1. No category

Entamoeba histolytica: an update
William Stauffera and Jonathan I. Ravdinb
Purpose of review
Over the past decade, since it was formally recognized that
Entamoeba histolytica and Entamoeba dispar were two distinct
species, studies in this field have made dramatic in-roads into
the understanding of E. histolytica and the pathogenesis of
invasive amoebiasis. Over the same period it has also become
clear that the true incidence of E. histolytica infection,
particularly in vulnerable populations such as low
socioeconomic children, is exceedingly high. Understanding the
epidemiology, pathophysiology, and the molecular and genetic
biology of the organism will not only lead to improved diagnostic
and treatment options but, ultimately, to the development of a
safe and efficacious vaccine.
Recent findings
The recent advances in the genetic and molecular sciences
have increased our understanding of the mechanisms that make
E. histolytica unique among enteric protozoa in causing invasive
disease. In addition, host factors, which predispose individuals
or populations to infection or disease, are beginning to be
elucidated. New diagnostic tools specific to E. histolytica are
being exploited by clinicians and researchers to identify and
treat patients as well as to add to the knowledge of the
epidemiology and natural history of this infection. The ultimate
goal – eradication of disease – is theoretically feasible since
humans and primates are the only reservoirs of E. histolytica.
Many talented and dedicated individuals are pursuing the
development of an effective and safe amoebiasis vaccine.
Summary
E. histolytica remains an important pathogen in many
populations of the world and although there has been
substantial progress into understanding the disease major
challenges still exist.
Keywords
Entamoeba histolytica, amoebiasis, amoebic abscess, amoebic
colitis
Curr Opin Infect Dis 16:479–485.
#
2003 Lippincott Williams & Wilkins.
a
Department of Internal Medicine, Division of Infectious Disease and International
Medicine, University of Minnesota, Minneapolis and Regions Hospital/HealthPartner’s Center for International Health and International Travel Clinic, St Paul and
b
Department of Medicine, University of Minnesota, Minneapolis, Minnesota, USA
Correspondence to Dr Jonathan Ravdin, MD, Nesbitt Professor and Chairman,
Department of Medicine, University of Minnesota, 516 Delaware Street SE, MMC
194, Minneapolis, MN 55455, USA
Tel: +1 612 625 3654; fax: +1 612 636 3055; e-mail: [email protected]
Current Opinion in Infectious Diseases 2003, 16:479–485
Abbreviations
ALA
GalNAc
PCR
amoebic liver abscess
N-acetylgalactosamine
polymerase chain reaction
# 2003 Lippincott Williams & Wilkins
0951-7375
DOI: 10.1097/01.qco.0000092821.42392.ca
Introduction
Entamoeba histolytica infects hundreds of millions of
people per year. While most individuals remain asymptomatic, perpetuating the natural cycle of the organism
through fecal excretion of infective cysts, an important
minority will suffer the severe morbidity associated with
invasive disease (approximately 50 million) with an
estimated 100 000 succumbing to the protozoa annually
[1 . .]. During the 1990s enough evidence had accumulated to support the formal separation of two morphologically identical species of amoeba: the non-pathogenic
Entamoeba dispar from the potentially pathogenic
E. histolytica [2–6]. Morbidity and mortality data (in
absolute numbers) which existed prior to this time
pertaining to cases of invasive disease were not greatly
affected by this reclassification because all invasive
disease was known to be caused by E. histolytica.
However, because most prevalence and incidence data
previously collected pertained to asymptomatic individuals, and it was clear that a majority of asymptomatic
individuals with cysts detected in their stool were
infected with non-pathogenic E. dispar, the true
prevalence and incidence of E. histolytica became a
matter of speculation. In 1997 an expert committee was
convened which issued several recommendations to be
given ‘highest priority’ in future research involving
E. histolytica. Top among these recommendations was a
call to investigate the true incidence and prevalence of
E. histolytica infection. The committee also felt it
imperative for proper planning and control strategies to
not only identify the prevalence of asymptomatic carriers
but to elucidate the likelihood of manifesting invasive
disease and the frequency of transmission to naı¨ve
individuals. Finally the report called for further immunologic, biochemical, molecular and genetic research
to assist in understanding the pathogenesis of this
organism as well as helping to pioneer new treatment
and prevention strategies, and ultimately, to the creation
of a safe and effective vaccine [7]. This paper will
discuss the most recent scientific literature and assist the
reader by placing these recent discoveries in the context
of previous work conducted in the study of E. histolytica.
