CLINICAL MICROBIOLOGY REVIEWS, Oct. 1992, p. 356-3690893-8512/92/040356-14$02.00/0Copyright © 1992, American Society for Microbiology

AmebiasisDAVID A. BRUCKNER

Department ofPathology and Laboratory Medicine, UCLA Medical Center,Los Angeles, California 90024-1713

INTRODUCTION ................. 356EPIDEMIOLOGY.................356LIFE CYCLE............o357Trophozoite.357Precyst...................357Cyst...... ...... ..... 357Metacyst.................358MetacysticTrophozoite..........358

PATHOGENICITY AS DETERMINED BY ZYMODEMES..........................0358PATHOGENESIS AND PATHOLOGY...................................o.oooo.oo360Asymptomatic Infection..................................000...o...361SymptomaticInfection..361

DIAGNOSIS .....362THERAPY................ ................ ............364AsymptomaticPatients.364Symptomatic Patients...............................364Extraintestinal Disease..................... ....... ..ooooooo..oooo. 364Follow-Up............................ ... ... ..........o364

SUMMARY AND FUTUREDIRECTIONS.364REFERENCES......................365

INTRODUCTION

Entamoeba histolytica is one of six parasitic amebae of thegenus Entamoeba that are known to infect humans (24, 38).Entamoeba coli, E. gingivalis, E. moshkovskii, and E. hart-manni are not associated with pathologic sequelae, but E.poleckh and E. histolytica are. L6sch in 1875 was the first todescribe the trophozoite form of E. histolytica and thepathology associated with the infection in a patient from St.Petersburg, Russia. Although Losch was able to infect a dogwith organisms obtained from the patient, he was not able tomimic the disease produced in humans and failed to recog-nize the relationship (59). Councilman and LaFleur (26), in aclassic monograph, described the clinical and pathologicalevidence of the association of E. histolytica with dysenteryand liver abscesses. Quincke and Roos in 1893 described thecyst form, and Schaudinn in 1903 named E. histolytica anddifferentiated it from E. coli (59). Early literature used thegenus names Endamoeba and Entamoeba to describe E.histolytica. In 1954, the International Commission on Zoo-logical Nomenclature ruled that E. coli was to be the typespecies and that Entamoeba was to be used in place ofEndamoeba to describe E. histolytica.Walker and Sellards (154) used prisoners in the Philippines

to carry out controlled experiments with E. histolytica. Theynoted that (i) transmission was most likely accomplished bycysts, not trophozoites; (ii) asymptomatic carriers wereprobably reservoirs and responsible for transmission; (iii)there were differences between individuals' disease risk; and(iv) there were differences in organism strain virulence.Boeck and Drbohlav (11) were the first to successfullycultivate E. histolytica by using Locke's egg serum medium.Other media frequently used to culture pathogenic strainsinclude those described by Robinson (123), Balamuth (3),and Jones (54); however, the first axenic cultivation was

accomplished by Diamond (32, 33). Culture techniques havegreatly aided our understanding of E. histolytica strains andtheir virulence differences in infected humans.The unclear pathogenic status of E. histolytica was noted

early in the literature with the terminology "small and largerace" E. histolytica. Burrows (19, 20) renamed the smallrace E. hartmanni on the basis of morphological criteria.Although clinical symptoms and pathological sequelae wereknown to vary considerably in areas where E. histolyticainfection was endemic, it was not until reports of infectionsin homosexuals appeared that the status of nonvirulent andvirulent strains took on renewed significance (44).

EPIDEMIOLOGYE. histolytica infections occur worldwide but are more

prevalent in the tropics. It has been estimated that approx-imately 480 million people, or 12% of the world's population,are infected and that annual mortality is 40,000 to 110,000persons (45). The prevalence of the infection differs from onearea to another, as does the severity of disease from patientto patient. Differences in prevalence figures often depend onthe detection methods used and the number of fecal exami-nations done. About 10% of those infected every year haveclinical symptoms (155). Of the approximately 48 millionpersons with clinical symptoms, 80 to 98% have symptomsrelated to the intestinal mucosa (diarrhea or dysentery);however, in only 2 to 20% of the population with clinicalsymptoms do amebae invade beyond the intestinal mucosa.Evidence of recurrence of invasive colitis or amebic abscessis unusual. DeLeon (27) monitored more than 1,000 patientswith amebic liver abscess for 5 years and found a recurrencerate of 0.29%.Humans are the major reservoir of infection with E.

histolytica, although natural infections in macaque monkeys

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AMEBIASIS 357

TABLE 1. Characteristics of E. histolytica in permanently stained smears

Characteristic Trophozoite Cyst

Size (diam or length)a 10-60 ,um; usual range, 13-20 p.m 8.5-19 Atm; usual range, 11-14 p.mShape In wet preparation, hyaline, fingerlike Usually spherical

pseudopodia; motility may be rapidNucleus (no. and visibility) 1; difficult to see in unstained preparations Mature cyst, 4; immature cyst, 1 or 2Peripheral chromatin Fine granules, uniform in size and usually Peripheral chromatin present; fine, uniform

evenly distributed; may appear beaded granules evenly distributedKaryosome Small, usually compact; centrally located but Small, compact; usually centrally located but

may be eccentric occasionally eccentricCytoplasm Finely granular, "ground glass" appearance Finely granularInclusions Noninvasive organisms may contain bacteria; Chromatoidal bodies may be present; usually

presence of erythrocytes diagnostic elongate, blunt, rounded, smooth edges, may beround or oval; the more mature the cyst, theless chance chromatoidal bodies will be present

a The organisms will be to 1.5 pm larger in wet preparations than organisms on permanently stained smears.

