Jpn. J. Infect. Dis., 53, 229‑237, 2000

Review

Current Problems ofAmebiasis in Japan andRecent Advances in Amebiasis Research
Tbmoyoshi Nozaki*
Department ofParasitology, National Institute ofInfectious Diseases,

Toyama 1‑2311, Shinjuku‑ku, TobJo 162‑8640, Japan(Received November 22, 2000. Accepted January 24, 2001 )CONTENTS:
1. Introduction

2. Epidemiology of amebiasis in Japan
3. Cu汀ent problems or amebiasis(1)Diagnosis(2) Treatment(3)Vaccine
4. Recent developments in basic research on amebiasis( 1 ) Virulence factors(2) Peculiar organellar stmcture(3) Prokaryote‑like energy metabolisms

5･ Remainlng problems ln amebiasis research

SUMMARY: Amebiasis is epidemic in two major populations in Japan: male homosexuals and institutionalized
people. Cu汀ently available diagnostic, chemotherapeutic, and prophylactic measures and their problems are

discussed. Recent advancesinbasic research on amebiasis are also described withnewfindings ofunlquemetabolisms and intracellular structures and organization. Discoveries and analyses of the unique features
presented by this parasitic protist help in our eluci血tion of the pathogenic mechanisms of the parasite and may

eventually lead to the development of new drugs and vaccines against amebiasis.

will also be discussed.

1. Introduction

The eukaryotic protozoan Entamoeba histolytica is a major

2. Epidemiology of amebiasis in Japancause of morbidity and mortality worldwide (I). There arean estimated 50 million cases of invasive amebiasis andCases ofAmebiasis have often been found in two groups

40,000‑ 1 1 0,000 deathanpually (1 )･ The most common clinicalin JapaT in male homosexuals (3‑7) and in institutionalized

presentation of amebiasIS is amebic dysentery and colitis;

people, lnCluding the residents of institutions f♭r the mentally

extraintestinal abscesses (i.e. , hepatiC, pulmonary,and cerebral)

retarded (8‑13)･ The high prevalen?e of amebiasis in maleare, however, also common and o洗en lethal. infection of thehomosexuals is aunlque CharacteristlC Of amebiasis in Japan.

human host occurs upon lngeStion of water or fわod contami‑

也 the early 1 980S, a high percentage of血e male homosexuals

nated bythe cyst form of the parasite. These cysts then excyst toform ameboid trophozoitesthat subsequently colonize the large

in the United States (U.S.) and Europe were reported to be
infected with E. histolytica. However, neither the presencebowel. Colonization of the human gut by E. histolytica mostof clinical symptoms nor positive serology co汀elated with thecommonly results in an asymptomatic intestinal infection

presence of the parasites in the stool (14,15). Furthermore,similar to that resulting Bom the closely related commensal

biochemical and molecular characterization of the isolates

E. dispar spp. (2). In approximately 5‑ 10% of those infected

kom the infected individuals showed that the amoebas fromwith E. histolytica, disease and symptoms including bloody

the male homosexuals were not E. histolytica, but exclusively

diarrhea, fever, tenesmus, and colitis develop. However, theE. dispar, a non‑pathogenic species morphologically ln‑

rest of the infected individuals remain asymptomatiC.lmmun0‑

distinguishablefrom E. histolytica (I 6‑1 8). It is now widely

logical, biologlCal, and genetic mechanisms that contribute toaccepted that the male homosexuals in westem countries arewhether or not a disease develops have not been elucidated.