Epidemiology
Some of the original epidemiologic descriptions distinguishing E. histolytica from E. dispar infections originated
from studies conducted in South Africa. A community
cross sectional survey of asymptomatic individuals found
through the use of culture and zymodeme analysis an
overall prevalence of the E. histolytica–E. dispar complex
of approximately 10%. On further analysis it was
479
480 Gastrointestinal infections
revealed that 90% of individuals were infected with the
non-pathogenic E. dispar and 10% asymptomatically
harbored E. histolytica [8]. In the same study it was noted
that E. dispar occurred more frequently in females than
in males while E. histolytica had an equal prevalence and
incidence in males and females. Further, it was also
interesting to note that although the prevalence of
E. histolytica infection was equal in all age groups and
between sexes, males and individuals over 16 years of
age had a much greater likelihood of invasive disease. It
was subsequently noted in this population that approximately 10% of asymptomatic carriers of E. histolytica
would develop invasive disease while a majority of
others spontaneously cleared their infection by 1 year
[9 .,10 . .]. It has been recognized that disease expression
varies geographically. For example, invasive disease in
Egypt is predominantly amoebic colitis [11] whereas in
South Africa there is an excessive rate of amoebic liver
abscess (ALA). In fact, in Hue City, Vietnam, an overall
estimated frequency of ALA was recently reported to be
as high as 21 cases per 100 000 inhabitants [9 .].
As part of a study investigating innate and acquired
resistance to amoebiasis, 230 Bangladeshi children (age
2–5 years) were enrolled in a 2-year observational study
[10 . .]. In this impoverished population 55% of children
acquired E. histolytica infection during the 2-year period.
Of these 55% who acquired infection, 80% of children
remained asymptomatic while 20% had an associated
diarrhea with 4% of these meeting the definition of
amoebic colitis. Further, 17% acquired additional
E. histolytica infections during the 2-year study which
were felt to be due to a second genetically distinct strain
(determined by polymerase chain reaction (PCR) for the
serine-rich E. histolytica protein) [10 . .].
A recent study [12] of low socioeconomic status school
children in Ecuador shed additional light upon the
epidemiology of E. histolytica infection. This crosssectional survey found asymptomatic E. histolytica infection present in only seven of 178 children. However, it
was interesting to note that more than 64% of children
showed high serologic titers implying current or recent
infection with E. histolytica [12]. This high seroprevalence
corresponds to another recent sero-survey conducted in
Mexico [13]. Further, this study summarized some of the
difficulties in conducting prevalence studies as it revealed
very non-concordant results between some previously
well validated tests: two antigen detection assays, the
Prospect Entamoeba histolytica Microplate assay and the
Entamoeba histolytica II assay; several methods of direct
microscopic exam; in-vitro culture; starch gel isoenzyme
electrophoresis; and serologic analysis by three separate
methods. This paper revisited the question of what
should be considered an adequate screening test (sensitive, specific, inexpensive), and what should be consid-
ered the true ‘gold standard’ in investigating individuals
for E. histolytica and E. dispar infection [12].
A related question arises frequently in clinical practice in
low prevalence areas which receive large numbers of
immigrants and refugees from endemic areas. Most
medical screening protocols in the United States for
immigrants and refugees arriving from the developing
world suggest that three ova and parasite examinations
are routinely collected [14]. This screening is conducted
to enhance the individuals’ health by treating intestinal
parasitic infections as well as to protect the public health
of the community. Entamoeba specifically is known to
spread within families, institutions (i.e. day care centers)
and occasionally may cause epidemics [15,16]. Despite
these recommendations it is known that stool ova and
parasite examinations are relatively insensitive and they
are unable to distinguish E. histolytica from E. dispar
infection. Further reducing the specificity is new
information indicating an inability of microscopy to
reliably distinguish E. histolytica [17] from another close
relative Entamoeba moshkovskii. Currently the standard at
most centers is to treat all asymptomatic individuals with
cysts in the stool with an anti-protozoal intestinal agent.