and pigs have been reported (50). Ingestion of food and drinkcontaminated with E. histolytica cysts from human feces anddirect fecal-oral contact are the most common means ofinfection. Transmission of E. histolytica by water is commonin Third World countries, where much of the water supplyfor drinking is untreated. The use of human feces forfertilizer is also an important source of infection (81). Onlythe cysts are infective, and most cases originate from asymp-tomatic human carriers who are cyst passers. The fact thatboth pathogenic and nonpathogenic isolates can producecysts may help explain the propensity for family clustering inthe transmission of this organism (41). Depending on envi-ronmental conditions, cysts may remain viable for as long as3 months. Viable cysts in water can be destroyed by hyper-chlorination or iodination (57, 81).Recognized high-risk groups include travelers, immi-

grants, migrant workers, immunocompromised individuals,individuals in mental institutions, and sexually active malehomosexuals. E. histolytica has been found in as many as32% of homosexual men in North America (1). Of specialinterest is the rarity of invasive disease in homosexual menand in patients with AIDS and AIDS-related complex.Almost all the strains of E. histolytica isolated from humanimmunodeficiency virus-positive and homosexual popula-tions have, through isoenzyme analysis, been classified asnonpathogenic (1, 18, 44, 157). Human immunodeficiencyvirus type 1 antigens, possibly resulting from phagocytosisof infected host cells, have been detected within E. histolyt-ica; however, transmission of human immunodeficiencyvirus type 1 from E. histolytica to human cells is not possible(17). The risk factors contributing to predisposition to infec-tion and the factors contributing to increased severity ofdisease are primarily speculative at present. There are a fewpublications concerning whether individuals are morereadily colonized by nonpathogenic or pathogenic isolates orwhether there are virulence differences in pathogenic iso-lates (34, 58, 95, 97, 98, 124). Evidence supports the viewthat there are two morphologically identical forms of E.histolytica, one nonpathogenic and the other pathogenic.This information will be discussed in the sections on patho-genicity as determined by zymodemes and pathogenesis.

LIFE CYCLE

The life cycle of E. histolytica was extensively describedby Dobell (35) and includes trophozoite, precyst, cyst,metacyst, and metacystic trophozoite stages. Lushbaugh

and Miller (75) have reviewed the ultrastructure of E.histolytica obtained by scanning and transmission electronmicroscopy.

TrophozoiteTrophozoites vary in size from 12 to 60 ,um in diameter,

with larger forms found in tissue and smaller forms found inasymptomatic carriers (45). The large tissue forms mayresult from increased cellular activity, as shown by increasesin the DNA and RNA content of the amebae (51, 77).Depending on environmental conditions, motility andpseudopod formation are rapid and unidirectional. However,movement rarely occurs in a straight line. The ectoplasm isclear, and the endoplasm is granular and may containbacteria and/or erythrocytes in various stages of digestion.On permanent stained slides, the trophozoite nucleus is

spherical and one-fifth to one-sixth the size of the trophozo-ite. The inner surface of the nuclear membrane is evenlylined with a rim of delicate chromatin. A small, compactkaryosome is generally located near the center of the nu-cleus. On permanent stained smears, the organism size maybe reduced by 1 to 1.5 ,um because of shrinkage by thestaining reagents (38) (Table 1).

PrecystIn the precyst stage, the trophozoite becomes approxi-

mately the same size as the cyst. The cytoplasm is cleared ofall food inclusions but usually contains diffuse glycogendeposits and occasional chromatoid bodies. The chromatoidbody is composed of ribosomes; however, its functional roleis uncertain (68, 81, 111). The precystic form is uninucleate,and the enlarged nucleus contains a karyosome that is moreor less eccentric.

CystA cyst wall develops around the precystic form, and the

single nucleus divides to form the mature quadrinucleatestage. Generally, glycogen and chromatoidal material disap-pear as the cyst matures. Once immature cysts are cooled toroom temperature, they cease to develop, and all the single-and double-nucleated cysts degenerate and die (35). Onpermanent stained slides, cysts ranges from 8.5 to 19 p.m indiameter. Again because of shrinkage caused by the dehy-dration reagents, cysts may be 1 to 1.5 p.m smaller thanorganisms seen on wet preparations. They are usually spher-

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I -

HK _

Origin

ME

OriginI

_ _ _ _

_ _ _ _

- m - - mm m m -

- m - - m__ _ _

- m m m_ _ _ _

------------------mmm m ---m

16GPI s _

Origin

- -_ m m m _

I- mmmmm minm

_ _

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16PGM zi

Origin a

Zymodeme

la--e

_ _- _ _ _ _

_ _ _ _ __

_ = xx2 > 5 ,> , X X X X X X R

FIG. 1. Zymodemes of E. histolytica identified by using GPI (EC 5.3.1.9), L-malate:NADP+ oxidoreductase (ME; oxaloacetatedecarboxylating) (EC 1.1.1.4.0), PGM (EC 2.7.5.1), and HK (EC 2.7.1.1). (Adapted from reference 131 with permission of the publisher.)Pathogenic zymodemes include II, II alpha, VI, VII, XI, XII, XIII, XIV, XIX, and XX. Nonpathogenic zymodemes include I, III, III alpha,IV, V, VIII, IX, X, XV, XVI, XVII, XVIII, and (not shown) XXI.

ical and contain four nuclei. The nuclear membrane isuniformly lined with peripheral chromatin. The karyosome issmall and usually centrally located within the nucleus.

MetacystDuring the process of excystation, the encysted ameba

containing four nuclei becomes very active, separating fromthe cyst wall. The quadrinucleate ameba escapes from thecyst wall through a tiny pore, and the nuclei clump together.

Metacystic TrophozoiteOutside the cyst, the nuclei within the quadrinucleate

ameba begin to separate from the surrounding cytoplasm andundergo division to form eight uninucleate metacystic tro-phozoites. The resulting trophozoites are always smaller (8,um) than the trophozoites seen in the bowel of an infectedhuman. The metacystic trophozoites continue to feed andgrow, finally achieving the size normally associated with thetrophozoite (Table 1).