infected with E. dispar, but not with E. histolytica. This is in

The aim of this review is to summarize the current situation

sharp contrast to the situation in Japan, where the maleregarding amebiasis in Japan, and to highlight the remainlng

homosexual population exhibited symptoms attributable toproblems of diagnosis, treatment, and inteⅣention. Recent

invasive amebiasis and seropositiveness to the E. histolytica

advances in basic research to understand the pathogenesis

antigens ( I 9,20). Furthermore, zymodeme analysis revealed

and biochemical and biologlCal peculiarities of this organismthat the isolates from Japanese male homosexuals have*Corresponding author: Tel: +81‑31528511 1 I 1 ext. 2733, Fax:

tlOn between seropositiveness to the E. histolytica antigens+81 ‑3‑5285‑1 173, E‑mail: nozaki@nih.go.jp

and that to Treponema pallidum hemagglutination test

Tmodemes indicative of E･ histolytica (2 1 )･ A high correla‑229

Lyophilization Laboratory, Tokyo, discontinued) and indirect(TPHA) was reported for Japanese.male homosexuals (5･ I 9),strongly suggestlng that amebiasIS in this population is aimmunonuorescence assay (Ameba‑spot IF, #72901,

sexually transmitted disease.

Biomerieux, Tbkyo), are commercially available.

The prevalence of amebiasis in institutionalized people,(2)Treatment
Aspreviously mentioned, E. dispar cyst passers requlree.g., the residents of institutes f♭r the mentally retarded, hasalso been reported several times sin.ce late 1980s in Japan(8‑1 3). A high prevalence of amebiasIS in institutions was also
reported in the U.S.,and believed to be associated with typicalno treatment. This may pose the medical care provider with aabnormal behavior of the retarded (22). Besides the residents

not have immediate access to laboratories where isoenzyme

of institutes and daycare centers, a small percentage of healthcharacterization and PCR are routinely performed. However,question: whether or not asymptotic cyst passers need to betreated, because most physicians and clinical lab workers docare workers are also found to be infected (e･!･, 2%, [12])･

consultation with these laboratories is strongly encouraged

There are also qulte a few cases of imported invasive amebiasis,

because asymptomatic E. histolytica cyst passers are awhich presumably constitutes up to 20% ofall cases in Japanpotential threat to public health, and there is also have a possi‑(e･g･, 【7])･ In ャontrast to the adult domestic cases, which arebility of future relapse. Freezing a Stool specimen nomallyalmost exclusIVely male, there is no reported bias on genderpreserves the integrlty Ofparasite DNA for at least a few

in the imported cases. However, it has been shown by fieldmonths for future identification of the infecting parasites bystudies conducted in Ⅵetnam that males have a higher riska variety of PCR‑based methods, and therefわre is strongly

of developing liver abscesses than females (Tannich etrecommended. After a diagnosis is made, the E. histolyticaal., unpublished), suggestlng that gender bias regarding acyst passers should be treated with one of the lumen‑actlng

susceptibilityto amebic liver abscess exists.

amebacides described below.

The commonly used anti‑ameba drugs metronidazole(Fragyl) and tinidazole (Fasigyn) have been proven to be

3. Current problems of amebiasis

effective agalnSt invasive amebiasis, including both intestinal
and extraintestinal amebiasis. Extensive clinical experience(1) Ding.nosis

A majontyof all diagnoses of amebic infection have been,and epidemiologic data showed that these compounds are notand still are, based on light microscopic demonstration ofcarci.nogenic or mutagenic in humans (23)･ Chloroquine,

hematophagous trophozoites in stool or liver asplrate. Physi‑

emetlne, and dehydroemethine have been successfully added

cians and lab teclmicians need to be careful not to overdiagnose

to metronidazole treatment for the rare patient not respond‑

E. histolytica by mistakenly identifying non‑pathogenic
amoebae, macrophages, or white blood cells. Unfortunately,
microscopy does not differentiate between E. histolytica anding to metronidazole or tinidazole alone (24,25). However,these compoundsare well absorbed from the intestinal Iumen,
and, therefore, are generally not effective in the treatment of

E. dispar. This is notusually a problem in diagnoslng lnVaSive

asymptomatic cyst passers unless ther叩y lS prolonged f♭r aamebiasis, since a demonstration of hematophagous tropho‑

minimum of I 0 days. With shorter courses of metronidazole

zoites in the specimen represents a definitive diagnosis.

