molecular recognition of E. dispar as a distinct species, researchers had collected E. histolytica from humans and had used this material to determine biochemical profiles of E. histolytica based on the electrophoretic patterns of four enzymes: (1) glucose phosphate isomerase, (2) phosphoglucomutase, (3) L-malate:NADP⁺ oxidoreductase, and (4) hexokinase.  These different patterns of enzymatic combinations are called zymodemes (= isoenzyme profiles).  There are 8 virulent zymodemes and 11 nonvirulent zymodemes of E. histolytica that have been identified in amebae collected from humans.  Based on survey data that include zymodeme analysis of amebae found in human stools, strains of E. histolytica possessing zymodemes recognized to be associated with virulence account for only 0-3% of human infections.  There is controversy surrounding the issue of whether or not E. histolytica trophozoites can transform from a nonpathogenic zymodeme into a pathogenic zymodeme.  Continuing research indicates that E. histolytica strains possessing different zymodemes are genetically different from each other.  Analysis of 3000 clinical isolates of E. histolytica detected E. histolytica with virulent zymodemes in 25 asymptomatic patients, while E. histolytica possessing nonvirulent zymodemes were never found in the feces of patients with symptoms of amebiasis.

    There are four explanations for the presence of organisms that are morphologically indistinguishable from E. histolytica in the feces of humans who are asymptomatic for amebiasis: (1) The organisms are E. dispar, a nonpathogenic ameba that is morphologically identical to E. histolytica; (2) The organisms are stages of one of the 11 nonvirulent zymodemes of E. histolytica; (3) The organisms are stages of a virulent zymodeme of E. histolytica that have only recently established an infection which has not yet resulted in tissue damage sufficient to result in symptoms; (4) The host is infected with E. histolytica but has developed protective immunity sufficient to prevent the development of clinical amebiasis.

    Direct contact between the cell membranes of E. histolytica trophozoites and host cells (i.e., cytoadherence) is necessary for the invasion of host tissues to occur.  Most E. histolytica trophozoites live in the lumen of the large intestine, barred from making direct contact with host cells by the presence of a protective layer of mucus that covers the apical surfaces of intestinal cells.  E. histolytica trophozoites have been found to possess a substance in their cell membranes that binds to mucins produced by colon mucosal cells (goblet cells), thus permitting these trophozoites to attach to the intestinal mucus layer.  Further research has demonstrated that this same substance in the cell membranes of E. histolytica trophozoites is also important in the in vitro adherence of E. histolytica trophozoites to cultured cells.  The ability of this substance to bind to cells could be inhibited by the addition of millimolar concentrations of either galactose or N-acetyl D-galactosamine to the culture medium.  Further investigations directed at explaining why these compounds blocked E. histolytica cytoadherence led to the discovery of a heterodimeric glycoprotein, often referred to as the Gal/GalNAc adherence lectin, in the cell membrane of E. histolytica trophozoites.  The heavy subunit (170 kDa) of this heterodimer has been demonstrated to be antigenic; it can stimulate T cells to produce gamma interferon, and 90% of human E. histolytica immune sera react with this molecule.  The Gal/GalNAc-inhibitable glycoprotein (= Gal/GalNAc-inhibitable adhesin) is important in the adherence of E. histolytica trophozoites to both the colonic mucus layer as well as large intestinal cells, and inhibition of this glycoprotein by mucins, antibodies, and other host produced substances and inflammatory cells may be important in host defense against E. histolytica.  Other substances that contribute to the adherence of host cells to E. histolytica trophozoites have been identified since the discovery of the Gal/GalNAc-inhibitable glycoprotein.  Several bacteria have been found that also adhere to the Gal/GalNAc-inhibitable glycoprotein of E. histolytica trophozoites.  Bacteria have been found to influence the virulence of E. histolytica trophozoites cultured in vitro, and it is possible that some of the ability of E. histolytica to cause disease in humans is influenced by interaction between trophozoites and intestinal bacteria.