PATHOGENICITY AS DETERMINED BY ZYMODEMESIn 1925, the pathogenic nature of E. histolytica was

questioned by Brumpt, who thought that there were twomorphologically similar strains of E. histolytica, one patho-genic and the other nonpathogenic (cited in reference 43).According to epidemiological studies, only 10% of theworld's population and a few homosexual males and AIDSpatients infected with E. histolytica develop disease (45, 68a,157). One explanation for this percentage is that most peopleare infected with nonpathogenic strains (43). Reeves andBischoff (122) were the first investigators to use enzymeelectrophoresis to differentiate "typical" from "atypical" E.histolytica. Typical E. histolytica were those organisms,including those associated with human disease, requiring

culture temperatures of 33 to 39°C, whereas atypical E.histolytica organisms could adapt to lower temperatures.This concept was further explored by Sargeaunt and associ-ates in a series of studies designed to differentiate E.histolytica from other intestinal amebae and to distinguishpathogenic strains (36, 134-138).A zymodeme is defined as a population of amebae in

which the electrophoretic mobilities of specific enzymesdiffer from those in similar populations. The enzymes usedto classify E. histolytica include glucosephosphate isomer-ase (GPI), hexokinase (HK), L-malate:NADP+ oxidoreduc-tase, and phosphoglucomutase (PGM) (Fig. 1, Table 2).Zymodeme analysis is performed by observing the migrationpattern of isoenzyme bands on a thin-layer starch gel or inpolyacrylamide gel electrophoresis (63, 82). Only one band isfound with L-malate:NADP+ oxidoreductase, whereas GPIand PGM show multiple bands that are labeled a through B.Blanc and Sargeaunt (10) have shown that the concentrationof starch in the growth medium can influence the appearanceof zymodeme patterns. Expression of y and 8 bands of GPIand PGM was observed more often when higher concentra-tions of starch were present in the culture media. HK ischaracterized by two bands, one migrating slowly and theother migrating faster. Pathogenicity is associated with thepresence of a fast-migrating HK band, a PGM I band, andno PGM a band. The only known exceptions are zymodemeXIII, which, although considered a pathogenic zymodeme,lacks a fast-migrating HK band, and zymodeme XXI (112).Organisms belonging to zymodeme XXI were recently iso-lated from a male homosexual. On the basis of clinicalhistory and serological tests, the isolate was considerednonpathogenic. The zymodeme pattern was unusual in thatthe isolate had a PGM a band and a fast-migratingHK band,a pattern which is often associated with pathogenic zymo-demes. Sargeaunt (130, 131) has shown a positive correlationbetween zymodemes and the clinical histories and serologies

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AMEBIASIS 359

TABLE 2. Differential characteristics of zymodeme patterns

Isoenzyme band Characteristicsor group Nonpathogenic isolates Pathogenic isolates

HK Fast migration Slow migrationPGM Beta AlphaL-Malate:NADP+ None None

oxidoreductaseGPI None NoneZymodeme groups II, II gamma, VI, VII, XI, XII, XIII,' I, III, III alpha, IV, V, VIII, IX, X, XV, XVI,

XIV, XIX, and XX XVII, XVIII, and XXI6a Zymodeme XIII is pathogenic but lacks the fast-migrating HK band.b Zymodeme XXI is nonpathogenic but has a fast-migrating HK band.

of more than 6,000 pathogenic and nonpathogenic E. his-tolytica patient isolates.

In order to perform zymodeme studies, nonaxenic culturetechniques must be used to isolate amebae from either feces,exudates, liver aspirates, or other suspect material. Themedia most frequently used are those described by Robinson(123) and Jones (54). Generally, three to nine subpassagesrequiring at least 2 days per subpassage are needed beforeenough organisms are obtained for enzyme electrophoreticanalysis. Although the cultures of amebae from patients arexenic, containing multiple bacteria, the bacterial enzymebands migrate faster than do those of E. histolytica. How-ever, it is still necessary to run bacterial and amebic controlswhen axenic cultures are not obtained. At present, zymo-deme analysis is not easily incorporated into routine clinicallaboratory work because of the expertise required, technicaldifficulties, and cost. Although microscopic diagnosis doesnot address the issue of pathogenicity, it is still the method ofchoice for detecting amebic infections. The ability to cultureorganisms from specimens determined by microscopic ex-amination to be positive is limited to approximately 50%(112, 144).By enzyme electrophoresis, 23 zymodemes of E. histolyt-

ica have been identified (Fig. 1). Of these, nine have beenassociated with pathology in the host. These include isolatesfrom stool in which the trophozoites contained ingestederythrocytes and isolates from intestinal ulcers and liverabscess material. Thirteen zymodemes have been obtainedfrom asymptomatic patients passing cysts.The zymodeme analysis data include information on iso-

lates obtained from various areas of the world (47, 96, 130,132). Even 9 years after the organisms were first isolated,Sargeaunt and co-workers have not detected changes inisoenzyme patterns with continued and repeated testing ofisolates. Interestingly, mixtures of nonpathogenic zymo-demes but no mixtures of pathogenic zymodemes have beenfound in the same specimen from the same patient. How-ever, two pathogenic zymodemes have been detected fromtwo specimen sources (stool and liver aspirate) from thesame patient (132). When specimens are cultured for E.histolytica, pathogenic strains always outgrow nonpatho-genic strains and all other amebae (112).

Clinically, patients who harbor pathogenic zymodemesgenerally have high antibody titers, whereas individuals withnonpathogenic zymodemes are either seronegative or havelow antibody titers (40, 135). Asymptomatic carriers ofpathogenic zymodemes and carriers of nonpathogenic zymo-demes have been found in many instances to self-cure within1 to 9 months (40, 44, 93). On the basis of this evidence, ithas been proposed that patients who have E. histolytica in

their stool specimens but no clinical signs of infection and noantibody titer need not be treated (40, 44, 66, 68a, 112).Jackson and Gathiram (52) conducted a seroepidemiologicalstudy in which they found asymptomatic individuals whowere serologically positive but harbored nonpathogenic zy-modemes. Whether these individuals were previouslytreated for invasive amebiasis was not addressed.

It has not been established whether the zymodeme pat-terns reflect genotypic or phenotypic traits. Sargeaunt (131)proposed that pathogenic and nonpathogenic E. histolyticaisolates represent distinct subspecies, whereas Mirelmanand co-workers and others (12, 14, 86, 87, 90, 97) havepresented evidence that zymodeme patterns may be a phe-notypic trait and that these patterns could change underenvironmental influences. If the latter were true, treatmentof only symptomatic patients or those who have a serologicalresponse could pose potential public health problems ifasymptomatic cyst passers who harbor pathogenic zymo-demes are not treated; they could serve as an importantreservoir for transmission.The reliability of differentiating between pathogenic and