therapy, patients may suffer a relapse of invasive amebiasis

However, asymptomatic cyst passers of either血e pathogenic

months later (24,25). The lumen‑acting drugs diloxanide

E. histolytica or E. dispar need to be differentiated, since cyst

furoate (Furamide), iodoquinol (Diodoquin), and paroPomy‑passers of E. dispar do not requlre treatment. In contrast,
asymptomatic E. histolytica cyst passers must be treated sincepassersandalso to prevent relapsesfrompersIStent Cysts a飴rthey are a potential public health hazard, and long‑tらminvasive amebiasis. Successful treatment of the cyst passers

camage could lead to later acute invasive disease.

with metronidazole only or with metronidazole and diloxanide

A conventional method of differentiating the two ameba
E. histolyticaand E. dispar is cultivationand propagation of

metronidazole was also reported for several Japanese casescin (Humatin) are oRen used to treat asymptomatlC Cyst

furoate on 5 consecutive days followed by 5 more days of

the isolated amoebae, followed by zymodeme profiling by(1 I,13). Whether or not treatment is effective for each cyst

isoenzyme electrophoresis. However, these procedures are

passer could depend upon biologlCal differences in thetime‑ and labor‑consumlng, and therefわre are routinely

virulence of the infecting strain and/or genetic differences in

performed in only a few laboratories in Japan, including thethe human immune background (see below).

Department of TroplCal Medicine and Parasitology, Keio
University School of Medicine. However, antlgen‑Capture

fraction of health care workers are infected, suggests thatenzyme‑linked immunosorbent assays (ELISA) using mono‑transmission to healthy individuals actually occur. As thisclonal antibodies that distinguish between E. histolytica andtransmission could pose a seriousthreat to public health, those

The fact that not only institutionalized people, but also a

E. dispar are commercially available (e.g., Techlab, Inc"

infected need to be identiRed and properly treated. To prevent

Blacksburg, Ⅵ1, USA). Altematively, to detect parasite DNA

transmission among homosexual males, people should bein the specimen, a method to specifically amplify either E.

made aware of transmission by oral‑anal sexual practices andhistolytica or E. dispar genomic DNA by polymerase chainof the relation between infection and sexual promiscuity.

reaction (PCR) is available in some laboratories (e.g.,(3) Vaccine

Department of Infectious Diseases, Tokai UniversitySchoolof Medicine, and Keio University).

E. histolytica exhibits a rather simple developmental transi‑tion in its life cycle, having only twoknown stages, tropho‑

Infection with the pathogenic E. histolytica is often, but

zoite and cyst. It has been suggested that a great proportion

not necessarily always, associated with positive serologlC

of proteins are expressed in both developmental stages,

tests, including gel diffusion precIPltln test, hemagglutina‑

providing loglStic identification of target molecules expressedin both stages. Thereare no insect vectors or secondary hosts

tion test, immunofluorescence assay, and ELISA. These tests

can be perfomed either at pnvate laboratories, universities,

involved in the transmission of amebiasis. Furthemore, asor national institutes, where reagents are available. Some kits,

E. histolytica is an extracellular parasite, an antibody is

including hemagglutination (E. histolytica HA, Japanpredicted to play a potential important role in vaccine‑induced230immunityto amebic infection. All these suggest that we havesulfatea very good chance of developlng a Successful amebiasis

I

vaccine. Several recombinant proteins have been tested fb∫

sulfatetheir efficacy ln the gerbil model, including serine‑rich E.
histolytica protein (SREHP) (26), Gal/GalNAc‑inhibitablelectin (27), and Peroxiredoxin (28). Animal studies using these

