Amebiasis: clinical and laboratory perspectives.

Volume 28, Issue 5,6 (1991)

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Amebiasis: Clinical and Laboratory Perspectives Maria Reitano, Ph.D., Joseph R. Masci, M.O., and Edward J. Bottone, Ph.D.

Critical Reviews in Clinical Laboratory Sciences Downloaded from informahealthcare.com by Nyu Medical Center on 01/06/15 For personal use only.

ABSTRACT Entamoeba hisrolyrica,the premier intestinal protozoan, has traversed time in its relentless quest for survival in its dichotomous role of parasite and pathogen. Enigmatic in its transition from human intestinal commensal to invader of human tissue, diverse in its pathogenicity for the human host, and intricate in its bacterial interrelationship in the bowel, E . hisrolyrica has become the focal point of intensive investigation in its basic biology underscoring human pathogenicity. This review will focus on facets of cell biology, pathophysiology, clinical, therapeutic, and epidemiologic, correlates, along with diagnostic modalities and future research trends. Key Words: parasite, protozoa, Entamoeba hisrolyrica, amebiasis, protozoan, amebae

1. HISTORICAL ASPECTS The most significant of the intestinal protozoa, Entumeba hisrolyrica is both a fascinating and elusive microbial entity that has been the focus of renewed interest regarding its mode of pathogenesis, the clinical spectrum of infection, and host response, both humoral and cell-mediated, that underscores containment of invasive infection. On a global scale, approximately 480 million people harbor the parasite and 10% of those infected go on to develop overt symptomatology. Furthermore, amebiasis, the disease state caused by E. hisrolyrica, ranks third among worldwide parasitic causes of death, trailing malaria and schistosomiasis. As a consequence of intensified investigative attention, especially within the last decade, a comprehensive review of the pathogenetic, clinical, diagnostic, and epidemiologic correlates regarding this intriguing protozoan is presented to render a state-ofthe-art perspective for this age-old scourge of humans as it tends its secrets at the close of the 20th century. Dysentery, caused by E. hisrolyrica, has been a topic of great concern, and documented cases date back to the time of Hippocrates (460-377 B.C.).’ Additional descriptions regarding the disease state can also be found in the Old and New Testaments (I1 Chronicles and Deuteronomy). Symptomatic disease caused by E. hisrolyrica is primarily a disease of the bowel, referred to as amebic dysentery. The initial association of pseudopodial protozoa as the causative agent(s) of gastrointestinal disease occurred when amebae were identified in

Maria Reitano, Ph.D., Assistant Professor, Department of Biology, St. Joseph’s College, Patchogue, N.Y.Joseph R. Masci, M.D., Assistant Director of Medicine, City Hospital Center at Elmhurst. Elmhurst, N.Y.;Associate Rofessor of Clinical Medicine, Division of Infectious Diseases, Department of Medicine, Mount Sinai School of Medicine, New York, N.Y.Edward J. Bottom, Ph.D., Director, Clinical Microbiology Laboratories, The Mount Sinai Hospital, New Yo&, N.Y.;Professor of Microbiology and Professor of Pathology, Mount Sinai School of Medicine, New York, N.Y.


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stool specimens from a child who died of infantile diarrhea.3In 1875, Fedor Losch4rendered the first specific description and identification of E . histolytica. Amebae were visualized in great numbers in the stools of a dysenteric patient who expired and, upon autopsy, the same organisms were found in ulcerated lesions of the intestine. Careful and detailed drawings of these organisms accompanied Losch's report, in which he designated them Ameba coli. Losch further attempted to study the in vifroeffect of various drugs on the viability of these organisms, and successfully reproduced dysentery in dogs fed stool specimens containing amebae. Based upon these early and imaginative studies, the role of ameba (E. histolytica) in the etiology of dysenteric disease was solidified. Kartulis5 and Hlava6 who reported on 150 and 60 cases of dysentery, respectively, conf m e d Losch's observations. In the U.S. the stellar publication by Councilman and LaFleur,' documenting the clinical and histologic presentation of 14 patients with intestinal amebiasis at Johns Hopkins Hospital, reaffirmed amebiasis as a specific disease entity and cited water as the source of infection. Subsequent studies were directed toward a better understanding of the specific nature of dysentery, including the recognition by Shigas of bacillary forms as a distinct cause of dysentery. Morphologic studies of amebae9 began to indicate a difference between amebae isolated from healthy patients as contrasted to those isolated from symptomatic individuals. Schaudinn'O concluded that there were two different kinds of ameba, pathogenic and nonpathogenic, although at that time the carrier state of E . histolytica was not appreciated. This conclusion eliminated the growing confusion regarding the role of amebae in individuals without dysentery, but conflicted with that of Quincke and Roos9 and Huber," who held that the cyst forms observed in stools of diarrheagenic patients were the etiologic agents of dysentery. Nearly a full decade was to pass before the work of Dobell'* and Craig," in the early 1940s, f i y established Entameba as the etiologic agent of amebic dysentery, and its morphology, clinical presentation, pathology, and therapy were clearly defined.

II. TAXONOMY AND BIOLOGY OF E. HlSTOLYTEA Protozoa are single-celled organisms that perform all necessary metabolic functions and undergo reproduction by fission. The phylum Protozoa is divided into four subphyla: Sarcodina (amebae), Mastigophora (flagellates), Ciliophora (ciliates), and the Sporozoa. Entameba histolytica, a member of the subphylum Sarcodina, is motile through extension of finger-like pseudopodia that serve as organelles of locomotion (Figure 1). Taxonomically separate from the free-living amebae, such as Naegluria and Acanrhumoeba that may also cause human disease, E . histolytica is the only proven pathogenic ameba that is host-bound in humans, causing both intestinal and extraintestinal disease.I4 E . histolytica also infects old-world primates. Stages characteristic of the life cycle of E . histolytica include a metabolically active, motile trophozoite, or vegetative form and an encysted form. The trophozoite may be encountered either as a small form ranging in size from 12 to 20 pm, usually associated with asymptomatic disease, or as a larger trophozoite up to 60 pm, usually implicated in both intestinal and invasive disease.I5 On the average, trophozoites measure 25 pm, with a single nucleus occupying approximately one fifth of that space. The nuclear morphology serves as the single most distinctive feature of the trophozoite, enabling the differentiation of E . histolytica from nonpathogenic amebae, including Entamoeba coli and Endolimar mna, which may also colonize the human gastrointestinal tract. Basically, the nucleus is a spherical organelle containing a single, compact karyosome that is often centrally located. The chromatin is evenly distributed around the nuclear membrane, imp*g a beaded appearance



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FIGURE 1. Light photomicrograph of E. hisrolytica trophozoite in culture. Motility is accomplished through the extension of a hyaline pseudopod. Phagocytized food particles are also evident.

to the inner lining of this enclosure. Sometimes, however, the chromatin is unevenly distributed around the inner periphery of the nucleus. Nuclear morphology is highlighted by phase microscopy or in preparations stained with trichrome and iron hematoxylin. The cytoplasm of the trophozoite is vacuolated and frequently contains ingested red blood cells, bacteria, and other debris. l6 Trophozoite reproduction is asexual, through binary fission. Cysts of E. histolyticu, as observed in fecal preparations, may be immature (precystic) and mature. Both forms are spherical in shape, but cyst size is variable.” The mature cyst may be less than 10 pm or may measure 12 to 15 Fm. Initially cysts of E. histolyticu were differentiated into a “small” and “large” race, respectively. However, antigen analysis has shown that “small race” E. histolytica are a new species, designated E . artmnni.I8 In E . histolyticu, the number of nuclei range from four in mature cysts to one or two in immature cysts. Except for size, the trophozoite nucleus and cyst nuclei are morphologically similar. Cytoplasmic components such as chromatoid bodies and glycogen vacuoles are characteristic of the cyst state (Figure 2). These morphologic features enable differentiationof E. histolyticu from nonpathogenic amebae. Mature cysts are infective upon passage from the body and, in a moist, cool environment, are able to survive for several weeks. The survival time for the fragile trophozoite, under optimal conditions, may approach several days (Table l).19-” Ingestion of the mature, quadrinucleated cyst through contaminated food and water, or by direct person to person passage, establishes infection.” Able to survive gastric acidity, the ingested cysts reach the lower part of the small intestine, where the cyst wall is enzymatically degraded through the action of alkaline digestive juices and the influence of amebic enzymatic activity. The quadrinucleated trophozoite is then liberated and undergoes asexual division resulting in


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FIGURE2. Trichrome stain of amebic culture showing spherical cysts of E . hisrolyticu, with characteristicnuclei containing centrally located karyosome and evenly distributed peripheral chromatin.