nonpathogenic strains of E. histolytica on the basis ofphenotypic expression of isoenzyme patterns and the stabil-ity of the isoenzymes has been questioned (14, 86, 87, 97).Many authors have published information concerning theinfluence of bacteria on the virulence of amebae (7, 14, 94,97, 107, 159, 160). During the process of axenization, An-drews et al. (2) and Mirelman et al. (90) converted anonpathogenic zymodeme to a pathogenic one in culture.This change was manifested not only by changes in themobilities of the HK and PGM bands but also by changes inin vitro and in vivo pathogenicity. The isolate was capable ofdestroying tissue culture monolayers and inducing liverabscesses in hamsters. The ameba-bacterium associationand the influence of bacteria on the virulence of E. histolyt-ica have been reviewed by Mirelman (88). The ability tochange from a nonpathogen to a pathogen or vice versa doesnot appear to be a universal trait among E. histolyticaisolates (87). Some isolates do not undergo pathogenicconversion. Mirelman suggested that the pathogenic state ofE. histolytica is interchangeable and that virulence is ex-pressed only under unique growth conditions. However, themechanisms or conditions that induce these changes areunknown. Through the use of polymerase chain reaction,Mirelman et al. (89) have shown that nonpathogenic isolatescontain extrachromosomal circular DNA that hybridizeswith DNA probe P-145, which has been used to characterizepathogenic isolates. They suggested that the extrachromo-somal circular DNA characteristic of pathogenic isolateswas induced to amplify when changes occurred in bacterial

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flora in the growth medium. If the pathogenic status of anisolate can readily change because of environmental influ-ences, then all E. histolytica isolates would have to beconsidered potential pathogens.

Sargeaunt and co-workers (129, 133) and Blanc et al. (9)have shown that genetic transfer between pathogenic strainscan occur both in vivo and in vitro, with new zymodemepatterns different from those of the parental strains beingdetected. The possibility that mutation is responsible for thezymodeme pattern changes was believed to be remote be-cause the frequency of mutation was postulated to beextremely low (9).

Recent work strongly supports the conclusion that thereare two species of E. histolytica, one nonpathogenic and onepathogenic (24, 25, 102, 120, 144, 145, 147, 149-151). Geneticstudies do not support the genotype interconversion theorybut rather provide considerable evidence that two stablegenotypes exist (7, 124). Nevertheless, issues of mutation,changes in the cell surface that affect virulence, and whetherpathogenic forms have a nonpathogenic stage in the intesti-nal tract need to be addressed.

Recently, Strachan et al. (144) have used immunofluores-cence methods with monoclonal antibodies to the HK isoen-zyme to differentiate pathogenic from nonpathogenic ame-bae. Others have shown that monoclonal antibodies directedagainst membrane protein or the galactose-specific adher-ence lectin can be used as a rapid method of distinguishingpathogenic from nonpathogenic isolates after the organismshave been cultured (101, 144, 150). DNA probes that selec-tively hybridized to DNA from either pathogenic or nonpath-ogenic E. histolytica have been developed (39, 149). Use ofmonoclonal antibodies and DNA probes was as effective asisoenzyme electrophoresis for determining the pathogenicityof the isolate. However, neither technique currently allowsone to determine the zymodeme pattern, which may beuseful for epidemiological purposes, and the question stillunanswered is whether E. histolytica can change its patho-genic status as a result of environmental changes. All ofthese studies were performed with organisms from culture.

PATHOGENESIS AND PATHOLOGY

E. histolytica is unique among the intestinal amebaeparasitizing humans because it is able to invade tissue. Afterthe host ingests cysts, metacystic trophozoites escape fromthe metacyst in the gastrointestinal tract and colonize thececum. At present, the mechanisms of colonization andpathogenicity are not well understood (43). Bacterial florahave always been thought to have a direct role in thevirulence of E. histolytica and its ability to colonize the host(14, 86, 106). Clinical syndromes associated with amebiasisvary with the host and the organism. Syndromes may rangefrom an asymptomatic infection to a disseminated fataldisease. When the infection disseminates to extraintestinalsites, it is found most frequently in the right lobe of the liver(113).Research on the mechanisms of pathogenicity has evolved

along two pathways: one considers pathogenesis to bedependent on export of soluble toxins, and the other consid-ers it to be dependent on cellular contact (31). It has beenpostulated that amebae can export pore-forming proteins(amebapores) that form aqueous pores in target cell surfacemembranes and also produce cytotoxic products that arereleased from the organism and cause the initial cytotoxiceffect on the host cell (62, 74, 76, 115, 161). This pore-forming peptide was recently purified by Leippe et al. (69).

Recent studies have focused on contact-dependent killing bythe trophozoite, including adherence, extracellular cytoly-sis, and phagocytosis. Host cell leukocytes also play a majorrole in pathogenesis (45, 152, 158).A number of E. histolytica adherence receptors have been

identified, but the galactose-specific lectin receptor has beenthe most thoroughly studied and is thought to be responsiblefor mediating attachment to colonic mucus and colonicepithelial cells (71, 78, 79, 83, 101, 104, 116). Human colonicepithelial cells and mucus contain large numbers of galactoseor N-acetylgalactosamine residues (23). Incubation of E.histolytica with colonic mucin prevents trophozoites fromattaching to and killing target cells such as those in theintestinal epithelium (23). E. histolytica also enhances mucussecretion, which correlates with its pathogenicity (22). Morevirulent strains stimulated mucus secretion and depletedgoblet cells of mucin, thereby making epithelial surfacesmore vulnerable to invasion.The binding to mucin was reversed by the addition of

galactose and was completely inhibited by monoclonal anti-bodies directed against the amebic lectin receptor (23, 103).Monoclonal antibodies directed against the galactose-spe-cific lectin have also been found to enhance adherence (102,105). Monoclonal antibodies to the galactose-specific lectinreceptor have been used to differentiate pathogenic andnonpathogenic strains, and the primary structure of thislectin has been sequenced (80, 102, 148). It has been notedthat a galactose-specific lectin receptor is present on non-pathogenic zymodemes; however, it is antigenically altered(102). The galactose-specific lectin receptor on pathogenicstrains is a potential target for therapy or vaccine produc-tion. Petri and Ravdin (103) were able to immunize animalswith galactose-specific adherence lectin and protect themfrom liver abscesses.

Leitch et al. (70) postulated that amebae may invade theintestinal epithelium in areas where the mucous blanket isthin and there are fewer galactose or N-acetylgalactosamineresidues to protect the host. Penetration of the mucousblanket was thought to be due primarily to mechanicalameboid movement and not to require enzymatic digestion.Talamas-Rohana and Meza (146) have shown that E. his-tolytica binds to a receptor on fibronectin, a glycoprotein, ofthe target cell. Whether this receptor contains galactose orN-acetylgalactosamine residues was not discussed. Once theorganism was bound to the fibronectin, there appeared to bechanges in its cytoskeleton, and the organism rapidly de-graded and internalized the fibronectin receptor.

