Advances in Amoebiasis Research Emphasizing Immunological and Oxidative Aspects
The purpose of this review is to report the scientific advances of amoebiasis, especially focusing the innate immune response and the oxidative stress related to the infection. Amoebiasis is a significant cause of morbidity and mortality, affects about 50 million persons and leads to approximately 100.000 deaths worldwide each year. As to the virulence, the different strains of Entamoeba histolytica and Entamoeba dispar can be characterized in several forms. The main ones are: the capacity of inducing liver abscess in hamsters, the erythrophagocytosis and the cytopathic effect on VERO cells. The tissue invasion by E. histolytica trophozoites induces a humoral immune response, which may persist for several years, being verified by the levels of antibodies. However, it is the cellular immune response that has been described as the most effective one. It still remains unclear whether, the oxidative stress generated at the inflammatory sites of amoebiasis gives rise to benefits or injuries to the host, or if both possibilities may coexist, depending on other factors such as, for instance, the type of strain and the profile of the host immune response. A better understanding on the relationship between parasite and the host immune response is crucial for the development of vaccines and for the improvement of therapeutic alternatives to amoebiasis.
June 17, 2010; Accepted: June 23, 2010;
Published: February 07, 2011
Amoebiasis is an important parasitic disease caused by the broadly worldwide
disseminated enteric protozoa Entamoeba histolytica, yet being more incident
in places that did not reach suitable sanitary conditions. In developed countries,
this protozoan is seen primarily in travelers to and emigrants from endemic
areas. The disease is a significant cause of morbidity and mortality, affects
about 50 million persons and leads to approximately 100.000 annual deaths worldwide
(WHO/PAHO/UNESCO Report, 1997).
Entamoeba histolytica infects people of both sexes and all ages; however,
populations at risk may vary with geographic location, host susceptibility and
differences in organism virulence (Pritt and Clark, 2008).
High levels of human infection are found in India, Africa and Central and South
America. In Brazil, for instance, the number of infected patients or individuals
presenting the symptoms of the disease varies by region, such a variation generally
running between 2.5 and 11%. Nevertheless, it is worthy to point out that levels
near 20% can be observed at the Amazonian region (Silva and
The purpose of this review was to show the scientific advances in the study of amoebiasis, especially focusing the innate immune response and the oxidative stress related to the infection.
The first scientific reports referring to amoebiasis arose on the XIX Century.
Through microscopic and clinical studies, Loesch (1875)
associated dysentery with the presence of trophozoites in the feces, which received
the name of Amoeba coli. Koch and Graffki (1887)
reported the tissue invasion by amoebae and Councilman and
Lafleur (1891) described the pathological process associated with the amoebian
invasion of the liver. These authors also described in details the amoebae that
are causative agents of lesions, designating them as Entamoeba dysenteriae.
At the beginning of the XX Century, the terminology of amoebae was a quite
controversial issue. Schaudinn (1903) attributed a new
name to E. dysenteriae, naming it as Entamoeba histolytica and
still described another specie of amoeba, the Entamoeba coli, which is
not pathogenic. Craig (1905) considered that the name
E. dysenteriae was preferable to E. histolytica. However, Walker
(1911) re-described the intestinal amoebae, confirming the name E. histolytica
and distinguishing it from E. coli through the number of nuclei inside
the cysts of both species.
Brumpt (1925) proposed the existence of two species
of the gender Entamoeba, which were morphologically similar and infected
men, being one of them pathogenic and the other non-pathogenic. Brumpts
studies were grounded on the verification that many of the patients apparently
infected by E. histolytica were asymptomatic, getting rid of the infection
in a spontaneous way. Brumpt called the non-pathogenic specie Entamoeba dispar.
However, given the identity between the morphological features of both species,
the idea proposed by him was not well accepted by the scientific community of
It was only in 1997 that, during the Congress of amoebiasis held in Mexico,
the World Health Organization (WHO), in association with the researchers of
the area, confirmed the existence of E. dispar (WHO/PAHO/UNESCO
MORPHOLOGICAL AND BIOLOGICAL FEATURES OF THE PARASITE
This protozoan is classified under the Sarcodina subphylum, shows an amoeboid
form and possesses pseudopodia for movement. Trophozoites usually measure 20
to 40 μm, being likely to reach 60 μm in the most invasive forms.