I ATP suJfurylase
Cholesteryl su一fate

thyl sulfate
P柑teOg暮yc a n s

recombinant proteins demonstrated the fbasibility of develop‑
lng a reCOmbinant parenteral vaccine to prevent amebic liverabscess (29‑38). Since E. histolyticafirst adheres to intestinal
epithelial cells to cause disease, mucosal anti‑amebic anti‑
bodies should play a critical role in defense. Stanley andcolleagues, therefわre, utilized an antlgen delivery andpresentlng System that is capable of stimulating mucosal lgA
antibody production･ They丘rst produced the dodecapeptide

repeat of SREHP fused to the cholera toxin B subunit. When
this antlgen Was Orally administered, IgA anti‑ameba anti‑

Serin ebodies were induced in the stool and in bile, and the numbers

Cysteineof antibody‑secretlng Cells producing anti‑SREHP antibodies

acetyトtransferase

were also slgnificantly increased in mesenteric lymph nodes

sefine AcetylCoA

and the spleen (32)･ They subsequently efPlored the use of
attenuated strains of Salmonella as carrlerS tO deliver the

SREHP or Gal/GalNAc lectin protein to induce mucosal andcell‑mediated anti‑amebic immune responses (33, 38, 39),

GIycolysis

showlng that in the gerbil model, an oral vaccine combining therecombinant amebic proteins andthe attenuated Salmonella
strain can provide protection agalnSt amebic liver abscess.

Successful utilization of the Salmonella and the nbrio strains

Fig. I. A schematic representation of the sulfur assimilatory cysteinebiosynthetic pathway in E. histolytica.

(35) for deliveヮof vaccine antigens provides the possibilityof a combinatlOn amebiasis/typhoid fever and amebiasis/cholera vaccine.

Although all of these studies appear promlS1mg, there arein Fig. I, E. histolytica possesses a sulfur assimilatory

still a number of important issues to be resolved. First, all ofcysteine biosynthetic pathway, where inorganic sulfur is fixed

these studies were conducted to assess protection agalnSt

into an organic compound, L‑CySteine, in the pathway that isamebic liver abscess with only one animal‑model gerbiL

absent in its.mammalian hosts (63‑66)･ Biochemical andConsequently, lt is largely unknown whether or not thereverse genetlC Studies of the major enzymes of this pathwayobserved protection in the gerbil reflects the potential successshowed that this pathway plays an important role in monitor‑of an ameba vaccine agalnSt human intestinal amebiasis.

1mg total cellular thiol concentrations and as a part of an

Second, the natural polymolphism of these target molecules

antioxidative mechanism (65). Since this pathway is absent

remains unknown. Therefore, lt is notknown whether immu‑

in mammalian hosts, it is a good target for developlng a new

nization with recombinant proteinsfrom one reference strain

chemotherapeutic･ Peroxiredoxin (formally called as 291?r

can protect gerbils against heterologous E. histolytica strains.

30‑kDa cysteine‑rich protein [67‑69]) is a ubiquitous antト

Third, currentlyused attenuated S.りphi strains still have, asoxidant conserved from prokaryotes to higher eukaryotes,

shown in previous clinical trials in humans, side effects suchand has also been shown to be involved, in amoeba, in an

as fever and diarrhea with vaccination. Thus, a truly immOcuous

antioxidative mechanism (70,7 1) together with NADPH:

host strain needs to be developed.

flavin oxidoreductase (70).

(2) Peculiarorganellarstructure
Entamoeba trophozoites appear to have a very prlmitive

4. Recent developments in basic research on amebiasis

cytoplasmic structtue (Fig. 2A)A The major cytoplasmic compo‑(1) Virulence factors

nents are the ingestive and digestive vacuoles, the residual

Quite a few virulence factors have been identified in thebodies, the nucleus, and a number of smaller membranous

last 1 0‑ 1 5 years. These factors include cysteine proteinases

vesicles. a ‑Glycogen is distributed throughout the cytoplasm.