    The recognition of the importance of direct trophozoite-host cell contact in triggering tissue damage in the host infected with E. histolytica has led researchers to focus on the molecular composition of the surface of E. histolytica trophozoites and its association with the virulence of this parasite.  E. histolytica trophozoites (as are most other eukaryotic cells) are coated with glycocalyx that is in close association with the underlying cell membrane.  Two glycolipids (lipophosphoglycans, LPG) have been detected that span the glycocalyx-cell membrane of E. histolytica trophozoites; one of these glycolipids is attached to a peptide.  During axenic in vitro cultivation, populations of cultivated E. histolytica trophozoites lose their glycocalyx layer and coincidentally experience a gradual reduction in virulence.  Studies have indicated that there is a direct relationship between the presence of LPG in E. histolytica glycocalyx and virulence of this parasite.  Techniques used to restore virulence in cultivated E. histolytica trophozoites result in the reappearance of surface LPG.

    The host cell that is in direct contact with the trophozoite of E. histolytica typically undergoes death.  The substances that contribute to cytoadherence of E. histolytica and host cells do not directly cause necrosis of the host cell however; other factors produced by the E. histolytica trophozoite have been identified that are believed to be capable of triggering host cell death.  The primary candidate for a substance in the cell membrane of E. histolytica that results in host cell death is ameba pore forming protein, also known as amebapore.  Amebapore is released from the trophozoite into the space between itself and the host cell, resulting in the formation of ion channels that induce rapid, lethal permeability of the host cell membrane to K, Na, and Ca.  The list of molecules, such as amebapores, that are candidates for substances that contribute to the contact-dependent killing of host cells by E. histolytica is growing.  Proteolytic enzymes (hyaluronidases, pepsin, trypsin, gelatinase, cathepsin, various hydrolases, collagenase, cysteine proteases) have been identified in trophozoites of E. histolytica, and these enzymes may assist the parasite in invading host tissues (by breaking down intercellular adhesive proteins) and killing host cells (by directly damaging cellular architecture/function and/or by initiating cellular "suicide", referred to as apoptosis by some researchers).  Most of these enzymes seem to be membrane-bound in E. histolytica, and are only able to function when the trophozoite is in direct contact with the host cell.  Contact- dependent cytolysis of host cells by E. histolytica trophozoites requires the presence microfilaments in the amebae, occurs at an optimum temperature of 37^oC, and does not take place in vitro at temperatures below 25^oC.

    Several investigations have been conducted to determine whether or not E. histolytica trophozoites are capable of secreting cytotoxic substances into host tissues and thus kill large numbers of host cells without making direct contact.  Although various substances with demonstrated cytotoxic abilities have been identified in sonicated preparations of E. histolytica trophozoites, none of these materials has ever been found in secretory material prepared from E. histolytica.

    Host inflammatory responses are initiated when the trophozoites of E. histolytica begin to invade tissue, and some investigations have been directed at determining the effects of the host inflammatory response on the progression of amebic tissue invasion.  Most of the research in this area has focused on the appearance of neutrophils in the intestinal wall during amebiasis.  E. histolytica trophozoites in axenic culture are capable of killing human neutrophils in vitro, with no subsequent damage noted in the trophozoites.  Neutrophils are attracted to E. histolytica trophozoites.  The release of neutrophilic granule contents can damage adjacent host cells, and it is possible that neutrophil destruction/degranulation in response to E. histolytica causes host cell death.  There are numerous mechanisms present in human cells, however, that are designed to prevent damage of the host's tissues from its own neutrophils, and the role of neutrophils in the progression of tissue amebiasis remains uncertain.  Eosinophils also appear as part of the host cellular response to E. histolytica trophozoites in tissues; Charcot-Leyden crystals , which form in fluids and tissues where eosinophils have undergone degranulation, are common in the feces of individuals with intestinal amebiasis.  Degranulation of eosinophils releases major basic protein (MBP), a substance that has been demonstrated to be cytotoxic to both parasite and host cells.  Despite the fact that eosinophils have been recognized to play an important role in the pathophysiology of other parasitic diseases, very little research has focused on the contributions of MBP and other eosinophil-derived materials to the development of amebiasis.

    Some other infectious agents that damage the mucosa of the large intestine, such as whipworms (Trichuris trichiura) and the ciliate protistan Balantidium coli, have been found in association with E. histolytica.  It is tempting to suggest that such organisms act synergistically with E. histolytica to invade the large intestinal mucosa and cause ulceration.