Pore-forming proteins that help destabilize the host celland bring about cytolysis have been studied by a number ofinvestigators, but no conclusive role for them in cytolysis byE. histolytica has been provided (62, 69, 76, 125, 161). Thepore-forming amebapore protein is transferred from thetrophozoite to the target cell, causing a disruption of thetransmembrane gradient and contributing to the cell's deathby the colloid-osmosis lysis mechanism. Tachibana et al.(145) postulated that the monoclonal antibody they used wasdirected against the pore-forming protein found only inpathogenic isolates.Luaces and Barrett (73) and Muller et al. (92) have

reported finding extracellular proteinases and hydrolases inE. histolytica. No direct role in host cell lysis has been foundfor these enzymes, but they might be involved in amebicinvasiveness at intercellular junctions, thereby allowingamebae to invade host tissues. Cysteine proteinases are themajor proteolytic enzymes detected in E. histolytica (30, 60,61, 121). Cysteine proteinase can degrade cellular attach-

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ment and matrix proteins such as collagen, laminin, andfibronectin. There is a direct correlation between the amountof proteinase activity and the pathogenicity of E. histolytica(30, 60, 100, 121, 150, 153). Pathogenic isolates secrete largeramounts of proteinase than do nonpathogenic isolates, andthe expression of mRNA is 10- to 100-fold greater in patho-genic than in nonpathogenic zymodemes (60, 121, 150).The effects of calcium and phorbol esters on lipases and

proteinases in the cytolytic event have been studied byLong-Krug et al. (72), Ravdin et al. (118, 119), and Weikel etal. (156). Release of intracellular calcium increased theactivity of phospholipases, and phospholipase increased thehemolytic activity of E. histolytica through the release offree fatty acids (126). Phorbol esters were noted to increasethe cytolytic activity of E. histolytica, whereas sphingosineinhibited cytolytic events. These two compounds are knownto stimulate and inhibit the activity of protein kinase C,which is thought to have a major role in the cytolytic event(29, 156). The definitive chronology of cytolytic eventsawaits further studies.

Asymptomatic Infection

Most human infections are asymptomatic, with the para-site acting as a harmless commensal organism in as many as90% of those infected (31, 45). Individuals harboring theorganisms may have either a negative or a weak antibodytiter and show no occult blood in stool samples, and cystscan usually be detected in the stool when examinations forova and parasites are done. If trophozoites are detected,they do not contain any phagocytized erythrocytes. Isoen-zyme analyses of organisms isolated from asymptomaticindividuals have usually shown that the isolates belong tononpathogenic zymodemes (130).Many asymptomatic individuals never become sympto-

matic and usually excrete cysts in the stool for only a shorttime, as shown by repeat examinations for ova and parasitesor by culture. This pattern is found in persons infected withpathogenic and nonpathogenic strains (1, 4, 16, 40, 44, 93).

Symptomatic Infection

About 10% of individuals infected with E. histolytica haveclinical symptoms, with intestinal and/or extraintestinal pa-thology. Although this proportion of infected individuals issmall, the disease represents a major public health problemin terms of morbidity and mortality in Third World coun-tries. Patients with noninvasive infection may exhibit non-specific gastrointestinal symptoms, including abdominal painand increased frequency of bowel movements, that may beintermittent or chronic. When amebae invade host tissues,only trophozoites are seen and the organism loses its abilityto encyst. Amebic invasion of the intestinal mucosa occursmost frequently (in descending order) in the cecum, ascend-ing colon, sigmoid colon, appendix, descending colon, andtransverse colon (56). The symptoms associated with inva-sive intestinal amebiasis are usually nonspecific. The onsetof symptoms is usually gradual, with abdominal pain, diar-rhea, dysentery, or weight loss occurring, depending on theseverity of disease. Almost all patients have occult blood inthe stools, and nearly one-third exhibit fevers. Histologicalexamination of tissue may be required to rule out malignancyand inflammatory bowel disease.When amebic colitis is detected, it is usually (70% of

cases) associated with segmentary ulceration of the colon(21). If ulceration with perforation occurs, it occurs mainly

in the cecum (53). Dissemination of amebic infection toextraintestinal sites most frequently involves the liver,lungs, pericardium, brain, and skin.

Colonic mucosal invasion begins in the interglandularepithelium, where the overlying mucous layer is noticeablythin (110). With initial microulceration, there is an outflow oftissue fluids, erythrocytes, neutrophils, lymphocytes, eosin-ophils, plasma cells, epithelial cells, and Charcot-Leydencrystals (108). The inflammatory response to amebic inva-sion is minimal and may be due to the lysis of tissue cells byE. histolytica. One or several ulcerations may be present,but if there are several ulceration sites, they are usuallyseparated. At first, the ulceration is superficial, and cellularinfiltration and tissue necrosis are limited to the invasionsite. Proctoscopic examination reveals an edematous andfriable mucosa. The ulcer has a pinhead center that iscomposed of eosinophilic granular debris, some inflamma-tory cells, and E. histolytica trophozoites. One can seenormal mucosal surface between ulcers (109). As the ulcerdeepens and progresses, it forms the classic flask-shapedulcer of amebic colitis, which extends from the mucosa andmuscularis mucosa into the submucosa. When ulcers co-alesce, the mucosa undergoes necrosis and sloughs. Amebaeare found on the advancing edges of the ulcer and usually notin the necrotic areas. Many times, the ulcers become sec-ondarily infected with bacteria. "Toxic megacolon" is theresult of total confluence of ulcerations and necrosis of thecolon. This form of amebiasis has a very high mortality rate.Ameboma, the result of chronic ulceration, is predomi-

nantly found in the cecum, sigmoid colon, and rectum (15).These tumorlike lesions are often mistaken for malignanciesand are sometimes present as palpable masses. Histologi-cally, the ameboma is nonfibrotic and contains granulationtissue with lymphocytes and giant cells (15).