In general, it has a single nucleus, which is very distinguishable when stained,
but rather clear in fresh preparations. Inside these preparations, the trophozoites
are pleomorphic and rapidly produce thick and hyaline pseudopodis, which seem
to slip over the blades surface (Silva and Gomes, 2005).
The ingestion of food occurs through pinocytosis (liquid particles) and phagocytosis
of debris. However, in the invasive forms of amoebiasis, erythrophagocytosis
and leukophagocytosis are also often verified. The process of phagocytosis starts
with the ligation of the Gal lectin/GalNAc (Okada et
al., 2005) and in order to promote degradation, amoebapores and cysteine
proteinases are secreted to the phagosomes (Que et al.,
2002; Andra et al., 2003).
The cysts are spherical, with approximately 10 to 16 μm of diameter and
present one to four nuclei. When mature, they have four nuclei. There is an
energetic reserve (glycogen) in a distinct vacuole inside the immature cyst,
becoming diffuse at mature cysts (Diamond and Clark, 1993),
whose cell wall is composed by chitin (Ravdin, 1995).
In the environment, they can survive for weeks or months, especially under favorable
conditions of humidity and temperature. They are subject to degeneration under
temperatures lower than 5°C and above 40°C (Tanyuksel
and Petri, 2003).
Although, being morphologically indistinguishable, several works showed biochemical
and genetic differences between E. histolytica and E. dispar (Tannich
et al., 1989; Gomes et al., 1997;
Srivastava et al., 2005). Besides, E. dispar
has been described as unlikely to cause the disease; in fact, trophozoites colonize
the intestine, but the patients remain asymptomatic (Tannich
et al., 1989; Mak, 2004).
The evidences of genetic differences led to the differentiation between the
two species, so that the commensal one was called E. dispar, while the
pathogenic was designated as E. histolytica (Burch
et al., 1991).
The parasites life-cycle is monoxenic and relatively simple. Infection
starts with the ingestion of cysts from water and contaminated food, which are
resistant to the action of gastric juice. During the process of excystation,
which takes place at the small intestine, the metacyst escapes from the cyst
wall through a tiny pore. The metacyst undergoes successive nuclear and cytoplasmatic
divisions and the resulting trophozoites migrate to colonize the large intestine.
In this place, they can leave the mucosa, deshydratate and become precysts;
subsequently, they secrete a cystic membrane and become cysts, which are initially
mononuclear. Then, the single nucleus divides to form the quadrinucleate stage,
being eliminated with the feces and so completing the cycle (Ravdin,
PATHOGENY, VIRULENCE AND IMMUNE RESPONSE
At the early stage of lesions, trophozoites inside the intestinal mucus adhere
to epithelial cells through mechanisms mediated by lectins. The lectin which
seems to be the major contributive factor to the adhesion is Gal/GalNAc (McCoy
et al., 1994; Lejeune et al., 2009).
Upon intimate contact, polypeptides called amoebapores are released by the
parasite. Amoebapores, that are constitutively present at the cytoplasm of trophozoites
(Gonzalez et al., 2008), are capable of inducing
apoptosis and necrosis of eukaryotic cells and also present an antibacterial
activity (Leippe et al., 1994).
The inhibition of expression of amoebapore in E. histolytica trophozoites
was responsible for a significant reduction of the lytic capacity on erythrocites
and renal cells in hamsters and also for the decrease of capacity of producing
zones of hepatic liquefative necrosis (Bracha et al.,
There are evidences of the main role of cysteine proteinases as a virulence
factor for E. histolytica, being involved in the breach of the mucus
barrier, which is crucial in the pathogenesis of amoebiasis (Moncada
et al., 2006; Lejeune et al., 2009).