    The effects of E. histolytica trophozoites invading the mucosa/submucosa of the large intestine, and the accompanying host inflammatory response, lead to the formation of an ulceration in the wall of the large intestine.  As the ulcer spreads deeper into the wall of the large intestine, the capillaries supplying blood to the mucosa-submucosa lose their structural integrity, resulting in bleeding into the intestinal contents.  Damage to local blood vessels and thrombosis of capillaries and venules results in further mucosal damage from ischemia.  The initial (primary) ulcer that develops is usually found in either the region of the cecum, appendix, and adjacent ascending colon or the sigmoidorectal area.  Other secondary ulcerations may develop, typically separated from the primary ulcer by areas of undamaged mucosa.  Intestinal ulcers caused by E. histolytica can progress deep enough in the intestinal wall to result in intestinal perforation and subsequent peritonitis.  Granulomatous inflammation, with eosinophilic and/or lymphocytic infiltration, develops in some amebic ulcers, resulting in the formation of a firm, nodular, fibrous mass known as an "ameboma" due to its resemblance to colorectal carcinoma.

    Once amebae have established themselves as aggressive tissue invaders in the wall of the large intestine, amebic trophozoites are able to spread to other tissue locations.  Spread to other organs takes place through direct movement of trophozoites from one area to an adjacent site or through into blood/lymphatic vessels and subsequent hematogenous dissemination.  The most common extraintestinal site invaded by E. histolytica trophozoites is the liver; other less frequent extraintestinal tissue locations for E. histolytica trophozoites include the skin (amebiasis cutis), lungs, and brain.  Many individuals who develop extraintestinal amebiasis have no prior history of intestinal amebiasis.  In extraintestinal sites, the lytic necrosis associated with the presence of the E. histolytica trophozoites is referred to as an amebic abscess.

    Clinical signs/symptoms of intestinal amebiasis in humans include diarrhea (with or without the presence of grossly visible blood/pus in the stool), abdominal cramps, vague abdominal discomfort, generalized malaise, weight loss, anorexia, and lethargy.  The severity of these symptoms varies, depending usually on the extent of intestinal damage; extensive intestinal lesions can result in severe illness manifested by dysentery, rapid weight loss, dehydration, fever, chills, and hypotension.  Abdominal pain may be severe enough to provoke nausea and vomiting, and may mimic that of appendicitis if the ileocecal region is involved.  In other cases, the symptoms may mimic those of peptic ulcer and/or cholecystitis.  Obvious dysentery is most frequently observed in cases where there is amebic ulceration in the sigmoidorectal colon.  The onset of clinical signs may occur slowly, with overt, clinically-significant disease being delayed for weeks to months after infection with the parasite is first established.  Death is common in cases of severe amebic dysentery, even in those cases that receive treatment.

    The clinical signs of amebic abscess are dependent on the location of the abscess.  The most common clinical finding in patients with hepatic amebic abscess is right upper quadrant pain, hepatomegaly, often accompanied by fever without jaundice.  Less frequently observed clinical symptoms of hepatic amebiasis include weakness, diarrhea, chills, nausea and vomiting, and jaundice.  Serum levels of enzymes that are frequently elevated when hepatic inflammation occurs are typically within normal limits in cases of E. histolytica liver abscess.

Detection of Infection and Diagnosis of Disease Caused by Entamoeba histolytica

    Infection with E. histolytica can be detected by (1) microscopic examination of a fecal specimen, (2) use of fecal antigen detection method, (3) serologic testing for presence of either antibodies against E. histolytica or antigens of the parasite, (4) cultivation of organisms from feces or other material collected from suspected case, (5) microscopic examination of processed aspirates, biopsies, and tissue specimens from intestinal ulcers and extraintestinal sites.

    Microscopic examination of a fecal specimen is the most common approach currently taken to detect E. histolytica infections in humans.  Amebic cysts can be detected in the feces of most infected individuals with uncomplicated intestinal amebiasis through the preparation of a direct fecal smear.  Concentration techniques, such as centrifugal flotation and ethyl acetate sedimentation can be useful in detecting small numbers of cysts in fecal samples.  The detection of amebic trophozoites in feces requires the use of special fecal specimen collection and processing; fecal smears must be made, permitted to dry, fixed in a suitable fixative solution (such as Schaudinn's fixative), and then are stained using an appropriate staining technique (such as Wheatley's trichrome technique).  Identification of parasitic stages in host feces requires knowledge of the comparative morphology of the parasitic/commensal protists of humans, in order to prevent both the misidentification of benign organisms as Entamoeba histolytica as well as the failure to recognize stages of E. histolytica that could be responsible for illness.