Perforation of the colon by an amebic ulcer occurs in asmany as 21% of patients with acute amebic colitis (15) andmay result in peritonitis and liver abscess. Perforationsoccur most commonly in the cecum (58). Peritonitis isfrequently the result of liver abscess rather than intestinalperforation, but the latter has a high mortality rate (91).Amebic infection of extraintestinal sites is the result of

previous intestinal infection. Liver abscess can occur con-currently with colitis; however, there is frequently no clini-cal history of recent E. histolytica intestinal infection orevidence of parasites in stool specimens. Onset may beacute, with abdominal pain and fever, or subacute, withweight loss; less than 50% of patients have fever or abdom-inal pain. Patients may have leukocytosis without eosino-philia, elevated alkaline phosphatase and transaminase lev-els, and a high erythrocyte sedimentation rate. E. histolyticatrophozoites infecting the muscularis mucosa frequentlydigest the walls of mesenteric venules, enter the circulation,and are carried to the liver via the portal venules (15). Thetrophozoites are detected more frequently in the right loberather than the left lobe of the liver, perhaps because ofincreased perfusion of the larger liver mass (15). Liverabscess is diagnosed more frequently in adults (ages 20 to 60years) than in children and more frequently in males than infemales (142).The liver abscess has a thin capsular wall with a necrotic

center composed of a thick fluid, an intermediate zone ofcoarse stroma, and an outer zone of nearly normal tissue thatis being invaded by amebae (15). The contents of the abscesshave been described as resembling anchovy paste because ofthe light brown color. Generally, the abscess is bacteriolog-ically sterile. Secondary bacterial invasion of an amebic

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TABLE 3. Laboratory diagnosis of amebiasis: specimen typea

Sample Fixative Examination Stain

Stool PVA, 10% Formalin, Schaudinn's Concentrate, permanently stained slide Gomori trichrome, iron hematoxylinfixative, SAF

Sigmoid colon PVA, Schaudinn's fixative Permanently stained slide Gomori trichrome, iron hematoxylinAspirate

Direct None Wet mount with or without enzymedigest

Fixed PVA, Schaudinn's fixative Permanently stained slide Gomori trichrome, iron hematoxylinBiopsy Formalin Routine histology PAS, H&EBlood None Serology" None

a PVA, PVA with either HgCl2 or CuS04; SAF, sodium acetate-acetic acid-Formalin; PAS, periodic acid-Schiff (organism stains intensely pink and has adistinct outline, but cytoplasmic and nuclear details are obscured); H&E, hematoxylin and eosin stain (cytoplasm is pink and nucleus is blue; in sections, thenucleus may not always be present).

b Standard serological tests include indirect hemagglutination, counterimmunoelectrophoresis, indirect fluorescent antibody, and enzyme immunoassay.

abscess occurs infrequently. Microscopic examination of theabscess fluid reveals granular eosinophilic debris with no orfew cells (67).

In animal models, amebae are not directly responsible forthe damage to the liver cells. The tissue destruction appar-ently results from lysis of leukocytes and macrophages bythe amebae (152). Neutrophils are attracted to amebae andare rapidly killed on contact with E. histolytica. Althoughthe neutrophils have little or no cytolytic effect on thevirulent organisms (46), the release of toxic intracellularproducts from these destroyed host cells promotes lysis andnecrosis of the hepatic cells.

If the amebic lesions heal, they invariably heal withoutforming scar tissue (15). Because of the cytolytic action ofthe amebae, leukocytes may be prevented from accumulat-ing in the abscess area, thereby inhibiting the initiation of acellular immune response. E. histolytica trophozoites inhibitthe mobility of monocytes but not leukocytes (42). Mono-cytes (macrophages) play a crucial role in any wound-healingprocess by stimulating collagen deposition (37, 64, 65).

DIAGNOSISE. histolytica infections can be diagnosed in the laboratory

by the detection of organisms in stool preparations, sigmoid-oscopy smears, tissue biopsy samples, or hepatic aspiratesamples or by serological tests (Table 3). Organism detectiondepends on proper specimen collection, processing, andexamination by trained personnel. Examination for ova andparasites in a minimum of three stool specimens, usingconcentration and permanent stain techniques, is the recom-mended standard for the detection and identification of theorganism (38). Reliance on inadequately trained personnelfor laboratory diagnosis may lead to missed infections or,more commonly, false-positive results because of misidenti-fication of leukocytes as E. histolytica. Although wet mountsof fresh specimens can be screened for motile trophozoitescontaining erythrocytes, most patients do not harbor tropho-zoites with erythrocytes. If fresh stool samples are exam-ined, they should be examined within 30 min after passage ofthe specimen, not 30 min after arrival in the laboratory. If thespecimen cannot be examined within 30 min, it should bepreserved in a suitable fixative such as polyvinyl alcohol(PVA). Although some textbooks advocate the use of directwet mounts to identify E. histolytica, the identificationshould always be confirmed by using a permanent-stainedslide. The importance of the stained slide cannot be over-emphasized, particularly when organisms are few in numberin the stool specimen.

Even if sigmoidoscopy is performed, a minimum of threestool specimens should also be collected and examined. Nofewer than six areas of the mucosa should be sampled, withexamination slides made at the bedside or in the examinationroom. Material scraped or aspirated from the mucosal sur-face can be examined by three methods. If there is no delay,a wet mount in 0.85% NaCl can be made and examined forameboid movement. It may be several minutes after theNaCl preparation has been made before the amebae exhibitany motility. Microscopic examination may be difficult be-cause of the need for low light intensity and difficulties indifferentiating the unstained amebae from cellular material.In wet preparations, other protozoa such as E. coli mayresemble E. histolytica (Table 3). Only careful examinationwill distinguish amebae from other host cells, such aspolymorphonuclear leukocytes, which mimic E. histolyticacysts, and macrophages, which may be mistaken for E.histolytica trophozoites (Table 4). The mucosal material mayalso be smeared on a slide and immediately immersed inSchaudinn's fixative. An alternative method of preparing apermanent fixed-slide specimen is to add 2 to 3 drops ofPVAdirectly to the mucosal material directly on the slide, mix it,and allow the slide to air dry. These two methods provide apermanent mount that can be stained with trichrome stain(38).