Table 1 Survival Time for E. histo/yticaTrophozoites and Cysts in Feces and Water Temwrature stage

Medium

37°C

20-25"C

5°C

Trophozoites cysts

Feces Feces Water

2-5 h 1-2d 2d

6-16 h 3-4d 10 d

48-96 h 14-40 d 42 d

Adapted from Reference 19.

eight small, uninucleate trophozoites that journey through the small intestine to their ultimate repository in the large intestine. On entering the bowel the trophozoite may embark on two overlapping courses. Initially, the trophozoites feed on bacteria and food particles, and continue to multiply until, for reasons not understood, encystment occurs. The young immature cyst, with a single nucleus, undergoes two nuclear divisions, giving rise to the mature, quadrinucleate cyst, which is shed in the feces. In asymptomatic carriage, colonization of the bowel is established without any apparent effect. Alternatively, however, the trophozoites are also capable of aggressive invasive interaction with colonic epithelial tissue, with ensuing ulcer formation attended by the potential for bowel perforation, peritonitis, and ameba formation. Extraintestinal dissemination of trophozoites to involve primarily the liver, and to a lesser extent the lungs, brain, and spleen, are noteworthy complications.



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The two different dimensions of E. histolyticu trophozoite activity, namely, invasive and commensal, are an enigma and of intense interest in current research. The impetus for such studies derives from an early hypothesis that two distinct species of Entumeba, virulent and avirulent, Factors that differentiate the two species include invasiveness primarily, in addition to morphology and geography. Ingested red blood cells are characteristic of invasive species that, as hypothesized, are restricted to endemic areas of disease. The worldwide distribution of trophozoites lacking ingested red blood cells, with a morphology similar to that of cysts, is characteristic of the noninvasive species of E. histolytica.

111. FACTORS RELATED TO THE PATHOGENESIS OF AMEBlASlS The clinical manifestations pronouncing the acquisition of E. histolytica are as much within the province of the parasite as governed by the host. While the recognition of asymptomatic gastrointestinal tract carriage of E. histolytica has been recorded since antiquity, little thought has been accorded the role of such amebic harmonious colonization in the subsequent expression of clinically apparent amebiasis. Unclear in this transition is the degree, if any, of responsibility on the part of the parasite as contrasted to host factors leading to disease. For instance, questions not elucidated are (1) is the commensal ameba the one accounting for disease, or is it the acquisition of a new strain that leads to disease?; (2) does the commensal strain, because of intrinsic or extrinsic (environmental) factors, undergo a structural or biochemical change leading to an agitated state in which mucosal invasion is a lateral event?; (3) if encystment characterizes the colonization state, then which indigenous tropism or environmental chemotactins drive the parasite to excyst and migrate (invade) beyond its colonic repository and perhaps never to encyst again? As a microbial pathogen of humans, E. histolyrica must follow the tenets laid down for other species whose mode of pathogenesis is invasion of mucosal surfaces. Thus, concepts of adherence, elaboration of “enterotoxins”, resistance to phagocytosis or intracellular degradation, and host immune surveillance, all play an intimate role in disease expression. These parameters, as they relate to E. hisrolyticu-inducedgastrointestinaldisease, have served as the focal point of recent intensive investigation and are reviewed below.

IV. BOWEL COLONIZATION BY E. HISTOLY’UCA As noted earlier, ingestion of cysts of E. histolytica is the inaugural step in the pathogenesis of amebiasis, either invasive disease or intestinal colonization. Once the cysts are acted upon by gastric hydrolytic enzymes, excystment takes place, liberating the trophozoite or vegetative form of the amebae. Irrespective of the outcome of liberating the trophozoite, disease or asymptomatic carriage, the parasite must persist in the large bowel and find its ecological niche. To achieve this desired state in the absence of a cell wall to provide adhesive factors, as often found in bacterial pathogens,24the limiting cytoplasmic membrane of the trophozoite must possess those adhesins necessary to establish colonization of the intestinal mucosa. The exterior-most surface of E. histolytica is encircled by a fibrillar coat termed a glycocalyx that is continuous with, and anchored to, the plasma membrane.25Numerous biological attributes have been suggested for the glycocalyx, including antigenicity (as evidenced by serologic reactivity), permeability of nutrients and exoproducts, cell to cell adhesion and interaction, chemotactic responses, phagocytosis of food particles such as bacteria and erythrocytes, and leukotoxic activity.


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Lushbaugh and MilleP showed cytochemically that the glycocalyx of E. histolytica trophozoites differs between strains derived from in vivo sources, as contrasted to that after axenic or monoxenic growth in culture. From tissue, the glycocalyx measured 20 to 30 p,m thick, and was comprised of high electronegative charge attributable to acid mucopolysaccharides, glycoproteins, or glycolipids. The preponderance of negative charge seemed to be associated with filamentous projections of the glycocalyx. These authors suggested that the increased size of the glycocalyx also noted with monoxenic-grown trophozoites may be induced by bacteria (as encountered in the gastrointestinal tract) and play a role in resistance to host humoral and cellular defense mechanisms. Interestingly, the glycocalyx of axenicgrown trophozoites was more compact and regular, and measured 5 p,m in thickness. Further supporting the role for the fully expressed glycocalyx in resistance to host defenses have been animal experiments showing decreased virulence subsequent to inoculation with E. histolytica trophozoites grown in the absence (axenically) of an accompanying bacterial flOra.26

In strains of E. histolytica in which virulence had been restored by the addition of cholesterol to axenic cultures or by two successive passages through hamster liver, the glycocalyx of these strains showed marked agglutinability in the presence of the lectin, Concanavalin A (Con A). These same strains showed increased toxicity toward host cells on direct contact, and were different antigenically from their avirulent cogeners. Increased agglutinability by Con A was associated with a well-developed glycocalyx, which had a higher capacity to bind the lectin because of the lack of a detectable repulsive charge at the glycocalyx s ~ r f ac e . ~ ’ To elucidate the mechanism of amebic adherence and its subsequent role in the lysis of target cells, Ravdin and GuerrantZ8studied the interaction of E. histolytica with Chinese hamster ovary (CHO) target cells and human erythrocytes. These investigators showed that the adherence of ameba to target CHO cells demonstrated a carbohydrate specificity and was a prerequisite to contact-mediated killing of the target cells. Lectin (Con A) binding of amebae supports their conclusion, as did inhibition of adherence with N-acetyb-galactosamine. Indeed, blocking of adherence uncoupled the amebic cytolethal process. Similar amebic carbohydrate receptors seem to be operative in adherence to human erythrocytes, irrespective of ABO blood group specificity. Prerequisite to amebic invasion of colonic epithelium is the adherence of E. histolytica to intestinal mucus and colonic epithelium. Adherence is achieved through an amebic surface protein with an affinity for the carbohydrates galactose or N-acetyl-D-galactosamineon the surface of target cells.28Through a series of imaginative approaches, Ravdin and colleagues29 and Petri et aL30 characterized the chemical composition of the carbohydrate receptors on the surface of cells that bind the E. histolytica adherence lectin. Using mutants of CHO cells deficient in an array of nitrogen- (N) and/or oxygen-linked (0)cell surface carbohydrates, Ravdin et al.2g,showed that the amebic adherence lectin bound preferentially to B 16 branched, N-linked carbohydrates lacking terminal sialic acid or fucose residues. Furthermore, using the technique of monoclonal antibody affinity chromatography, Petri and colleaguesm defined the subunit structure of the galactose/galactaseN-acetylgalactosaminebinding lectin for the amebic surface protein as a 170-kDa subunit enjoined by disulfide bonds to a smaller 35-kDa subunit. Inhibition of amebic binding by monoclonal antibody directed against the heavier unit supported its role as the major determinant mediating amebic adherence. Correlative to these observations, an earlier study by Petri et al.3‘ showed that individuals recovering from amebic liver abscess develop humoral and cell-mediated immune responses. In fact, the human immune sera contained blocking antibody against the galactoseor N-acetylgalactosamine-bindinglectin of E. histolytica that prevents the in vitro adherence