(40144), Gal/GalNAc inhibitable lectin (45‑49), amebapore

The rough endoplasmi? reticulum (ER) is not present in theusual form, but is funct10nally replaced by shorter lengths of(50‑54), and phosholipase (55‑57), and details on each factor
should be consulted in the onglnal references.

helically arranged ribosomes. These helices coalesce into

Antioxidative mechanisms of E. histolytica are also a part

bundles as larger crystalline arrays, fomlng the chromatoidof thevirulence function, as the parasite has to detoxify thebodies. The smooth ER is also poorly developed. Both typlCal

host oxidative stress in order to suⅣive in oxygen‑rich tissues

Golgiapparatusand mitochondria are absent. The trophozoites

and organs, including the liver, lung, and brain. ln contrast to

also lack peroxisome and hydrogenosome. E. histolytica hasother eukaryotic cells, E. histolytica lacks glutathione andlong been thought to be an ancient eukaryote due to a lack ofglutathione‑dependent enzymes (58). L‑Cysteine is the major
thiol in E. histolytica and is presumed to play an importantmitochondrion, a major SOurCe OfATP in most eukaryoticrole in antioxidative defenses (58,59). L‑Cysteine has beenshown to be essential in its attachment to the matrix (60),genes encoding proteinsthat in other eukaryotesare localized
in the mitochondria, i.e. , pyridine nucleotide transhydrogenase

elongation, motility, and growth in vitro (6 1 ,62). As shownand the chaperonin cpn60, unequlVOCally proved that且cells. However, a recent demonstration of two E. histolytica

231

lop.mFig･ 2･ (A) Transmissioヮelectronmicrograph of the E･ hisEolytica trophozoite･ A bar represents 10 LLm. (B) A schematic

representation of a varlety Of cellular trafficking events in the E. histolyEica trophozoite.

histolytica secondarily lost mitochondrial function during

species. Furthermore, Rab5from E. histolytica, for example,

evolution (72)･ Althoughthe ameba does.not apparently havedoes not reverse the a growth defect of a yeast rab5lmutant,a stmcture that resembles mitochondria, lmmunOlocalizationand is involved in phagocytosis, whereas, in yeast andrevealed that a residual organelle called a crypton or mitosome

mammalian cells, Rab5 is located in endosomes and isis present in血e cytoplasm (73, 74). Ghosh et al. have recently

involved in vesicular transport from the plasma membrane toshown that this residual organelle contains double‑stranded

early endosome and in the homotypic fusion of early

DNA and is bound by a double membrane (75), suggestingendosomes during endocytosis (78). Al母y et al. showed thatthat this organelle shares some structural similarities with

E･ histolytica trophozoites possess a unlque nOn‑acidified

mitochondria. The real function of this mitochondria‑like

rapidly exchanging endocytic compartment (86). These data

organelle will likely be revealed by further investigation. In

suggest that Entamoeba has developedunlque machinery ln

addition to the lack of mammalian‑like mitochondria, anprotein tra氏cking, especially in the endocytic and phagocytic

anaerobic organelle called hydrogenosome, present in an

pathways, that significantly differs from that of othereukaryotes, although E. histolytica mostly shares identicalanaerobic protozoan Trichomonas, is also absent in E.
hLytolytica. Furthemore, amoebas are not able touse hydrogenproteins for vesicular transport from ER to Golgi.
(3) Prokaryote‑like energy metabolisms
E. histolytica is a unlque eukaryote with respect to energy

as an electron acceptor, suggesting that hy血ogenosome is not

functional even if it is present. However, pyruvate:ferredoxin
oxidoreductase (POR) from E. histolytica showed a close

metabolism (87,88).Asa mitochondia‑lacking, and anaerobic
or microaerophilic eukaryote, E. histolytica largely depends

phylogenetic association with the FOR of the enterobacteria
and TI Vaginalis, which targets its POR to hydrogenosome(76)･ Moreover, a gene encoding iron hydrogenase, demon‑

onglycolysis and fermentation for its energy production.
E･ histolytica lacks several typical eukaryoticglycolytic

strated in hydrogenosome of TTrichomoァas (77), is present inenzymes. Pentose phosphate shunt enzymes are completely

Entamoeba,and its recombinant protein lS enZymatically active

missing. Another unusual feature of glycolysIS is the utiliza‑

in vitro.