It is the authors’ experience that there is poor
compliance with this approach as it subjects asymptomatic patients to prolonged multiple daily dosing of
medications that are difficult to administer and carry
substantial adverse effects. It is therefore suggested that
positive stool samples be confirmed with stool antigen or
serum serology studies for E. histolytica prior to initiation
of treatment [18,19]. It remains to be determined if in
addition to the stool ova and parasite examination other
diagnostic screening examinations (i.e. stool antigen
testing) would be a more sensitive, specific and costeffective addition to the routine medical screening of
immigrants and refugees.
Pathogenicity and clinical disease
When the host comes into contact with the protozoa the
result, in a majority of cases, will be establishment of a
non-invasive infection [8]. However, some individuals
will acquire invasive disease predominantly in the form
of amoebic colitis or ALA. The parasite–host interactions
which determine the course of disease, although largely
still a mystery, are beginning to come to light. The three
virulence factors that are best understood include a
parasite surface protein (lectin), the amoebapore, and the
cytoproteins (cysteine proteases).
Generally, a cyst of E. histolytica is ingested by a human
host from fecally contaminated food or water. Excystation takes place in the intestinal lumen to produce
trophozoites. The trophozoite surface protein (lectin)
recognizes the sugars galactose and N-acetylgalactosamine (GalNAc) on the host cell surface [20,21]. The
Entamoeba histolytica Stauffer and Ravdin 481
interface of the galactose/GalNAc lectin with the host
mucins lining the intestine [22] is the defining moment
of the infection. If the parasite lectin attaches to the host
mucin glycoproteins that line the intestinal lumen, a
non-invasive gut infection ensues. The life-cycle then
continues as the trophozoites reproduce by clonal
expansion in the mucin layer. Subsequently, the
galactose/GalNAc lectin, along with mucin glycoproteins
or other gut bacteria, initiates the developmental pathway leading to encystment [23].
On the other hand, if the trophozoite penetrates the
mucin layer and the lectin attaches directly to a host cell
surface N-acetyl-D-galactosamine, a cascade of events
occurs ultimately leading to death of the host cell [20]
and progression of invasive disease. Once the trophozoite is committed to invasion, replication and encystment will not occur and the life-cycle will not be
successfully completed. Therefore, invasive disease, as
determined by the lectin protein, must be considered a
maladaptation of the organism as it is disadvantageous to
the protozoa’s survival. A complete review of the lectin
protein is beyond the scope of this paper but interested
readers are referred to an excellent recent review of the
structure and function of this protein [1 . .].
E. histolytica trophozoites kill (in vitro) a wide variety of
tissue cell lines including neutrophils, T lymphocytes
and macrophages [24–26]. As mentioned, the killing of
host cells is contact-dependent, initiated via the
galactose/GalNAc lectin [20,27]. It has been shown that
when the amoeba engages the host cell the cytoskeleton
undergoes changes that are vital to the ability of the
trophozoite to kill [1 . .]. A pore-forming protein complex
is inserted into the host cell by the trophozoite that is
similar to the human complement system (amoebapores)
and is likely involved in pathogenicity [28]. Killing
ability is also dependent on calcium and cytolysis. It has
been shown that within seconds of contact with the
trophozoite the host cell calcium will precipitously rise
20-fold with cell death occurring within 15 min [29].