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of trophozoites to cultured CHO cells at 4°C. On the basis of these observations, this investigative group concluded that the humoral response to the binding lectin may be associated with protective immunity against invasive amebiasis. The early interactions between the ameba and host tissue resulting in adherence (colonization) of intestinal mucosa are still uncertain. For instance, what is the nature of the ameba surface upon excystment in the small and large bowel? Does movement through the bowel under peristaltic pressure allow significant time for the trophozoite to feed on bacteria and other nutrients and thereby acquire (synthesize) the glycocalyx necessary to establish colonization? Is this an orderly process? Is the ameba trophozoite focal in its movements, ingesting bacteria in a progressive manner as it approaches the mucosal surface? In this fashion the trophozoite could begin to elaborate its glycocalyx by simultaneously “clearing a path” to receptor sites on the mucosal surface. Indeed, Wittner and Rosenbaum3*have shown that virulent amebae require a direct association with viable bacteria. The provocative thought in these interactions is the possibility that certain bacterial species interact with the ameba surface and provide a mantle surrounding the trophozoite, which facilitates not only ingestion, but adherence to other bacteria and perhaps also to the mucosal surface itself. Bracha and c011eagues,~’-~~ in a series of illuminating experiments, showed that E . histolytica trophozoites are quite selective in their interactions with bacteria. Carbohydratebinding activity on the surface of trophozoites bound bacterial species such as Escherichia coli containing mannose-binding surface components; attachment occurred through mannose receptors on the trophozoite surface. Bacterial species lacking mannose-binding capability attached to the ameba surface only through the intervention of Con A as a ligand between the bacteria and ameba. Opsonization of bacteria also facilitates adherence, although not mediated through Fc receptors on the ameba surface. E . histolytica trophozoites may also bind to E. coli serotype 0 5 5 , which lacks mannose-binding capacity. In this instance, binding could be inhibited by galactose, lactose, and N-acetylgalactosamine, which is contained in the E. coli lipopolysaccharidemoiety. Thus the expression of the ameba surface components, such as its cytoplasmic membrane-associated carbohydrate (N-acety1galactosamine)-binding protein or its glycocalyx containing mannose-binding components, remain critical in the early host-parasite interaction following excystment. In the susceptiblehost, therefore, ingestion of cysts of E . histolytica may lead to symptoms, depending on the nature of the bacterial flora colonizing the human host. If mannose-binding species are present, these will attach to the surface of the ameba trophozoites as prerequisite to phagocytosis. Under such a premise, in the absence of the stereospecific bacterial species in the gastrointestinal tract of a given host, the liberated trophozoite could pass harmlessly through the intestinal tract and be eliminated. Does specific bacterial deprivation result in a “nutritional immunity” against E. histolytica? Conversely, does persistence of E. histolytica in the bowel occur in those individuals whose bacterial flora presents a suitable substrate for the maintenance of parasitism? Strength for this assumption is derived from the failure of cell-free filtrates of the requisite bacteria to support in vitro cultivation of amebae or to enhance ~irulence.~’ Once having established residence in the bowel, several factors conspire to determine the host outcome. In Ravdin’s definitive review of the pathogenesis of disease caused by E. h i ~ f o l y f i c ahe, ~postulates ~ a sequence of events beginning with colonization of the gut by a virulent amebic strain, and culminating in a contact-mediated disruption of the intestinal cells and mucosa under the influence of amebic enzymes. The results of these events are colonic ulcer formation and deep tissue and/or distant organ, especially liver, invasion. Germane to this concept of E. histolytica pathogenesis is the definition of a “virulent” strain as contrasted to a strain that establishes colonization but renders the host asymptomatic. Early studies postulated that virulent E . histolytica could be differentiated from avirulent


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strains on the basis of morphologic criteria. Light and electron microscopy studies, however, failed to reveal any distinct morphologic differences to account for the state of amebic virulence. To obtain more objective criteria to define concepts of virulence as ascribed to a given strain of E. histolytica, several investigatorshave studied various virulence-associated biologic characteristics of axenically grown trophozoites. Features associated with virulent E. histolyticu, but not carrier strains, include Con A aggl~tination,~~ distinct isoenzyme electrophoretic pattern,37more extensive phagocytosis of erythrocytes,27 production of liver abscess in hamsters by intrahepatic inoculation of trophozoites,38 contact-mediated cytolysis of target cells39and in vitro cultured mammalian cells,4oincluding polymorphonuclear leuk o c y t e ~ and , ~ ~production of soluble enterotoxic s~bstances.'~ Augmenting many early empirical observations was the inception of newer test systems that enabled better discrimination between virulent (invasive) and avirulent (noninvasive) E. histolytica. Among the more innovative approaches was the electrophoretic examination of isoenzymes of E. histolytica from symptomatic vs. asymptomatic individual^.^^ Working with 85 strains of E. histolyfica,Sargeaunt et al.37compared the electrophoretic mobility pattern of three enzymes: glucosephosphateisomerase (GPI); phosphoglucomutase 0; and L-malate:NADP+ oxido-reductase (ME) and distinguished four isoenzyme p u p s (zymodemes), of which 18 of the strains comprising group 2 (ME, GPI, and fast-migrating PGM) were recovered only from symptomatic patients including amebae recovered from liver pus. Since this description, and assessing for an additional enzyme (hexokinase), 22 zymodemes of E. histolytica from various parts of the world have been recognized, of which 9 are associated with subjects with a m e b i a ~ i s The . ~ ~ studies of S a r g e a ~ n textended ,~~ by Meza and colleagues,u identified a subpopulation of asymptomatic individuals who nevertheless harbored amebae with pathogenic isoenzyme patterns. This result is not so startling considering that humans are a reservoir for both virulent and avirulent E. histolytica. Perhaps the true value in zymodeme assignment to isolates of E. histolytica resides not only in identifying virulent clones but also in the recognition that the designationE. histolytica should be conserved for those strains that meet the many virulence criteria. In this way if any asymptomatic individual harbors phenotypically virulent strains, decisions to treat such a carrier should not be based on presence or absence of symptoms, but rather on the potential the strain has for in vivo invasive transformation and for passage to other persons through fecal excretion. Facilitating differentiation would be the establishment of a system of diagnostic markers of E. histolytica virulence for routine testing. As numerous virulence markers are identified, perhaps a nucleic acid probe derived from E. histolyfica strains showing a panorama of virulence markers could be developed and adopted for routine usage in the identification of virulent clones, as has been the case for many bacterial and viral pathogens. Despite the evolving process of defrning virulent E. histolytica, little knowledge exists regarding the intrinsic switch mechanism(s) that govern invasion vs. colonization and vice versa. Equally vague is the role of the host-provided environmentin triggering the equilibrium driving the transition. What has been carefully elucidated, however, are those events that transpire once the ingested and successful trophozoite has adhered through a surface lectin to the mucosal border. Based on some of the early cinemicrography studies conducted in Mexico (cited in Reference 45) and extended by Ravdin et al. ,39 immediately following adherence by a virulent trophozoite, extensive cytopathic alterations of the epithelial cells occur at the contact site. To account for the rapid dissolution of colonic tissue, multiple mechanisms seem operative, including contact-dependent cytolytic activity, secreted enzymes, and cytotoxin production. The impressive event, however, propelling cytolysis is mucosal adherence of trophozoites,



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followed by cell death and subsequent phagocytosis of disrupted cells by the inciting ameba.39 Cytolytic activity is enhanced at 37°C and is inhibited at 25°C.28This observation has been correlated with the presence in the amebic trophozoite of microfilaments composed of actin, which serve to enhance mucosal adherence.28Further evidence supporting direct contact as the initiation of cytolytic activity derives from the serum inhibition of numerous exoenzymes e.g., trypsin, pepsin, gelatinase, caseinase, fibrinolysin, etc. produced by invasive and noninvasive E . hisrolytica,46and including cytotoxin production by virulent strains.42 Intimate cell-ameba contact is thus necessary to ensure the molecular integrity of amebic enzymes operative in cytolysis. We have seen that there are numerous factors that bear on and describe E. histolytica virulence. Perhaps two important attributes of invasive E . histolytica that decide the early host-parasite interaction and outcome are production of a cytotoxic enterotoxin and their ability to kill and phagocytize host neutrophils. Dating back to the 189Os, numerous investigators postulated that a toxin elaborated by E. histolytica secondary to colonization accounted for the destruction of the mucosal surface. In fact, when Schaudinn characterized E. histolytica he named it for its cytolytic effect on tissues. lo Since these early descriptions, more definitive work by Jarumilinta and Maegraith46 demonstrated that E . histolytica produces several exoenzymes, such as amylase, gelatinase, hyaluronidase, and trypsin, which, however, appear not to play a significant role in clinical amebiasis. Thus, although viable ameba trophozoites show numerous tissue-destructive activities in a variety of tissue-culture monolayers such as HeLa cells, chick embryo cells, Chang’s liver cells, and rabbit kidney cells, cytoxic activity in cell-free supernates was difficult to establish prior to the cultivation of E . histolytica in a bacteria-free (axenic) medium.47 succeeded in efforts Utilizing axenic strains of E. hisrolytica, Lushbaugh and c011eagues~~ to demonstrate, characterize, and partially purify a toxic substance from cell-free extracts of sonicated E. hisrolytica. This substance was cytotoxic in tissue culture, producing a cell rounding and dislodgement of cells from the monolayer, and had cytotonic activity, as evidenced by induction of fluid secretion in ligated rabbit ileal loops. Further studies by these investigators showed that the cytotoxin was a neutral cysteine proteinase inhibited by human serum and present in greater quantities in trophozoites of highly virulent amebae.48 Complete p ~ r i f i c a t i o nand ~ ~characterization studies50have shown that the cysteine protease degrades native collagen and other components of the extracellular tissue matrix,49cartilage proteoglycan, and kidney glomerular basement membrane collagen. Lucaes and Barrettso have purified and characterized the major cysteine proteinase of E . histolytica. Empirically, it had been noted that in amebic ulcers or in amebic liver abscesses there was a paucity of polymorphonuclear leukocytes unless a concomitant bacterial infection was present. To account for these observations, Jarumilinta and KradolfeP’ studied the interaction of cultured E . histolytica on leukocytes of various mammalian species. When admixed together on a glass slide and observed microscopically, a rapid chemotaxis of leukocytes toward the amebae took place, culminating in the articulation of the leukocyte pseudopodia with the ameba surface. Within 1 to 2 min of contact, the leukocytes began to undergo morphologic alteration, beginning with a loss of motility and surface activity, rapid loss (liquefaction) of cytoplasmic granules, loss of nuclear architecture, and cell rounding. Xnterestingly, when poorly motile ameba were used in similar experiments, chemotaxis was diminished and adherent leukocytes did not undergo the morphologic aberrations noted when actively motile amebae were used. The morphologic changes were noted with all leukocyte species tested. To further elucidate the “head-to-head” confrontation of two professional phagocytes,


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Guerrant and co-workers4’studied the interactions of human polymorphonuclear leukocytes (PMNs) against two strains of E . histolytica exhibiting different degrees of virulence. These authors showed that the less virulent amebae were surrounded by 2 3 0 0 leukocytes per ameba fragmented, and were readily ingested by an oxygen-independent phagocytosis. Polymorphonuclear (21,OOO PMNdamebae) contact with virulent amebae, however, resulted in loss of their motility, degranulation, and death, with occasional phagocytosis by the amebae. Based on these observations, these investigators concluded that two components of ameba cytopathogenicity were operative: first, an extracellular, contact-dependentcytolytic process as first reported by Jarumilinta and Kradolfer,” and second, leukophagocytosisthat required intact amebic microfilament production.