The proteolitic enzymes secreted by the parasite breach the mucus and the epithelial
barrier, thus facilitating the penetration inside the tissue (Que
and Reed, 2000). The combination of these molecules and some other possible
unknown factors lead to the formation of ulcers and to the subsequent migration
of amoeba to the liver and other sites (Stanley, 2001).
He et al. (2010) clonated and characterized
a cysteine proteinase (EhCP4), with the specificity of a unique substrate and
nuclear localization. It is related to the exposure to colon cells, being activated
and liberated outside the cell. The mapping of the substrate led to the conception
of an inhibitor, which mitigated the infection in model of intestinal amoebiasis.
It is a possible target of efficient drugs for the treatment of amoebiasis.
As to the virulence, the different strains of E. histolytica and E.
dispar can be characterized in several forms. The main ones are: the capacity
of inducing liver abscess in hamsters, the erythrophagocytosis and the cytopathic
effect on mammalian cells cultured in monolayers. It is relevant to consider
the biologic characteristics or functions of the parasite, since they may be
related to pathogenic mechanisms verified during the development of invasive
amoebiasis (Gomes et al., 1997).
Carranza-Rosales et al. (2010) ascertain that
animal models animal models may pose ethical issues and are time-consuming and
costly. In view of this, they developed a new model of experimental infection
and report the infection of precision-cut hamster liver slices with E. histolytica
trophozoites. The infection time-course, including tissue damage, is shown to
be similar to the findings in the animal model. This new model to study Amoebic
Liver Abscess (ALA) is simple and reproducible and employs less than 1/3 of
the hamsters required for in vivo analyses.
As to the interaction with the immune system, the Lipopeptidophosphoglycan
(LPPG), a molecule exposed on the surface of E. histolytica, was described
as being capable to be recognized by Toll-Like Receptor (TLR) 2 and TLR4 of
the leukocytes, being likely to trigger an inflammatory response (Maldonado-Bernal
et al., 2005; Wong-Baeza et al., 2010).
The LPPG leads to the release of cytokines from human monocytes, macrophages
and dendritic cells, increasing the expression of costimulatory molecules. The
LPPG also activates NKT cells, induces antibody production and anti-LPPG antibodies
prevent the development of the disease development in animal models of amoebiasis.
Because LPPG is recognized by both the innate and the adaptive immune system,
it may be a good candidate to develop a vaccine against E. histolytica
infection (Wong-Baeza et al., 2010).
The tissue invasion by E. histolytica trophozoites induces a humoral
immune response, which may persist for several years with the production of
low levels of antibodies after healing. These levels are not necessarily related
to the host defense, such data suggesting that humoral immunity does not afford
protection against E. histolytica infection (Perez
and Kretschmer, 1994; Manrique et al., 2002).
The morphological and immunohistochemical results disclosed by a recent study
performed by Costa et al. (2010) suggest that
both the complement system and the antibodies may destroy trophozoites in livers
of hamsters, which were experimentally infected with E. histolytica
and E. dispar. This first comparative study also showed a higher in
situ resistance of E. histolytica against antibody response and complement
activation. Yet being demonstrated that the complement system is not enough
to avoid the development and progress of the liver injuries, this system, in
association with antibodies, was responsible for a partial control in vivo.
Cellular immune response has been demonstrated to be crucial for the infection
control, as shown by studies that focused the passive transfer of T cells and
experiments of selective immunosuppression. Cellular immunity suppression of
laboratory animals was shown to lead to severe clinical symptoms, as well as
tissue invasion (Wang et al., 1992; Perez
and Kretschmer, 1994).
There are evidences showing that amoebas are destroyed in vitro by T
lymphocytes obtained from ALA healed patients and that CD8+ cells are responsible
for the parasite lysis. In order to perform their amoebicid activity, lymphocytes
require not only the contact cell to cell, but also the action of some cytokines
such as IL-2 (interleukin- 2) and INF-γ (gama-interferon) (Schain
et al., 1992).