Histologic examination of tissue biopsy sections can bedone with periodic acid-Schiff and hematoxylin-eosin stains(Table 3). In histologic material, all the nuclear detail maynot be seen in one organism, and sequential sections mayhave to be examined to identify the organism. The amebaemust be differentiated from normal host cells such as histi-ocytes. Hepatic aspirates can be examined for the organism,but this examination is rarely performed and rarely donecorrectly. Aspirated material should be collected in a num-ber of different containers as it is obtained from the abscess.The amebae are sparse in necrotic material from the centerof the abscess, but they are more abundant on the marginalwalls. Therefore, they are more commonly found in the lastportions of aspirated material. Enzyme liquefaction has beenused to facilitate the search for organisms by freeing theorganisms from aspirate material (5). Demonstration of theorganisms is often very difficult because they may be trappedin viscous pus or debris and will not exhibit typical motility.

In extraintestinal amebiasis, organisms may or may not befound in the stool; therefore, one of the many types ofavailable serological tests may be necessary for diagnosis(48, 99). Serology should not be used solely for the diagnosisof intestinal amebiasis, because antibody titers may be low

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TABLE 4. Differential characteristics of host cells and E. coli, commonly mistaken for E. histolytica, in wet preparations

NucleusDiamorlength ~~~~~~~~~~~~CytoplasmicCell Diam or length Motility Visibility (ratio of appearance Inclusions(Lm) No. nuclear material to (stained)

cytoplasm)

E. histolyticaTrophozoites 12-60; usual Progressive, with hyaline 1 Hard to see in un- Clear differentiation Presence of erythro-

range, 15-22 fingerlike pseudopodia; stained prepns (1: of ectoplasm and cytes diagnosticmay be rapid 10-1:12) endoplasm; vacu-

oles usually small,if present; lookslike ground glass

Cysts 10-20; usual None 1-4 1:2-1:3 Clear Chromatoid bodiesrange, 12-15 (stained) may be

present; usuallyelongate withblunt, smooth,rounded edges;round or oval

E. coliTrophozoites 15-50; usual Sluggish, nondirectional; 1 Often visible in un- Granular, little dif- Bacteria, yeast cells,

range, 20-25 blunt, granular stained prepn ferentiation into other debrispseudopodia ectoplasm and en-

doplasm; usuallyvacuolated

Cysts 10-35; usual None 1-8 >16 nuclei seen oc- Clear Chromatoid bodiesrange, 15-25 casionally (stained) may be

present, less fre-quently than in E.histolytica; splintershape with rough,pointed ends

Host cellsPolymorphonuclear Avg, 16 None 1 2-4 segments; if Granular None

leukocytes lobed nucleus frag-ments may mimicthe 4 nuclei foundin E. histolyticacyst (1:1)

Macrophages 20-60; may be Sluggish 1 Large, may be irreg- Coarse; may be Usually contain in-5-10 ular in shape (like highly vacuolated gested debris,

monocyte); may polymorphonu-mimic E. histoljt- clear leukocytes,ica trophozoite; and erythrocytescan also ingesterythrocytes

and/or impossible to interpret, particularly with patientsfrom an area of endemicity where a high prevalence ofseropositivity already exists. Asymptomatic cyst passersusually have negative serological test results, but there isgood correlation between infection with pathogenic zymo-demes and positive serological tests (130). Positive indirecthemagglutination, indirect fluorescent-antibody, and immu-nodiffusion tests give results that correlate well with thepresence or absence of an amebic liver abscess. An impor-tant major problem common to most serological tests is thepersistence of antibody for prolonged periods (up to 2 years)after diagnosis and therapy. These tests should therefore beinterpreted with caution. However, indirect fluorescent-antibody titers decrease within 6 to 12 months of successfultherapy. Serological tests can also be used to rule outamebiasis in patients with inflammatory bowel disease (49).Although numerous methods for the serological detection

of amebiasis have been reported in the literature, the use ofthese methods in clinical laboratories is severely limited bythe unavailability of commercial reagents or test kits (38).

Newer diagnostic methods being proposed include the use ofpurified antigens and assays for immunoglobulin M and Aantibodies (6, 28, 55, 102, 117, 139). The Centers for DiseaseControl offer amebic serological tests. Submission regula-tions vary from state to state; therefore, laboratories shouldcheck their county or state public health department forguidelines.

Recently, culture techniques with Robinson's medium,Diamond's medium, Jones's medium, and Locke's egg yolkserum medium in conjunction with isoenzyme electrophore-sis have been used to differentiate infections caused bypathogenic and nonpathogenic E. histolytica strains (85-87,136). At present, this technique is not practical for routineclinical laboratories. Results usually are not available for 4days or more. Even though a patient may be diagnosed byclinical history, serological tests, or zymodeme analysis asharboring a nonpathogenic strain, the question of whether ornot the strain will revert to pathogenicity in vivo has beenraised (42, 85-87, 89, 130, 131).To provide clinically relevant information to physicians

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for treatment of patients infected with pathogenic strains ofE. histolytica, methods using monoclonal antibodies, puri-fied antigens, or DNA probes may be required (8, 13, 39,102, 127, 143, 144, 147, 151). Although there have beennumerous reports of using techniques such as enzyme im-munoassay to detect antigens in specimens, none of thesemethods are routinely used in clinical laboratories (84). Ifzymodeme patterns are found to remain stable, these tech-niques could be incorporated into a routine diagnostic labo-ratory, provided that they can be used directly on clinicalspecimens rather than after the isolation of organisms byculture. Zymodeme patterns could be very useful for epide-miological purposes in tracking organism transmission andpotential differences in organism invasiveness, virulence,and therapeutic susceptibility. The use of purified antigensand RNA or DNA probes for routine diagnostic testingwould offer significant improvements over current serologi-cal or direct detection methods. The serological techniquescommonly used for diagnostic purposes are not sensitive orspecific enough to be solely relied on clinically. Reliableprobes would help to eliminate false-positive test results dueto misidentification of E. coli or host cells such as polymor-phonuclear leukocytes or macrophages. Commercial kitsthat make use of purified antigens or DNA probes are notavailable at this time. For a more comprehensive review oflaboratory diagnostic techniques, see Garcia and Bruckner(38).

THERAPY

Two classes of drugs are used in the treatment of amebicinfections. Luminal amebicides, such as iodoquinol anddiloxanide furoate, act on organisms in the intestinal lumenand are not effective against organisms in tissue. Tissueamebicides, such as metronidazole, chloroquine, and dehy-droemetine, are effective in the treatment of invasive ame-biasis but less effective or ineffective in the treatment oforganisms in the bowel lumen.