    Antibodies against E. histolytica can be detected using a variety of conventional methods.  Many individuals with mildly invasive intestinal amebiasis will not have detectable levels of antibodies against E. histolytica.  Parasite- specific antibody may not appear in the fluids of infected hosts for as long as a week after the onset of clinical signs of amebic abscess, but after this amount of time most individuals with extraintestinal amebiasis have detectable levels of anti-E. histolytica antibodies in their bloodstream.  The levels of specific immunoglobulins directed at E. histolytica are not useful in distinguishing current infections from past, resolved infections in most instances.  Recently, a serologic test designed to detect IgA against E. histolytica in the saliva of infected humans has been made commercially available.

    Cultivation of E. histolytica stages is not routinely performed by most diagnostic laboratories.  The cost, in both time and money, of attempting to cultivate the parasite is much higher than the relative cost of other diagnostic procedures, and in many cases these efforts are either unsuccessful or yield inconclusive results.

    Imaging techniques, such as ultrasound and CT scan, permit viewing of damage done by E. histolytica in some extraintestinal locations, such as the liver, but do not provide a specific identification of the organism involved in producing such damage.  CT scans may be useful in assessing the progress of treatment of amebic abscess.

Control and Prevention of Infection with Entamoeba histolytica

    Almost all cases of infection with E. histolytica result from the ingestion of viable cysts of this parasite.  Cysts are found in the feces of infected hosts and in/on materials contaminated with the feces of infected hosts.  Infection with E. histolytica can be prevented by blocking human oral contact with feces and/or objects contaminated with feces.  Sanitary disposal of human fecal material is paramount in preventing E. histolytica infection in humans.  Water used for drinking by humans should be protected from fecal contamination.  Municipal water treatment should include measures (including sedimentation, filtration, chemical treatment, etc.) that are adequate to kill and/or remove cysts of E. histolytica from the water supply.  Filtration, using either sand or diatomaceous earth, is effective in removing most E. histolytica cysts from water supplies.  Small quantities of water (mainly intended for personal use) can be made safe from E. histolytica by (1) boiling, (2) treatment with iodine (8 drops of 2% tincture of iodine/quart of water; 12.5 ml of saturated aqueous solution of iodine crystals/liter of water) or other approved materials (1 tablet of tetraglycine hydroperiodide/quart of water), (3) use of a portable filter with a pore size of less than 1.0 µm.  People should be educated about the risk that is posed to their health by water of unknown purity, including ice and water used in food preparation.  Travellers and other individuals with relatively short term exposure to E. histolytica should be aware that locally-produced pasteurized nondairy beverages, such as beer, have a relatively low risk of infectious disease transmission.  Sexual practices that can result in fecal-oral contact (such as oral contact of an individual with the penis of a male sexual partner previously engaged in unprotected anal sex) carry a substantial risk of acquiring infectious diseases, including amebiasis, and education directed at high risk groups for this behavior is essential in preventing disease transmission in this manner.

   Cultivation of fruits and vegetables should, if possible, not involve the use of human fecal material/untreated municipial waste as fertilizer.  Vegetables and fruits possibly contaminated with human feces should be, at the least, thoroughly washed before consumption.  If the water used to wash fruits and vegetables is itself contaminated with human fecal material, then washing may actually result in the transmission of E. histolytica.  After washing, fruits and vegetables should be allowed to dry completely before being eaten, since desiccation is lethal to E. histolytica cysts.  Immersion in 5% acetic acid (a concentration of acetic acid that is approximately the same as that of vinegar) kills cysts of E. histolytica without adversely affecting flavor and appearance of fresh vegetables.  Cooking of vegetables to a temperature of 50^oC (122^oF)is a good way to kill any E. histolytica cysts that might be present on them.

    Human hands that are contaminated with cysts of E. histolytica can transmit these stages to other surfaces, and individuals involved in food handling/processing should practice effective personal hygiene, including washing their hands regularly with soap and hot water, to reduce the risk of transmitting E. histolytica and other fecal-borne pathogens to other people.