V. CLINICAL PERSPECTIVES OF AMEBlASlS Disease caused by E . histolytica is of enormous global significance. Amebiasis occurs in all parts of the world, but is most common in warm climates, with prevalence exceeding 40% and incidence approaching 100% in some areas.” Children bear a disproportionately large share of the burden of morbidity and mortality in many underdeveloped countries. Crowding and lack of indoor plumbing predispose to infection, while malnutrition is associated with increased seventy of disea~e.’~ A variety of human and animal studies address possible relationships between diet and amebic disease, but data are somewhat ~ o n f lic tin g . ~ ~ Specifically, high-carbohydrate diets may foster growth of the parasite, while low-protein diets have been associated with an increased risk of invasion. Milk-containing diets appear to be associated with lower disease rates. In contrast to the situation in developing countries, the overall prevalence of amebic infection in the U.S. is low. Only 0.6%of 400,OOOindividuals surveyed by stool examination were found to be i n f e ~ t e dHowever, .~~ in many developed countries amebiasis has been seen more frequently in recent years, due both to increasing travel and immigration and to a high prevalence of infection among male homosexuals. Clinically, the presence of cysts of E. histolytica in the stool of a patient with diarrhea or other enteric complaints does not necessarily establish ca~sation.’~ Nevertheless, as asymptomatic infection represents a large reservoir of organisms, treatment with drugs active against the cysts, including iodoquinol, diloxanide furoate, or paromomycin continues to be standard pra~tice,~’ and presumably reduces the likelihood of invasive disease. A. Clinical Manifestations

Less than 20% of individuals infected with E . histolytica develop symptoms. Most experience a mild diarrheal illness. More severe intestinal infection comprises a spectrum from dysentery to megacolon, bowel perforation, peritonitis, and death. The reasons for this clinical variability are poorly understood and appear to represent a combination of host factors and organism virulence properties. The clinical features of symptomatic amebic infection have been described in several reports of a large series of hospitalized patient^.^^'^^ Although severe disease may be overrepresented in these reports, they do stress the breadth of findings underscoring amebiasis. 300 consecutive adult patients with amebiasis presented In one report from South with diarrhea and 99% with dysentery. Abdominal pain and back pain were recorded in 85 and 66% of patients, respectively, while abdominal tenderness, either localized or generalized, was noted in 83% and fever in 38%. In 48% of the patients the duration of symptoms was less than 1 week, while 37% were symptomatic between 2 and 4 weeks. Between 1955



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and 1974, the overall mortality was 1.9% in over 3000 patients treated with a variety of amebicidal agents. In a more recent study from B a n g l a d e ~ hdescribing ~~ 85 children and adults with invasive disease, the age distribution was bimodal, with peaks at age 2 to 3 and above 40. In children the sex distribution was equal, but a preponderance of cases existed among females between the ages of 21 and 40 and of males over 40. Dysentery was present in 49 patients (58%). When compared with control patients with other etiologies of their diarrheal illnesses, patients with amebiasis were significantly more likely to have dysentery, abdominal pain, and a history of measles. The duration of illness was longer, averaging 16.9 d , compared with 8.6 d among controls. Mortality was greatest in patients over 40 years of age (69%), and was 29% overall, compared with 9% in the control group. A syndrome similar to ulcerative colitis has been described in some patients after recovery from severe amebic dysentery.60 Intestinal amebiasis may ensue in the absence of histological abnormalities or may be associated with a spectrum of pathological features. Severe colitis, sometimes accompanied by ileitis, and deep ulcers throughout the colon, often with mucosal necrosis, is a typical postmortem finding in patients dying of intestinal disease.59 Trophozoites within the ulcers are usually seen in untreated patients. Peritonitis, with or without perforation of the bowel, may occur. The immunologic response to infection with E . histolytica is complex and only partially understood. Although severe invasive disease appears to be related to some immunodeficiency states, including pregnancy, malnutrition, and corticosteroid therapy, the exact role of the immune system in limiting invasive disease and confemng resistance to recurrent infection is unclear. Both humoral and cellular factors appear to be involved in host defense, but as recurrent attacks of invasive disease may occur,6' either immunity is incomplete or strain variation does exist. Humoral immunity is stimulated readily by invasive infection. Serum antibody can be detected in 80 to 100% of patients with invasive intestinal or extraintestinal disease, although titers do not correlate with clinical severity or likelihood of recurrence.62Serum containing antibody has been shown to be amebicidal through activation of complement by either the alternate63or classical pathwayw and pathogenic strains of the organism may be relatively resistant to complement-mediated lysis. In vitro studies indicate that monocyte-derived human macrophages are capable of killing amebic trophozoites independent of antibody.65This killing appears to take place by an oxidative, hydrogen peroxide-dependent, as well as a nonoxidative mechanism. Cytotoxic T lymphocytes may also be amebicidal by a contact-dependent mechanism%and lymphocytes from patients recovering from amebic liver abscess can kill trophozoites, but only after stimulation with a soluble protein preparation from axenically grown amebae.

B. Extraintestinal Infection Extraintestinal spread may complicate invasive amebiasis in up to 40% of and may involve a variety of sites. Liver involvement with the formation of abscesses that may extend into structures adjacent to the liver, such as the pleural or pericardial spaces or lung parenchyma, accounts for the majority of extraintestinal complications. Liver abscess is the most common extraintestinal manifestation of infection with E . histolytica, although the exact incidence of this complication is not known. Amebic liver abscess was found in 5.8% of all patients at autopsy in one series from and in 30% of cases of fatal amebiasis at In general, although amebic infection oc-


368


curs in both sexes equally, amebic liver abscess is associated with a striking male predominance.70-72 While most cases of amebic liver abscess present in the second through sixth decades of life,7oit has been suggested that young children and infants have a particularly high incidence of hepatic as well as other extraintestinal complications.73 Immunosuppression,particularly therapy with corticosteroids, may play a role in the development of amebic liver abscess in some patients. In addition to possible host factors favoring the development of liver abscess, evidence exists that certain strains of E. histolytica are more associated with severe and invasive infection. Sargeaunt et al.74found that isolates of E. histolytica from 38 patients with liver abscess and 42 patients with dysentery all belonged to zymodeme types I1 or XI. In contrast, 57 of 67 asymptomatic cyst passers had isolates belonging to zymodemes I or 111. The signs and symptoms of amebic liver abscess are variable, but usually include fever and abdominal pain of either abrupt or insidious onset. Dysentery and other evidence of intraluminal disease is usually lacking in these patients.” Symptoms may also include nausea, vomiting, night sweats, anorexia, cough, pleuritic chest pain, and right shoulder pain. Weight loss may be a prominent feature of chronic cases. Presenting complaints and findings in these patients are sufficiently nonspecific to lead to a mistaken initial diagnosis in the majority of cases.7’ Physical findings may also be nonspecific. Hepatomegaly is present in almost all patients with more than 10 d of illness, but is usually absent in more acute cases. Although epigastric or right upper-quadrant tenderness occurs in the majority of patients, peritoneal signs are rare except in advanced cases. Fever is almost universal and, in one study, was the only manifestation of infection in 9 of 67 patients.70 Laboratory studies reveal a moderate peripheral leukocytosis in almost all cases. Alkaline phosphatase is typically elevated, even in early disease, although abnormalities of transaminases and bilirubin are encountered only in aggressive cases.7oIn 48% of patients reported, chest X-ray was abnormal, revealing atelectasis, infiltrate, or effusion in the right lower lung field, or elevation of the right hemidia~hragm.~~ The constellation of fever, right upper-quadrant tenderness, or right pleuritic chest pain in an individual who has been in an endemic area within the previous year should raise the suspicion of amebic liver abscess, even if bowel symptoms are lacking. Similarly, unexplained leukocytosis and elevation of alkaline phosphatase should lead to consideration of the diagnosis, despite the absence of localized findings on physical examination. Patients with pyogenic liver abscesses present with many of the same clinical features as those with amebic liver abscess. Since pyogenic abscesses usually require drainage and protracted courses of antibacterial therapy, while amebic abscesses generally respond to amebicidal therapy without drainage, distinguishing between these two conditions is of paramount importance. Fortunately, the distinction can usually be made on the basis of laboratory tests and imaging studies. Occasionally, however, aspiration may be necessary for diagnosis. Reports comparing pyogenic and amebic liver abscesses show that the latter are more frequently encountered in males born outside the U.S. and who are below the age of 50.72.7s*76 Symptoms are quite similar among the two, although abdominal pain, pulmonary symptoms, nausea,7sdiarrhea,72and right upper-quadrant tenderness75may be more common in patients with amebic abscesses. In patients with pyogenic infection, laboratory studies often reveal a leukocytosis with a shift to the left, decreased albumin, and elevated bilirubin, lactic dehydrogenase, and hepatic tran~aminases.~’ Length of hospital stay, time to defervescence, and mortality all tend to be substantially greater in patients with pyogenic a b s c e s s e ~ . ~ ~ . ~ ~ . ’ ~