Cysteine proteases are known to be important to the
pathogenesis of E. histolytica and function by degrading
extracellular proteins and through the disruption of cell
monolayers [30]. Approximately 20 genes, a majority of
which are not expressed, have been identified that code
for the cysteine proteases. Interestingly it was also
recently demonstrated that the differential expression of
these 20 genes varies between E. histolytica and
E. dispar, explaining at least one mechanism that allows
E. histolytica to invade the host [31]. In the final stages,
when the host cell dies, it undergoes apoptosis as the
nuclear chromatin condenses, the membrane forms blebs
and the internucleosomal DNA fragments [32,33].
Activation of caspase 3, a distal effector molecule in
the apoptotic pathway, seems to be a crucial step in cell
killing (in vitro) and in the formation of ALA (in vivo)
[32,34]. It has further been suggested, based on a study
of the ability of E. histolytica to kill and phagocytose host
cells, that virulence may be directly proportional to the
amoeba’s ability to cause apoptosis and then phagocitize
the dead cell as this process seems to substantially limit
the host inflammatory response [35 .].
Molecular biology and genetics
Molecular biology and genomics research is initiating
revolutionary advances in medicine. Amoebiasis research
is no exception with extraordinary insights being made
into the understanding of the organism ranging from its
teleologic origins to discoveries of novel gene products
and functions. Molecular phylogeny has revealed that
Entamoeba species (histolytica and dispar) are close to
Dictystelium discoideum on one of the lowest branches of
the eukaryotic tree [36 . .]. Although amoebae were
thought to lack organelles (mitochondria, endoplasmic
reticulum and Golgi apparatus) evidence to the contrary
is coming to light. In addition to recently described
nuclear-encoded mitochondrial genes and a remnant
mitochondrial organelle [37,38], a study [39] found that a
51 kDa protein of E. histolytica has a significant similarity
to the amino acid sequence of the calreticulin-like
protein of spinach leaves (77% homology) and of the
calreticulin precursor of D. discoideum (60% homology)
indicating an equivalent system to the eukaryotic
endoplasmic reticulum and the Golgi apparatus [39].
The recognition that the similar but non-pathogenic
E. dispar, a much more successfully commensual
parasite, differs from the potentially invasive E. histolytica
has led to great opportunities from a genomics standpoint to compare species and elucidate the specific
genes and proteins that make E. histolytica pathogenic. A
joint project between the US Institute of Genomic
Research (Rockville, Maryland, USA) and the UK
Pathogen Sequencing Unit based at the Sanger Institute
(Cambridge, UK) is endeavoring to sequence the
complete E. histolytica genome and for comparative
purposes, to partially sequence four other closely related
intestinal protozoa (E. dispar, E. moshkovskii, E. terrapinae, E. invadens). It has become clear through this effort
that E. histolytica has a small genome (approximately 20
MB) that is very repetitive and contains densely packed
coding sequences with closely spaced genes lacking
introns [1 . .,40–42]. It appears the sequences encoded in
the genome arrangement are proteins involved in highly
conserved functions and cellular processes. However,
there are also areas of the genome that express amazing
genetic polymorphisms [43]. It has been proposed that
these polymorphic loci may assist in determining
geographic origins of isolates and may even assist in
ascertaining routes of transmission [44]. Certain unique
features that are proving to be a challenge in decoding
482 Gastrointestinal infections
the genome include a high adenosime-thymidine content which makes it refractory to large insert cloning and
uncertain ploidy across the genome.
Host defense and the potential for a vaccine
Although the human host responds to E. histolytica by
activating both cell-mediated and humoral immunity
mechanisms, the organism has developed multiple
methods of rendering many components of the human
immune system ineffectual. Further, as with many
infectious diseases, it appears that certain host responses
worsen the pathologic sequelae of disease. On the other
hand, some components are effective at limiting the
disease process and producing protection against future
infection. These components, particularly mucosal IgA,
are currently being exploited in the pursuit to develop
an effective vaccine.
Host defense
Both cell-mediated and humoral immune responses to
E. histolytica develop with either asymptomatic or
symptomatic infection. E. histolytica has developed
multiple immunomodulatory effects on the host response that allows it to be a successful pathogen.