    Per rectum transmission of E. histolytica from person to person can be avoided by proper heat/chemical sterilization of surgical instruments and colonic irrigation/enema apparatus, by the use of disposable gloves/proper cleansing of hands between serial rectal examinations, and by following hygienic guidelines designed to prevent infectious disease transmission during anal intercourse.

Nonpathogenic Amebae Found in Humans

   Several other species of amebae are known to inhabit the human digestive tract, yet unlike E. histolytica these species do not cause disease in humans.  These commensalistic amebae typically live in the large intestine, where they feed on intestinal contents.  They are important because these amebae can be misidentified during fecal examinations as stages of E. histolytica.  Nonpathogenic amebae in humans are transmitted via the same routes that Entamoeba histolytica is transmitted, and the presence of these organisms in a person indicates that ingestion of a substance contaminated with feces has occurred.

   Entamoeba hartmanni
        Entamoeba hartmanni is very similar in morphology to E. histolytica; in the past, E. hartmanni was referred to as "small race" E. histolytica, due to the fact that it appeared to be a smaller form of E. histolytica.  E. hartmanni is smaller than E. histolytica, with trophozoites of E. hartmanni measuring 4-12 µm and cysts being 5-10 µm in diameter.  The chromatin lining the nuclear membrane of E. hartmanni is relatively coarse and the karyosome is large and typically eccentric in location.  E. hartmanni trophozoites do not ingest erythrocytes.  Chromatoidal bodies are usually very numerous and very large in E. hartmanni cysts, frequently obscuring the cyst nuclei.  E. hartmanni has never been demonstrated to cause disease in humans.

    Endolimax nana
        Endolimax nana is a common cosmopolitan ameba that lives as a trophozoite in the lumen of the large intestine of humans and other primates.  E. nana is smaller than Entamoeba histolytica, with trophozoites that are 8-10 µm across through their widest point and spherical-ellipsoid shaped, mature quadrinucleate cysts that are 5-14 µm across.  Endolimax nana trophozoites do not ingest erythrocytes.

   Entamoeba coli
        Trophozoites and cysts of Entamoeba coli typically have dimensions that are significantly greater than  those of E. histolytica; cysts are 10-30 µm in diameter and trophozoites are 15-50 µm across.  The mature cysts of Entamoeba coli contain eight nuclei (rather than the four seen in mature Entamoeba histolytica cysts), the chromatin located along the nuclear membrane is coarse and unevenly distributed, and the karyosome is usually large and eccentric in location.  Entamoeba coli is common in a wide range of mammals, including primates, rodents, and domestic/wild ungulates.

  Iodamoeba bütschlii
        The trophozoites and cysts of Iodamoeba bütschlii are both uninucleate, with a nucleus that has a large karyosome and negligible chromatin distributed along the nuclear membrane.  I. bütschlii cysts have a prominent mass of glycogen that stains dark brown with iodine (hence the genus name).  Trophozoites of I. bütschlii range from 8-20 µm across, and cysts measure 5-18 µm across.  I. bütschlii lives in the intestine of other mammals, including pigs and nonhuman primates.

Free-living Amebae and Disease

        Most amebae are free-living aquatic organisms that typically live among microscopic organisms/non-living organic material in stagnant bodies of water, soil, etc.  Some genera of free-living amebae, such as Naegleria, Acanthamoeba, and Balamuthia have species that have been identified as causing opportunistic disease in humans and animals.  The free-living amebae that have been recognized as causative agents of opportunistic infections have uninucleate trophozoites and cysts in their life cycles.  In aquatic environments, trophozoites of Naegleria will develop flagella.  When they are found in hosts, the trophozoites of these amebae are found invading tissues away from the gastrointestinal tract and do not form cysts in animal tissues.

    Acanthamoeba spp. have been found in corneal ulcers in humans with contact lenses.

 A few free-living amebae have been found to house potentially pathogenic microorganisms within their cytoplasm.  The etiologic agent of legionellosis (Legionnaire's disease), Legionella pneumophila, has been found surviving and multiplying within the cytoplasm of free-living amebae living in the water associated with building cooling systems.

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