E. histolytica induces an intense inflammatory response at the intestinal
mucosa. Epithelial cells start a defensive answer by producing pro-inflammatory
cytokines such as IL-1, IL-8, chemotactic factors for macrophages and neutrophils
and induce production of nitric oxide and tumor necrosis factor alpha (TNF-α)
During the cellular lysis mediated by the amoeba/cell contact, there is a powerful
chemotactic action for neutrophils, which may be destroyed by virulent amoebas
at the site of inflammation (Ravdin and Murphy, 1992;
Manrique et al., 2002).
By investigating molecular factors involved in amoebiasis, Blazquez
et al. (2006) showed a relevant role of TNF-α in vitro,
as it acts as a chemotactic agent for trophozoites, attracting them to the sites
where this cytokine can be found.
Another study demonstrated that expression of the inflammatory enzyme cyclooxygenase-2
(COX-2) in trophozoites and macrophages is relevant to the ALA formation, suggesting
that, besides macrophages and neutrophils, trophozoites themselves are involved
in the inflammatory process related to extra-intestinal amoebiasis (Gutierrez-Alarcon
et al., 2006).
The MLIF (monocyte locomotion inhibitor factor) was identified as a peptide
produced by E. histolytica, being capable of inhibiting the locomotion
of human mononuclear phagocytes and the respiratory burst as well, facilitating
amoeba invasion (Rico et al., 1992; Kretschmer
et al., 2001). Later, a similar study showed that E. dispar
in axenic cultures does not present the monocyte locomotion inhibitor factor,
such a lack being likely to consequently facilitate its elimination by leukocytes
(Silva-Garcia et al., 2003).
Stanley (2003) proposed a model for induction of inflammation
and tissue damages in amoebic colitis. Some stages were highlighted, as exposed
||Adherence of trophozoites to the intestinal epithelial cells
||Activation of the virulence program (amoebapores and cysteine
||Cell damages and liberation of IL-1β precursor
||Cleaving IL-1β by the cysteine proteinases of amoebas
||IL-1β activates Nuclear Factor Kappa Beta (NF-κB),
leading to the liberation of inflammatory mediators such as IL-8 and COX-2
||Neutrophils are attracted by chemotactic substances
||Macrophages liberate TNF-α
||Substances liberated by leukocytes and amoebae cause tissue
damages (cysteine-proteinases, other enzymes and free radicals)
||Amoebae invade the tissue
There are evidences showing that innate immunity plays a fundamental role at
the healing of amoebic colitis, as the patients who were treated with high doses
of corticosteroids, or, in other words, with powerful inhibitors of NF-κB,
presented the disease in its most severe form (Stanley,
The relevance of innate immunity is also evidenced by a recent study in
vivo, demonstrating that an inflammatory response induced by bacilli Calmette-Guérin
and lipopolysaccharide protected the challenged animals, avoiding an invasive
amoebiasis (Shibayama et al., 2008).
Nevertheless, it is important to emphasize that the infection can be aggravated
by exaggerated immune responses from the host, since elements resulting from
activated leukocytes, such as free radicals, may contribute to give rise to
lesions (Stanley, 2003; Santi-Rocca
et al., 2009).
The interaction of amoeba isolates of low pathogenicity with a variety of gram-negative
bacteria, mainly E. coli strains, which are readily ingested by the amoebae,
significantly increased the ability of the trophozoites to ingest and destroy
epithelial cells, to secrete cytopathic substances and to cause hepatic abscesses
in hamsters (Mirelman et al., 1983).
A recent study in vitro showed an increased pathogenicity of trophozoites
in xenic cultures (Pysova et al., 2009), corroborating
the data from Furst et al. (2002) and Costa
et al. (2007) who showed that E. dispar, yet being considered
non-pathogenic, may, in association with the specific microbiota, produce hepatic
injuries in hamsters. In xenic cultures, E. dispar trophozoites induce
severe hepatic lesions and still present lytic activity for VERO cells.
AMOEBAE-LEUKOCYTES INTERACTION AND OXIDATIVE STRESS
Molecular oxygen is indispensable for the life of the most part of organisms.
However, considering the chemical characteristics and the metabolic pathways
of its use, some possible reactions may produce deletery effects to life itself.