369

Amebic liver abscesses are not true abscesses but rather areas of necrosis surrounded by a wall composed of fibroblasts, macrophages, and lymphocytes. Neutrophils are present only in small numbers and amebic trophozoites are usually present only at the periphery of the lesion. Diagnosis can be confirmed with a combination of serological data and noninvasive imaging of the liver using one of several effective techniques. Stool examination is recommended, although usually unrewarding. For instance, Greenstein et reported finding ameba in the stool of only one of 1 1 patients with amebic liver abscesses. Aspiration of a liver abscess for diagnosis is rarely necessary and usually reserved for patients who are not responding as expected to medical therapy. Techniques used to provide detaiIed images of the liver and other intraabdominal structures are invaluable, as chest X-rays revealed either right lower lung field atelectasis, infitrate, or effusion or elevation of the right hemidiaphragm in only 48% of patients with amebic liver abscess.75 Scanning of the liver after administration of a variety of radiolabeled substances, either alone or sequentially, has proven to be an extremely useful technique for identifying amebic abscesses that appear as “cold” areas on the scan. Cuaron and Gordon77reported their results of radionucleotide scans of 2500 patients with proven amebic liver abscesses, and showed that 83% of patients had solitary abscesses, with the majority (79%) located in the right lobe, usually in the external or posterior portions. Scans using Gallium 67 citrate may occasionally provide additional diagnostic information as amebic abscesses are characterized Cholescintigraphic scanning with by cold centers surrounded by a hot rim of Tc99m HIDA may aid in the identification of amebic abscesses, particularly if rim enhancement is demonstrated.79 Using sonography, Ralls et al.,80who studied 143 amebic liver abscesses in 106 patients, identified the following features suggestive of the diagnosis: lack of significant wall echoes; round or oval lesion configuration; less echogenicity than normal liver parenchyma with low-level homogeneous echoes on high gain; location contiguous to the liver capsule; and distal sonic enhancement. Thirty-eight percent of abscesses had four of these features and an additional 53% had all but one or two. In a subsequent however, distal sonic enhancement and location contiguous to the liver capsule were not specific findings. Ralls et al.,*’ in a blinded study comparing sonographic findings in amebic and pyogenic liver abscesses, found that amebic abscesses were significantly more likely to appear round or oval and to have low echogenicity and homogeneous internal echoes with hgh-gain settings. Although neither feature was diagnostic, when coupled with clinical data they allowed for correct identification of amebic abscesses in 86% of cases. Sukov et however, found no specific features that reliably distinguished amebic abscess between 3 and 10 cm from neoplastic, inflammatory, or benign cystic lesions. Sonography correlated well with radionuclide scans in determining size, number, and location of the abscesses. Computed tomography (CT) provides another effective means of detecting liver abscesses, although, analogous to sonography, it cannot establish an etiological diagnosis with certainty. Radin et al.,83 in a retrospective series describing CT findings in 23 patients with amebic liver abscesses (17 single, 6 multiple), found that 74% were located in the right lobe and that the majority were smooth, round or oval in contour, and had an enhancing wall. In addition, in eight patients an incomplete rim of edema was seen and several abscesses had internal septations. Abnormalities outside the liver, including right pleural effusion, perihepatic collections, gastric or colonic involvement, and retroperitoneal extension were seen in 18 patients (78%). Mathieu et al.,84 on the other hand, found no CT features to be useful in distinguishing amebic from pyogenic abscesses. The diagnostic yield with magnetic resonance imaging (MRI) is comparable to that of CT


370


and sonography in the detection of amebic liver absces~.’~ As with the other modalities, lesions tend to appear as round or oval areas with decreased density compared with normal liver parenchyma and smooth lesion contours.34These findings, however, are not specific. In addition, diaphragmatic disruption was detected in some cases. The sensitivity of the above commonly used imaging studies appears to be comparable. Rubinson et a1.86found that CT, and gallium and technetium sulfur colloid scanning were 88 to 90% sensitive in the detection of amebic or pyogenic liver abscesses and that sonography was 75% sensitive. Since the specificity of these studies are also comparable, they should be employed in the diagnosis of amebic liver abscess largely according to their availability and cost. It should be noted, however, that CT and MRI may be more effective in the diagnosis of extrahepatic extension of disease. The initial therapy of choice is metronidazole given orally or intravenously. Katzenstein et al.70 found that 75% of patients became afebrile by 48 h of therapy with 750 mg of metronidazole three times per day and that most of the remaining patients responded within 6 d. Four of 55 patients studied required surgery, 2 because of apparent treatment failure, and 2 because of rupture of the abscess. Furthermore, only 1 of 96 patients treated with metronidazole, chloroquine, or both, required therapeutic aspiration and none required surgery.” The median time to deverfescence was 3 d. Because of these favorable results, therapeutic aspiration or drainage is reserved for those patients with bacterial superinfection or with large abscesses adjacent to the pericardiurng7 or who fail to respond to reasonable trials of medical therapy.71Percutaneous aspiration, when necessary, is best performed under sonographic or CT guidance. Indwelling catheters are rarely necessary for adequate drainage. In contrast to “sterile” amebic liver abscess, aspiration or surgical drainage remains necessary in most cases of pyogenic liver abscess.75 There is no consensus regarding optimal therapy for patients who do not respond rapidly to metronidazole. This is true, in part, because of the lack of a standard definition of treatment failure. Thompson et al.71were unable to predict metronidazole failures on the basis of age of the patient, size or location of the abscess, or clinical manifestations of infection on initial evaluation. Patients who had failed to respond to therapy by 72 h, however, were significantly more likely to suffer treatment failure. Recommendations derived from these data suggest that patients be hospitalized for the first 72 h of therapy and that chloroquine and emetine be substituted for metronidazole in patients failing at that point. The importance of early response to therapy in predicting overall prognosis has not been confirmed in other series, although delayed defervescence may be a clue to bacterial superinfection.75 After successful therapy, abscess cavities usually resolve gradually over a period of months. Ralls et al.,88on serial sonographic studies, found a median time to resolution of 7 months. However, a case of nonresolution following apparent parasitologic cure has been described.” Foci of extraintestinal amebic infection may occasionally occur in sites other than the liver. This usually results from extension of an amebic liver abscess into adjacent structures in the thorax or abdomen, such as the pleura, lung, pericardium, subphrenic space, or peritoneal cavity. Thoracic extension is associated with an overall mortality of 11%90 and is most devastating when the pericardium is involved. Rarely, amebic abscesses may appear in sites as remote from the liver as the brain. Diagnosis and management of these unusual infections may present unique challenges. Pleuropulmonary involvement of some degree complicates over 40% of amebic liver abscesses.68An intact amebic liver abscess adjacent to the diaphragm may cause a serous pleural effusion or atelectasis, almost always on the right side.68Less commonly, a liver abscess may rupture and extend directly or via lymphatics through the diaphragm into the