Further, it is clear, as with many infectious diseases,
the host responses may not only provide defense for the
host but also are a key ingredient in the disease process
[45,46]. Initial resistance to E. histolytica is dependent on
natural or innate mechanisms. Beyond the physical
barriers of the mucin and structures of the colonic
epithelium the organism must also overcome the
complement system. It has been recognized that the
complement pathway is of limited defense for the host,
particularly the alternative pathway as the trophozoite
has ‘acquired resistance’ to the C5b-9 complex. The
organism has acquired this ‘resistance’ through expressing an epitope on the GalNAc lectin heavy subunit that
has sequence similarity and antigenic cross-reactivity to
the human CD59 (a leukocyte antigen that prevents the
assembly of the C5b-9 complex), effectively making it
invisible to the host alternative complement system [47].
Amoebic cysteine proteases also degrade C3a and C5a
complement components [48].
In-vitro data have demonstrated a role for cell-mediated
response, especially interferon-g, tumor necrosis factor-a
and interleukin-4, in the activation of macrophages and
neutrophils to kill E. histolytica trophozoites [49].
Although neutrophils seem to be essential in regard to
initial host defense, it is interesting to note that
E. histolytica produces a chemoattractant for neutrophils
[50] and will kill these cells on contact [24]. In addition,
it has also been demonstrated that amoebas will lyse
inactivated monocytes/macrophages [25] and new evidence suggests that tumor necrosis factor-a may, at least
in part, act as a chemoattractant to these cells [51].
Perhaps most importantly, both non-invasive and invasive E. histolytica infection has been shown to elicit
mucosal immunoglobulin A and serum immunoglobulin
G response to the lectin protein. The importance of this
finding lay in the fact that mucosal immunoglobulin A
response directed at the carbohydrate-recognition domain of the galactose/GalNAc lectin protein has shown
to be protective against infection [36 . .,52]. An interesting observation was made recently in a study of
Bangladeshi children [10 . .], which confirmed the
protective effect of mucosal anti-lectin immunoglobulin
A but found that a serum immunoglobulin G response to
the same protein seemed to be associated with a higher
likelihood of future invasive disease [10 . .]. In a
prospective study of adult patients cured of ALA and a
large number of controls (family members or neighbors),
it was found that high levels of intestinal anti-lectin
immunoglobulin A antibodies are present in ALA
patients for over 18 months and that they are highly
immune to new infections by E. dispar [52], which
contains functional lectin molecules that are antigentically cross reactive with the E. histolytica lectin [53]. By
stool culture criteria, new E. dispar infections were
fivefold more common in the control population than E.
histolytica infections, highlighting the immunity observed
in the ALA patients. A cysteine-rich portion of the
galactose-inhibitable lectin heavy sub-unit (a portion of
LC3) appears to be one of the main epitopes on the
lectin as it recently was shown to induce humoral
(immunoglobulins A and G) immunity in asymptomatic
infected patients as well as in ALA patients [54]. In
addition, epitope specific mouse monoclonal antibodies
against the cysteine rich region of the galactose–lectin’s
heavy sub-unit have been shown to inhibit adherence to
target cells [52,54–56]. Hence, the cysteine rich portion
of the heavy subunit of the lectin would appear to be a
potential vaccine target. In considering this protein as a
target it is imperative that the epitope does not vary with
genetically distinct stains of E. histolytica. It is therefore
of interest to note a study that demonstrated the
galactose/GalNAc lectin heavy subunit isolates from
three distinct areas of the world (Bangladesh, Georgia,
and Mexico) retain remarkable sequence conservation
[57 .].
Vaccine development
The recognition of an extraordinarily high prevalence of
E. histolytica infection in certain vulnerable populations
of the world suggests that an effective and safe vaccine
would not only prevent considerable mortality but that it
would provide a valuable tool in decreasing morbidity in
developing nations. This would be particularly true in
children in developing nations as the epidemiology
indicates that children in poverty are at an extremely
high risk for developing infection. As humans are the
only known reservoir for the parasite, other than
Entamoeba histolytica Stauffer and Ravdin 483
primates such as baboons, a safe and effective vaccine
could theoretically lead to eradication of the protozoa.