tion of pyrophosphate instead of ATP in several enzyme

Vesicular transport in E. histolytica is believed to share areactions: phosphofructokinase (89), phosphoenolpyruvatecommon basic machine? with that in other eukaryotes (Fig･carboxytransphosphorylase (90), pyruvate phosphate dikinase2B)･ Common proteins lnVOIved in vesicular transport and(9 I ), and acetate kinase. E. histolytica also possesses fermen‑shared by both E. histolytica and other eukaryotes include

tation enzymes that resemble those or anaerobic bacteria.

small GTP‑binding proteins (Raゎ [78‑80] and Arf 【8 1 ]), ER

These enzymes include POR, ferredoxin, and alcohol dehy‑

chaperons BiP (8 1 ,82) and putative disul丘de isomerase, signal

血ogenase･ Phylogenetic analyses strongly suggest that theserecognition particle (SIp54) (83) and ER membrane protein

amebic genes encoding fermentation enzymes are laterally

Sec6 1 , which is involved in transport from the cytosol to thetransferred from bacteria that amebae have phagocytosed (76).

ER lumen, Golgi protein Erd2 (84), and lysosomal vacuolar

This soICalled "net hypothesis" is supported better than "treeATPase (85). More than ten R礼b genes have been identはed

hypothesis", which proposes that these amebic genes wereand characterized from E. histolytica (78‑80). All of these
Rab genes obtained by sequence homology or from EST

present in a common eukaryotic ancestor that lived underanaerobic conditions, but were lost from other eukaryotes

databases show high identities to Rabsfrom other eukaryotes.

because fermentation enzymes are not necessary for growth

However, domainsknOwn to interact with regulatory proteins,in the presence of oxygen (76).

including GTPase activatlng protein and guanine nucleotide
exchange factor, Vary slgnificantly from Rabs from other232the development of new chemotherapeuticand prophylactic

5･ Remalnlng Problems in amebiasis research●●measures. Considering the rapid occurrence of multidrug

Why does disease occur only in 5‑ 10% of those infected?

resistant strains of protozoanand prokaryotic pathogens, e.g. ,This has been a long‑lasting question since the first descrlP‑

ofamebiasis also see l93,94]), and we still do not have a

Plasmodium and Mycobacteria, drug‑resistant strains of E.
hislolytica will probably emerge in the future. Therefore, weneed to continue to look for parasite‑Specific cellular events

proper answer to it. There are three possible explanations. 1.

not only to elucidate pathogenic mechanisms of the parasite,

Commensal theory. E. histolytica isusually viewed as an
innocuous gut commensal. However, with a yet‑unconflmed
stimulus (e.g., nature of gut flora, dietary implications), it

but also to eventually develop measures to intervene in trans‑

tion ofamebiasis in 1875 by Lesh (Losch) (【92], for history

mission of the disease.

turns to a tissue‑invading formthat lost its capacity for cyst

ACKNOWLEDGMENTS

production. Thus far there is no direct evidence supporting

this hypothesis of a possible conversion &om a non‑virulent

The author thanks Yumiko Saito‑Nakano for providing

to a virulent type, such as an identification of differentiallyexpressed pathogenic factors. 2. Host immune theory. It

unpublished results ofE. histolytica Rab, Tomoyoshi Yasuda

depends on the genetic background of host immune systems

discussions. A part of the work described in this review waswith regard to whether amebiasis develops in the infectedsupported by a grant for reseaqch on emerglngand re‑emergmg

individual. It was reported that individuals witha certain typeofhumanleucocyteantigen (HLA) in Mexican mestizo (DR3)

infectiousdiseasesfrom the Ministry of Health, Labour andWelfare and a grant from the Japan Health Sciences Founda‑are more s†sceptible toamebic liver abscess (95)･ Although notion.

for electron micrographs,and Tsutomu Thkeuchifor our helpful

such assoclation was fわund between HLA types and amebic

colitis (96), the contribution of the host genetic background

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