371

pleural space or lung parenchyma. In addition, if the abscess communicates with the airways, secondary lung abscesses may form. These complications may occur from days to months after the onset of symptoms of liver infectionw and may occur in 8% of cases,68most commonly as a complication of abscesses in the superior portion of the right lobe of the liver. Pleuropulmonary amebiasis may induce hepato-bronchial fistula (47%), pleural effusion and empyema (29%), lung abscess (14%), and consolidation (10%). As expected, pleuritic chest pain, often radiating to the shoulder, cough, and hemoptysis are common complaints. Occasionally, as such symptoms may not be prominent, pleuropulmonary amebiasis may be difficult to distinguish from liver abscess. Sudden extension into the pleural space may present as respiratory distress and shock, often with abrupt worsening of chest pain. Expectoration of dark brown material, once considered a favorable sign in patients with amebic liver abscess, may represent spontaneous drainage of the liver abscess through the bronchial tree and is associated with a very good prognosis.w The diagnosis of pleural or pulmonary extension of an amebic liver abscess should be considered in patients from endemic areas with otherwise unexplained elevation of the right hemidiaphragm, right pleural effusion, or evidence of atelectasis or consolidation at the right lung base. One or more of the liver-imaging studies discussed above, coupled with serologic tests, may be used to establish the diagnosis. Disruption of the diaphragm has been visualized by sonography in some cases of thoracic amebiasis," and might also be identified by CT and MRI. In patients receiving at least 10 d of therapy with metronidazole for amebic liver abscess, the mortality of pulmonary amebiasis is less than 15%.69It is generally recommended that pleural effusions resulting from extension through the diaphragm be aspirated and drained by closed t h o r a~ o t o m y . ~Surgery '. ~ ~ may be necessary when adequate drainage cannot be obtained or when secondary bacterial infection occurs. Massive hemoptysis or persistent drainage complicating a hepatobronchial fistula are also potential indications for surgery. Direct extension of an amebic liver abscess into the pericardium is an unusual but catastrophic event. This complication is seen most often in association with abscesses of the left lobe of the liver and may be heralded by the development of a serous pencardial effusion manifested as an enlarging cardiac silhouette on chest X-ray. Rupture into the pericardial space may occur in 1.3% of cases of liver abscess6' and may occur gradually or abruptly at any point during the course of the illness. In many cases, signs and symptoms of pericarditis with effusion may mask a liver abscess.68 Conversely, the signs and symptoms of liver abscess may be so prominent in some cases that subtle signs of pericardial involvementare overlooked. As in the case of pleuropulmonary extension of amebic liver abscess, diagnosis rests on a high index of clinical suspicion in a patient from an endemic area presenting with signs and symptoms of suppurative pericarditis, with or without clinical evidence of amebic liver abscess. Serological tests for amebiasis and imaging studies of the liver and pericardium will lead to the diagnosis. Overall, pericardial extension of an amebic liver abscess is associated with a 30 to 60% mortality .68,w Chronic constrictive pericarditis may develop in patients surviving this complication."* Although small pericardial effusions not associated with frank rupture into the pericardium may resolve with medical therapy, significant effusions, particularly those associated with hemodynamic compromise, should be drained via pericardiocentesis when feasible. When adequate drainage cannot be established in this manner, surgery may be necessary. Chemotherapy with metronidazole alone, or in combination with chloroquine or emetine, should be continued for at least 10 d. In some patients, constrictive pericarditis may subsequently


372

Critical Reviews in Clinical Laboratoly Sciences

become hemodynamically significant, even after adequate acute therapy, and may require surgical stripping of the pericardium. Rupture of an amebic liver abscess into the peritoneal cavity is another rare and potentially disastrous complication, and may present in an indolent fashion or as an acute intraabdominal catastrophe. Amebic peritonitis occurred in approximately 2% of cases of liver abscess in a series reported from India9*and was associated with a mortality of 42%.Presenting findings in these patients ranged from months of nonspecific abdominal pain (68%) and fever to abrupt onset of clinical peritonitis with board-like rigidity of the abdomen in 25% of cases. Diarrhea, constipation, abdominal distention, melena, vomiting, and jaundice were present in a minority of these patients. Hepatomegaly was reported in 63% and masses in the epigastrium or right iliac fossa in 26%. Because of the presence of extrahepatic masses, a mistaken diagnosis of pancreatic pseudocyst was made in several cases. Elevated bilirubin and blood urea nitrogen, and decreased hemoglobin were frequent abnormalities. Considering the nonspecific nature of the findings, amebic peritonitis should be considered in any patient known to have an amebic liver abscess who develops increasing generalized abdominal pain or evidence of peritoneal imtation as well as in individuals presenting with peritonitis of unclear etiology, with or without hepatomegaly. Controversy exists over the best management of amebic p e r ito n iti~ .In~ ~older series, surgical mortality was extremely high,92reflecting, in part, that many of these patients were debilitated with advanced infection at the time of presentation. With recent improvements in amebicidal therapy, it is possible that limited surgery or percutaneous drainage, in combination with parenteral metronidazole, is the best current approach.69 E . histolytica is not restricted by body compartments or-topography and can be found on rarer occasions invading the brain, genitourinary system, and other sites. Amebic brain abscess is an uncommon occurrence, which is thought to result from hematogenous dissemination of organisms from the bowel,@ which prior to the advent of effective amebicidal therapy, was an almost uniformly fatal event. Because of improved therapy and diagnostic imaging studies, however, recovery from amebic brain abscess can be achieved.93Focal neurological signs, with or without evidence of meningeal irritation, are common and abscesses are often multiple and appear as irregular, nonenhancing lesions.93Metronidazole crosses the blood-brain barrier and therapy with this agent may eliminate the need for surgery, unless drainage is necessary to relieve significant pressure effects. Corticosteroids may be contraindicated in this condition.93 Amebic abscesses in the renal parenchyma and perinephric spaces have been reported occasionally. It has been suggested@that these collections may form as a result of direct extension from a liver abscess or by hematogenous or lymphatic dissemination. Optimal therapy is not known. Rupture of amebic liver abscess into the bowel has been described with formation of hepatoduodenal fistula.58*”This complication should be suspected when air is seen on roentgenograms of a liver abscess, although this finding may also be caused by superinfection with gas-forming organisms. Further, thrombosis of the inferior vena cava with extension into the right atrium has occurred in the course of amebic liver abscess.95 Cutaneous amebiasis is a rare complication of intestinal infection. Painful ulcers found to contain trophozoites on scraping or biopsy have been described, most commonly located on the perineum or around thoracic or abdominal fistulous trackm

VI. CIRCUMSTANTIAL DISEASE SYNDROMES E . histolytica is an uncommon cause of travelers’ diarrhea, accounting for less than 9%



373

of cases in recent surveys of travelers to Latin America, Africa, and Asia.%sg7The likelihood of acquiring amebic infection correlates with length of stay in the endemic area. Amebiasis is particularly uncommon in short-term travelers. Indeed, none of 326 American students traveling to Mexico for an average of 4 weeks had positive stools or serum antibody tests for amebic infection on return, including 180 with enteric In a survey of 251 expatriates in Bangladesh, the incidence of infection was less than 7% in those staying for less than 1 year and greater than 26% in those staying 3 years or more.* Infection, when it does occur in travelers, usually results from exposure to contaminated water or food'O0 and, therefore, reflects the region visited as well as the traveler's activities. Visitors to underdeveloped areas are advised to reduce their risk of infection from enteric pathogens, including E. histolytica, by drinking only bottled, processed, or boiled water and to avoid using ice. Since ground-grown vegetables may be contaminated, they should only be eaten cooked or after washing with detergent or vinegar.'" High carriage rates of E . hiaolyticu as well as major outbreaks of symptomatic amebiasis have been reported periodically from chronic-care institutions, particularly residential facilities for the mentally retarded.'02.'03 Fecal incontinence among retarded patients along with poor handwashing practices among staff may lead to extensive person-to-person transmission in such an environment.IoZControl of outbreaks in this setting may be difficult, as segregation of infected patients is not always effective and periodic mass chemotherapy has been suggested as an alternative approach.Io3 Infants and children, especially those living in warm climates or under unsanitary conditions, are particularly prone to infection with E . histolytica, with increased complications and mortality rates. Children hospitalized with amebic dysentery had a 9% rate of peritonitis, compared to 3% in adults, and an overall mortality of 27%.58As in adults, transmission occurs primarily by the fecal-oral route. Amebiasis in infants may represent person-to-person transmission from infected household members and should prompt thorough in~estigation.'~ Spread of infection in the daycare setting appears to be less common than with other enteric pathogens such as Shigella, rotavirus, and Giardia lamblia. '04 As in adults, intestinal amebiasis in children may produce no symptoms or a broad spectrum of disease, including a diarrheal illness of variable severity as well as colitis, dysentery, peritonitis, and death. In addition, hematochezia without diarrhea, dysentery with appendicitis, and exacerbation of ulcerative colitis, have been described.lo' Amebic liver abscess may also occur in children. As in adults, findings include fever and abdominal pain and distention, as well as leukocytosis and roentgenographic abnormalities at the right lung base. Intestinal symptoms, rare in adults with this infection, were noted in 4 of 6 children, all under the age of 3 years.'" Invasive and disseminated amebiasis are associated with a poor prognosis in young children.73*'" The prevalence of infection with E . histolytica among homosexual men rivals that seen in the most highly endemic countries in the world, exceeding 20% in some series.1o7Infection presumably spreads primarily by the fecal-oral route and has been shown to correlate with anal-oral contact in these individuals.Io8Other factors contributing to high carriage rates in this group probably include large numbers of anonymous sexual contacts, many of whom are asymptomatic cyst passers.'@ Spread to other segments of the population through food contamination and other routes magnifies the significance of carriage rates among homosexual men even further. Although invasive amebiasis has been described in homosexual men,"' it appears to be quite unusual given the high prevalence of infection. This low rate of invasive disease has been explained in part by Allason-Jones and c o - w ~ r k e r s , 'who ~ ~ found that E . histolytica isolates from homosexual men attending a sexually transmitted disease clinic in London all

374


belonged to nonpathogenic zymodemes. In addition, infection in these patients did not correlate with gastro-intestinal symptoms, raising the speculation that treatment of infected homosexual men who do not have invasive disease may not be necessary. Additional conf i a t o r y data will be needed, however, before this approach can be recommended.