Asymptomatic Patients

Asymptomatic patients who are cyst passers may betreated with diloxanide furoate, iodoquinol, or paromomycin(38, 81, 112, 128, 140, 141). Like iodoquinol, diloxanidefuroate is a luminal amebicide. Diloxanide furoate is avail-able only from the Centers for Disease Control on specialrequest. Although known as a luminal amebicide, diloxanidefuroate is readily hydrolyzed in the intestinal tract andabsorbed into the bloodstream. For asymptomatic patientswho are passing both cysts and trophozoites in the stool, therecommended treatment is metronidazole plus iodoquinol(38, 81, 128, 140, 141). From zymodeme analysis, it has beendetermined that many asymptomatic patients harbor non-pathogenic zymodemes, and the use of drug therapy in theseindividuals is controversial. As discussed above, methods todifferentiate pathogenic from nonpathogenic E. histolyticastrains are not easily incorporated into routine clinicallaboratory use. Therefore, from a public health viewpoint, itis best to treat all infected patients.

Symptomatic Patients

The treatment of symptomatic patients varies with theclinical stage of the infection. Different therapeutic ap-proaches are employed for intestinal and extraintestinalamebiasis (38, 81, 114, 128, 140, 141).

Mild intestinal disease due to amebic dysentery should betreated with metronidazole plus iodoquinol or with metro-nidazole plus diloxanide furoate. Patients should be warnedto refrain from ingesting alcohol when taking metronidazole,for they may have a reaction similar to that to disulfiram.Symptoms may be abrupt and severe and include abdominalpain, nausea, vomiting, and headache. The safety of iodoqui-nol taken during pregnancy has not been determined. Incases of dehydration, fluid and electrolyte replacement maybe necessary.

Extraintestinal Disease

A number of combination therapies have been used fortreating extraintestinal amebiasis (38, 81, 114, 128, 140, 141).These include metronidazole and dehydroemetine with chlo-roquine. Chloroquine is useful for treating amebic liverabscess because it is concentrated in the liver; however,chloroquine is not useful for intestinal amebiasis. Dehydro-emetine can be toxic to the heart, so patients should bemonitored throughout therapy. Although surgical drainageof larger abscesses may be necessary, aspiration of small andmoderate-sized abscesses can usually be avoided. Smallerabscesses resorb, and their disappearance can be monitoredby scanning procedures (computerized tomography, liverscan, or ultrasound). Extraintestinal therapy should be fol-lowed with a luminal amebicide such as iodoquinol ordiloxanide furoate.

Follow-Up

All treated patients should be followed up clinically andwith laboratory tests. Patients who had positive stool sampleresults should have follow-up stool examinations at 2 to 4weeks posttherapy. The stool examination must include apermanent stained slide. Patients with extraintestinal ame-biasis should show a clinical response after therapy and canbe monitored with the scanning procedures mentionedabove. Although antibody titers in serum may remain ele-vated for prolonged periods and may be of little use infollow-up, indirect fluorescent-antibody titers have beenshown to decrease within 6 to 12 months of successfultherapy.

SUMMARY AND FUTURE DIRECTIONS

Although approximately 12% of the world's population isinfected with E. histolytica, only 10% of those infected willdevelop clinical symptoms, and of this group, only 2 to 20%will have invasive disease. It has been postulated that thelarge number of asymptomatic people are infected with anonpathogenic strain that cannot be distinguished morpho-logically from pathogenic strains. It has been shown thatpathogenic and nonpathogenic strains can be differentiatedby the use of isoenzyme (zymodeme) analysis, monoclonalantibodies, and molecular probes, and the results correlatewith clinical symptoms and serological tests. Whether theisoenzyme patterns are a phenotypic or genotypic trait willhave to be answered by future research. It is possible totransfer genetic material between organisms, thus transform-ing one pathogenic zymodeme into a completely differentpathogenic zymodeme, and the presence or absence ofspecific bacterial flora has been associated with the intercon-version of nonpathogenic and pathogenic strains in culture.Mirelman (87) found that nonpathogenic E. histolytica iso-lates contain extrachromosomal DNA that hybridizes with

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pathogen-specific DNA probes. However, through the use ofrecombinant DNA technology, two genetically distinctforms of E. histolytica have been found to exist. Geneticanalysis has shown substantial differences between the non-pathogenic and pathogenic forms. Until our understanding ofthe pathogens is elucidated, there will continue to be ques-tions concerning the influence of bacterial flora and geneticexchange between isolates. Because of the uncertainty ofisoenzyme stability, the expression or repression of patho-genic genes, or transfer of genetic material between organ-isms, the designation of an organism as nonpathogenic orpathogenic should not be solely relied on for therapeuticpurposes.Although recent research has helped to elucidate the

molecular mechanisms of organism attachment, cytolysis,and phagocytosis of host cells, pathogenesis is still not wellunderstood, even though E. histolytica has been studied forover 100 years. Most recent work cited has dealt with thegalactose-specific lectin receptor of E. histolytica. Preven-tion or alteration of attachment may eliminate the pathoge-nicity of this organism. The sequencing of this receptor andothers involved in attachment may lead in the near future toa vaccine or a specific therapeutic agent that will prevent orreduce the pathological sequelae associated with pathogeniczymodemes.

Diagnosis of E. histolytica infections depends primarily onexaminations for ova and parasites and on serological tests.There are no commercially available or practical and reliablemethods for differentiating pathogenic from nonpathogenicstrains for routine use in clinical laboratories. Currently,there are no antigen tests for routine clinical laboratory use,and monoclonal antibodies and DNA probe tests have beenused only with culture isolates to detect pathogenic zymo-demes.With the current interest in developing genomic libraries

(51) and in using amplification methods to selectively look atspecific functions of the organism, our knowledge of E.histolytica will be considerably enhanced. Amplificationmethods and serological tests that are sensitive and specificenough to directly detect very small numbers of organismsare currently needed. In the near future, clinical laboratorieswill be evaluating a number of new test products for diag-nosing infections caused by pathogenic E. histolytica.

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3. Balamuth, W. 1946. Improved egg yolk infusion for cultivationofEntamoeba histolytica and other intestinal protozoa. Am. J.Clin. Pathol. 16:380-384.

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