Since it appears acquired immunity to E. histolytica is
correlated with mucosal immunity, it follows that a
vaccine that induces gut-associated lymphoid tissue
would be a primary candidate. Several potential oral
vaccine candidates have been reported in the literature
[58 .]. A recent study of oral immunization of gerbils with
a live-attenuated Salmonella vector expressing a portion
of the galactose/GalNAc lectin [59] was shown to protect
animals from ALA. Another innovative study [60]
utilized Yersinia outer protein E to orally deliver heavy
chain antigen (170CR2) through the Yersinia type III
secretion pathway. This novel approach not only
induced a significant antibody response but was shown
to be protective against ALA in the gerbil model [60].
Finally, it was recently found through an in-vivo study
conducted in mice [61] that an injectable codonoptimized DNA vaccine targeting a portion of the
galactose-lectin heavy subunit (including the carbohydrate recognition domain), stimulated a Th1-type
galactose-lectin specific cellular immune response as
well as inducing development of serum antibodies that
recognized a recombinant portion of the heavy subunit.
Investigation is now underway to assess this vaccine via
an oral delivery route.
Diagnosis and treatment
Traditionally E. histolytica is diagnosed by microscopy of
the stool through identification of either cysts or motile
trophozoites. As previously mentioned, this technique is
neither sensitive nor specific. The ‘gold standard’ has
been considered amoebic culture and zymodeme determination although it is known to be far from 100%
sensitive. Recent advances have made new diagnostic
tools available to the clinician including serologic antibody tests and the commercial availability of stool
E. histolytica-specific antigen testing [62]. Antibody tests
are generally used to confirm positive stool ova and
parasite examinations or antigen tests as the currently
available serologic tests are unable to differentiate acute
infection from past infection.
In addition to the clinically available tools, other
diagnostic tools have become available for research
including PCR, sputum antigen testing, and serum
antigen testing by enzyme-linked immunosorbent assay
[63–65]. Most recently real-time PCR, able to distinguish E. histolytica from E. dispar, has been pioneered
allowing large numbers of samples to be run with high
sensitivity (0.1 parasite per gram of stool) and specificity
[66,67].
available furazolidine [67]. As E. dispar is the predominant intestinal organism in most settings, asymptomatic
intestinal carriage of amoebae should only be treated if
the organism has been confirmed to be E. histolytica, or if
this can be inferred from clinical events such as recent
amoebic liver abscess. Invasive disease responds to
nitroimidazoles such as metronidazole or tinidazole,
which have no effect on intraluminal carriage [36 . .].
When these are necessary they should be given before
paromomycin, because the diarrhea that is a common
side effect of paromomycin may make it difficult to
assess the patient’s response to therapy [36 . .]. Occasionally therapeutic aspiration of an ALA or surgical
intervention for complications of colonic disease may be
necessary.
Conclusion
Amoebiasis is one of the most common and longest
known parasitic infections of mankind. Over the last
several years it has become clear that in vulnerable
populations, such as in low socioeconomic children
residing in the tropics, the incidence rates greatly exceed
previous estimates. In fact, E. histolytica is likely one of
the major causes of diarrhea in young children and has
significant negative health effects on this group. Further,
it is now clear that invasive disease manifestations
(colonic versus ALA) vary geographically being driven
both by differences in the organism and through genetic
susceptibility of the host. Significant progress has also
been made in the understanding of the mechanisms that
make these intestinal protozoa unique in its ability to
cause invasive disease in humans. The Gal/GalNAc
lectin protein, which is principally responsible for
invasion, has been further characterized using newly
available molecular techniques. Also, the genetic mysteries of the organism are beginning to be revealed,
helping to explain the pathogenic differences from
closely related commensal organisms such as E. dispar.
Several recent trials of vaccine candidates have been
performed as well as verification of new diagnostic
techniques that may be used both clinically and in
research. Although extensive work is still needed to
understand this organism and create an effective vaccine,
substantial progress has been made over the last several
years.
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