VII. LABORATORY DIAGNOSIS Diagnostically, major deficiencies exist in the clinical and laboratory recognition of infection due to Entamoeba h i s t o l y t i ~ aFurther, .~~ it has been suggested" ' that stool examination of parasites be performed for all patients presenting with dysenteric or nondysenteric ulcerative colitis. However, direct identification of trophozoites and cysts of E. histolytica, or the application of diagnostic assays and their interpretation, requires highly competent and experienced individuals, who are not often encountered in routine diagnostic laboratories. Three readily applicable methods exist for the diagnosis of E. histolytica infection. These embody the microscopic recognition and identification of trophozoites or cysts, amebic cultivation, and serologic assay for antigen or antibody. Of these, the most definitive mode of diagnosis is the microscopic detection of the parasite in representative specimens obtained from intestinal and/or extraintestinal sites of involvement. Fecal specimens are evaluated routinely for the presence of E. histolytica. Because of intermittent cyst excretion, however, three stool specimens should be examined over a period of 7 to 10 d.'13 According to Thomson et a1.,Il4 as many as 6 fecal specimens may be necessary to rule out amebic infection, in view of the fact that the predictive value of parasite recognition is between 50 to 80%when only 3 specimens are evaluated. Further, the presence of substances such as laxatives, antacids, barium sulfate, and antidimheals may obscure the microscopic observation and recognition of E. histolytica. I I s The administration of antibiotics may suppress but not eradicate E. histolytica gastrointestinal carriage. ' I 6 Other limitations imposed by microscopic evaluation include submission of poor-quality specimens (a warm, fresh stool is optimal), and confusion of E. histolytica with other protozoa, white blood cells, especially monocytes, or artifacts present in fecal material. Furthermore, parasite detection and recognition in extraintestinal specimens such as abscess fluid is less optimal by comparison with fecal specimens.'I7 Specimen screening may be enhanced through evaluation of wet mount preparations augmented by the addition of vital dyes such as Loffler's methylene blue"' or more commonly, Lugol's iodine. Both reagents highlight the salient morphological characteristics, i.e., nuclear structure inclusive of the centrally situated karyosome, even distribution of chromatin granules around the nuclear membrane, and the presence of blunt-ended chromatoid bars in the cytoplasm. Wet mount preparations for preliminary screening are made by mixing a selected aliquot of specimen with a drop of saline and/or stain and examining the slide systematically in its entirety, using first low-power magnification (10 X ), then high power (43 X ). Concentration of fecal specimens greatly assists microscopic detection of cysts, especially when present in diminished numbers. Two popular concentration techniques include the formalin-ether sedimentation technique"' and the zinc sulfate flotation technique. 12' The former method has been recently modified by the substitution of ethyl acetate for the highly flammable diethyl ether. lZ2 Details regarding the precise step-by-step implementation of these methodologies may be found in standard parasitology reference texts. IL9 For the detection of fragile trophozoites, unpreserved specimens should be examined within 2 h following collection.I5 However, methods for the preservation of specimens exist which arrest morphologic deterioration of both trophozoites and cysts. Preservation of fecal



375

specimens is critical and mandated for specimens which cannot be immediately evaluated, and is useful for long-term maintenance of specimens to be utilized for quality control, teaching, and training purposes. The preservatives most frequently used include polyvinyl alcohol (PVA),123merthiolate-iodine-formaldehyde(MIF),'24formalin, and phenol-alcoholformaldehyde (PAF).125 Often direct microscopic examination of wet mount preparations supplemented with vital stains or iodine does not elucidate the fine points of nuclear and cytoplasmic morphology necessary to differentiate among different amebic species. Critical examination of amebic morphology is markedly enhanced through the examination of permanently stained smears. Specimens are prepared by fixation with PVA (since fixation with formalin or MIF is not optimal for tropho~oites'~), and stained for cellular detail by the Wheatley modification of Gomom's trichrome stain126or Heidenhain's iron hematoxylin.127 Additional stains that may be used include iron hematoxyiin phosphotungstic acid and chlorazol black of which the latter is applied only to fresh and not PVA-fixed smears. In vitro cultivation of E . hisrolytica is used for diagnostic and research purposes. Culturing provides an environment conducive to excystment and multiplication of trophozoites, which are then readily observed microscopically. Additionally, cultivation has provided large numbers of trophozoites necessary to study the biochemical, pathologic, and antigenic properties of E. histolytica. I3O Three basic culture systems, xenic, monoxenic, and axenic are used for E. histolytica. Xenic cultures are characterized by the presence of other organisms, usually bacteria. Media for xenic cultivation of E . histolytica include the modified Locke-Egg (LE) medium,131a medium formulated by Robinson,132and the TYSGM-9 (Trypticase-yeast extract-serumgastric mucin) medium.'33 These media are all utilized in conjunction with a rice starch supplement,134which provides a nutritive stimulus for the multiplication and growth of E . histolytica. In addition, additives such as antibiotics and/or bacteriostatic dyes are included in these cultures to suppress bacterial overgrowth and nutrient depletion. In the axenic cultivation of E . histolytica, the most commonly utilized medium is TYIS-33 (trypticase-yeast extract-iron-serum).135 However, the establishment of viable cultures of E . histolyrica in the absence of a concomitant microbial organism(s) is extremely difficult and therefore infrequently applied. The dismal success rate with axenic cultures is explained by the requirement for bacteria in the growth of E. histolytica.136As discussed, the association of bacteria and other microbial entities, such as Trypanosoma cruzi, with E. histolytica are necessary in the pathogenesis of certain amebic strains.I3' In routine diagnostic laboratories, cultivation of E . histolytica is not often used as a diagnostic adjunct, largely because of poor or equivocal Edelman and S p i n g a ~ n ' ~ ~ noted that excystment occurred in only two thirds of specimens from patients with localized intestinal infection. These authors attributed excystment failure to insufficient numbers of cysts present in the original inoculum, andlor to the inappropriate nature and quantity of bacterial and fungal organisms present in the specimens to enhance growth. However, the desirability of a cultural approach to diagnosis may be implemented through Endameba media, a commercially prepared product (Difco Laboratories).

VIII. SEROLOGY Serologic techniques based on the assessment of anti-ameba antibody production or detection of amebic antigen are used widely in the diagnosis of amebiasis. Methodologies used to assay for antibody production include indirect hemagglutination (IHA), indirect fluorescent antibody (FA), counterimmunoelectmphoresis(CIE), latex agglutination, and enzyme-linked

Y

Antigen-antibody reaction in gel under electrical current, driving antigen and antibody toward each other Agglutination of latex or bentonite particles coated with E. histolyticu antigens Binding of antibody to a fixed E. histolyticu antigen with subsequent detection of complex through enzyme-linked reagent Binding of E. hisrolyricu antigen to fixed antibody with subsequent detection through enzyme-linked reagent Detection of E . histolyticu using fluoresceinlabeled antibody to E. histolyricu

CIE

Highest

High

High

Moderate Comparable to IHA High

High

High

Moderate

Moderate

Moderate Comparable to IHA High

High

Extraintestinal disease

Direct examination of specimens by ELISA for the detection of E. histolyticu antigen is the method of choice

Limitations of these techniques include differences in antigens used, titers of antibody considered positive, and persistence of antibody

Comments

MA, Indirect hemagglutinationassay; F A , indirect fluorescent antibody assay; CIE, counterimmunoelectrophomis; LA, latex agglutination; BF, Bentonite flocculation; ELISA, enzyme-linked immunosorbent assay; DFA, direct fluorescent antibody.

DFA

ELISA, monoclonal

ELISA

LA, BF

FA

Agglutination of erythrocytes sensitized with E. histolyticu antigen in presence of specific antibody Detection of antibody to E. histalyticu using fl uorescein-labeled antiglobulin

Principle

IHA

for antibody detection'

b

Symptomatic intestinal disease

Sensitivity/specificity

Table 2 Serologic Techniques Used in the Diagnosis of A r n e b ~ a ~ i s ~ ~ ~ ~ ~ ~ ~ *


0,

c

(3

3

(D

2.

-. 3

f?.

(3

z.

-. 3

OI

4

W



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immunosorbent assay (ELISA). Overall, these methods are of limited value for localized gastrointestinal disease, but are almost 100% effective in the diagnosis of extraintestinal a m e b i a ~ i s . ' ~However, *'~ in comparing one serodiagnosticassay to another, much confusion arises due to lack of standardization of titer interpretation with each assay, as well as inherent errors within the titer ranges employed. 14' The resultant variability in reported sensitivity and specificity values for these assays must be taken into consideration when reviewing the literature on comparative studies. Complement fixation (CF) was the first test used in the serologic diagnosis of amebiasis. ~,'~~ Positive results were achieved in 84 to 100% of cases of hepatic a b s ~ e s s . ' ~Although CF is highly specific, this diagnostic approach is rarely used because of technical difficulties and the availability of other easier and more sensitive tests such as IHA.'" In extraintestinal disease, IHA sensitivity approaches 95 to 100%; IHA in localized intestinal involvement is less optimal, with a reported sensitivity generally approaching 85%.'17 A limiting feature of this technique is the prolonged elevation of positive M A titers in some individuals 11 years following cure. Antibody persistence makes MA ideal for epidemiological surveys but is of less value in rendering diagnostic and prognostic indicators for acute disease. Indirect fluorescent antibody techniques are comparable to IHA in sensitivity and specificity for both intestinal and extraintestinal involvement.'41 In patients with acute amebiasis, therapeutic intervention results in a rapid fall in IFA anti-amebic antibody titer, thus making this test of value in gauging the efficacy of therapy.146 Particle agglutination tests using bentonite and latex beads have also been developed, but are generally less sensitive, although equivalent to IHA in the diagnosis of both amebic hepatic involvement and symptomatic intestinal illness.'41 Detection of precipitating antibodies in the sera of patients with amebiasis is another diagnostic approach initially developed by Atchley and colleagues.'41 Analogous to hemagglutinating antibodies, precipitating antibodies are detectable several years after cure. Overall, however, the precipitation in gel technique is inexpensive and simple to perform and its sensitivity, while not comparable to IHA, is adequate.149 Counterimunoelectrophoresis (CIE) is the prototypic method for enhancing the diagnosis of amebiasis. Th~stechnique, which combines features of immunoelectrophoresis and gel precipitation, is based upon the electrophoreticmigration of positively charged antigen toward the anode in an electric field and migration of antibody, due to the electroosmophoreticflow of water, to the cathode. Thus by the careful apposition of two wells in a suitable gel support, antigens and antibodies may be electrically directed to flow toward each other and interact over an optimal concentration gradient, with resultant formation of a visible precipitate in the supporting matrix. CIE provides useful information regarding the change in antibody titer and number of visible precipitin bands concomitant with therapy. Initially, the sera of patients with active disease may show up to 10 distinct precipitin bands indicative of an antibody response to E. hisfalyticu antigens derived from secreted exoproducts and/or lysing trophozoites. Following a response to antiamebic therapy, antibody production to distinct antigens diminishes, as evidenced by a disappearance of several precipitin bands.'29 Convalescent sera drawn 2 to 5 years later show only two or three major precipitin bands, which may be regarded as residual, perhaps even lifelong, reactivity. Therefore, careful analysis of precipitin band patterns may permit differentiation between current and old infection. Furthermore, because of the utility of an electrified field to drive reactants toward each other, test results are obtained more rapidly than by double diffusion in a gel substrate with no compromise in sensitivity.


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A second-generation development for the serodiagnosis of invasive amebiasis, enzymelinked immunosorbent assay (ELISA), may be used to detect either anti-ameba antibodies or amebic antigen in stool or other body fluids. The sensitivity of these assays, when using a polyclonal antibody for antigen detection, is 82%, with low specificity. With the advent of monoclonal antibodies directed against major amebic surface epitopes, however, while the sensitivity has remained unchanged, a specificity of 98% has been reported.lS1 Assessment of clinical specimens for ameba antigens, either as the whole trophozoite or cyst, or internal antigens subsequent to ameba lysis, is achieved primarily through ELISA152 and direct fluorescent antibody techniques (DFA).’ As these tests are not readily available, the efficacy vis i vis the direct microscopic detection of the parasite in fecal and extraintestinal sources remains to be determined. Serodiagnosis of amebic infection is requisite in both diagnostic, prognostic, and epidemiological studies. However, as these assays may be subject to false positive and false negative results, skilled judgment is essential in the laboratory application and utility of these serological tests.

IX. THERAPY Antimicrobial agents currently used in the treatment of amebiasis are classified as luminal, bowel wall active, or tissue active, depending on their pharmacokinetic properties. Different drugs or combinations of drugs are used for asymptomatic intestinal disease and those with extraintestinal infection based on their sites of action. Luminal drugs are poorly absorbed and therefore reach therapeutic levels in the colon. They are not effective in the treatment of extraintestinal infection because of poor blood and tissue levels, but are efficacious in asymptomatic or mild intestinal infection and to eradicate occult foci of intestinal infection in patients with extraintestinal amebia~is.~’ Diiodohydroxyquin has generally replaced other 5-hydroxyquinolones for the treatment of luminal infection. Five percent of an oral dose is absorbed; excretion is through the kidneys, with a half-life of 12 h. It is thought to act by chelation of ferrous ions, which are essential for growth of E. histolytica. 153 Side effects include headache, nausea, rashes, and pruritis ani. Optic neuropathy may occur with prolonged use. Diloxanide furoate is structurally similar to chloramphenicol and may act by blocking protein ~ynthesis.”~ It is converted to diloxanide by intestinal e ~ t e r a s e s .As ’ ~ ~less than 10% of an oral dose is absorbed, cure rates in cyst passers exceed 90%.67The most common side effect is flatulence. Paromomycin, as with other aminoglycosides, is virtually unabsorbed after an oral dose. Significant absorption may occur in individuals with disrupted colonic mucosa. Because of its broad antibacterial spectrum of activity, paromomycin may produce intestinal overgrowth of resistant bacteria or fungi.’54 Bowel wall-active agents include erythromycin, chlortetracycline, oxytetracycline, and tetracycline hydrochloride. They are incompletely absorbed and hence are active in bowel wall infection.ls3 These drugs, however, are no longer recommended in the therapy of amebiasi~.~’ Tissue-active agents are selective against amebic trophozoites in sites such as liver and lung. However, adequate intraluminal levels for eradication of intestinal cysts, may not be achieved. Metronidazole, a tissue-active agent, may be administered parenterally or orally. Absorption is almost complete after an oral dose. The half-life is approximately 8 h and clearance is both hepatic and renal. Levels obtained are excellent in bile, cerebrospinal fluid, bone,



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and a variety of other sites. Therapeutic levels have been documented in brain and liver abscesses. 155.156 The amebicidal action of metronidazole is thought to result from electron trapping due to its low redox potential, with production of cytotoxic intermediate comp o u n d ~ . Side ’ ~ ~ effects include nausea, vomiting, metallic taste, and, rarely, leukopenia, sensory neuropathy, seizures, and other central nervous system syndromes. Disulfiram (Antabuse) reactions have been described in patients who consume alcohol. Because metronidazole has been shown to produce tumors in mice and rats, concern has long been expressed over a possible carcinogenic effect in humans. Clinical studies, however, have failed to substantiate this concern. 158 Metronidazole is currently recommended in the treatment of mild to severe intestinal amebiasis and amebic liver abs~ess,~’ as well as for other forms of amebiasis. Emetine/dehydroemetine are among the most potent amebicides.Ia The mechanism of action of these alkaloid compounds appears to be inhibition of protein synthesis.Is9Both drugs are administered by intramuscular injection with resultant therapeutic levels in the liver and other tissue Cardiac toxicity, including arrhythmias, EKG changes, chest pain, and dyspnea have limited the usefulness of both of these compounds, although these side effects are less frequent with dehydroemetine. Both agents are currently regarded as alternatives to metronidazole in the therapy of severe, or even refractory, intestinal or extraintestinal infections.57 Chloroquine is an antimalarial that has been effective in the therapy of amebic liver abscess for 40 years.’53It is currently used as an adjunct to therapy for patients treated with emetine or dehydroemetine.57 Due to the availability of metronidazole and other effective amebicides, indications for surgical intervention in amebiasis are few. Patients with severe intestinal infection complicated by toxic megacolon may require colectomy, and liver infection extending into the pericardium may be an indication for urgent surgical intervention. In the majority of cases, however, even advanced intestinal or extraintestinal infection may be managed medically.

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158. Beard, C. M., NoUer, K. L., O’Fallon, W. M., Kurland, L. T., and Dockerty, M. B., Lack of evidence for cancer due to use of metronidazole, N. Engl. J . Med., 301, 519, 1979. 159. Krogstad, D. J. and Cedeno, J. R., Problems with current therapeutic regimens, in Arnebiusis: Human Infection by Entumoebu hisfolyticu, Ravdin, J . I., Ed., Churchill Livingstone, New York, 1988, chap. 45. 160. Cedeno, J. R. and Krogstad, D. J., Susceptibility testing of Enturnoebu hisrolyticu, J . Infect. Dis., 148, 1090, 1983.

Excellence in assisted reproductive technologies: clinical and laboratory perspectives.

Novel concepts in male factor infertility: clinical and laboratory perspectives.

Amebiasis.

Biocontainment laboratory risk assessment: perspectives and considerations.

Extraintestinal amebiasis.

Laboratory diagnosis of mucormycosis: current status and future perspectives.

Novel perspectives on laboratory techniques and sport performance.

Pleuropulmonary manifestations of hepatic amebiasis.

Treatment of asymptomatic amebiasis in homosexual men. Clinical trials with metronidazole, tinidazole, and diloxanide furoate.

Informatics and the clinical laboratory.

Letter: Cerebral abscesses in amebiasis.

Current concepts in parasitology. Amebiasis.

Systemic manifestations of invasive amebiasis.

Clinical laboratory directors.

Alagille syndrome: clinical perspectives.

Horner syndrome: clinical perspectives.

Prevalence of amebiasis in inflammatory bowel disease in University Clinical Hospital Mostar.

Endemic amebiasis in an Arkansas community.

Luminal agents in the treatment of amebiasis.

Primary Pulmonary Amebiasis Complicated with Multicystic Empyema.

Multiple cerebral abscesses complicating hepatopulmonary amebiasis.

Evaluation of laboratory perspectives on hereditary cancer panels.

Clinical and Laboratory Diagnosis of Intestinal Tuberculosis.

Clinical and laboratory assessment of nutritional status.