Such a destructive aspect is not properly due to molecular oxygen, since this
latter shows low reactivity and does not appear as a direct causative agent
of oxidative lesions. Nevertheless, the intermediary products from its metabolism,
known as reactive oxygen species, are involved in several kinds of oxidative
events inside the cells, generating the oxidation of cellular structures (Halliwell
and Gutteridge, 1996).
During the process of cellular oxidation, a great part of the oxygen consumed
is reduced to water, but about 2 to 5% of this oxygen may suffer a sequential
univalent reduction and form superoxide anions (O2¯), hydrogen
peroxide (H2O2) and hydroxyl radical (OH¯) (Alessio,
A free radical is defined as any chemical specie presenting one or more unpaired
electrons or, in other words, an electron that is the unique to occupy an atomic
or molecular orbital (Halliwell and Gutteridge, 1996).
The concept of oxygen as being capable of forming free radicals and producing
toxic effects is very ancient. By 1960, it was proposed that living organisms
may also produce endogen free radicals, since they present an enzymatic complex
likely to eliminate superoxide anions, which is known as antioxidant enzyme
superoxide dismutase (SOD) (Fridovich, 1995).
The superoxide radical (O2¯) is formed after the first reduction
of O2. It is produced during the maximum activation of neutrophils,
monocytes, macrophages and eosinophils. Despite being considered as not very
reactive in water solutions, an excessive production of this radical has been
observed to cause secondary biological lesion to systems that generate O2¯
(Halliwell and Gutteridge, 1990).
The hydroperoxil radical (HO2●) represents the protonated
form of the superoxide radical or, in other words, possesses the hydrogen proton.
There are evidences that hydroperoxil is more reacting than superoxide, given
its bigger facility in triggering the destruction of biological membranes (Halliwell
and Gutteridge, 1990).
The hydroxyl radical (OH-) is more reactive in biological systems. Its extremely
rapid combination with metals or other radicals at the site where it was produced
confirms its high degree of reactivity. Therefore, if hydroxyl is produced close
to DNA, modifications of purine and pirimidinic bases may occur, leading to
DNA inactivation or mutation. Besides, hydroxyl can inactivate several proteins
(enzymes and cellular membrane) by oxidizing its sulphidril groups to disulphide
bridges. It can also trigger the oxidation of polyunsaturated fatty acids of
cellular membranes (lipoperoxidation) (Halliwell and Gutteridge,
According to Eaton (1991), the hydrogen peroxide (H2O2),
yet being not characterized as a free radical due to the lack of unpaired electrons
at the last layer, is a deletery oxygen metabolite, since it takes part at the
reaction that produces OH¯. The high toxicity is related to the lipoperoxidation
of membranes, especially in association with iron, Fenton and Haber-Weiss reaction.
The free radicals can attack all the main classes of biomelocules, among which
lipids are the most susceptible ones. The polyunsaturated fatty acids of cellular
membranes are rapidly affected by oxidizing radicals. Lipoperoxidation, as a
reaction of self-propagation at the membrane, is very harmful (Halliwell
and Gutteridge, 1990).
The body system of antioxidant defense plays the main role of inhibiting or
reducing damages caused to the cells by the reactive oxygen species. There is
a wide range of antioxidant substances, which can be classified in accordance
with their source and/or location as antioxidants from nutrition habits and
intra and extracellular antioxidants. The action mechanism still allows to classify
them as preventive antioxidants (those which prevent formation of free radicals),
scavengers (those which prevent attack from free radicals to the cells) and
antioxidants for repair (which remedy damages to the DNA molecule and reconstitute
the injured cellular membranes) (Jacob, 1985).
The SOD has been reported as a significant antioxidating mechanism in eukaryotic,
prokaryotic, strictly aerobic and microaerophilic organisms. In order to play
its functional role, this enzyme requires a metal as co-factor, which can be
copper (Cu-SOD), manganese (Mn-SOD) or iron (Fe-SOD) (Fridovich,
This enzyme acts as a catalyzer for the dismutation of superoxide anion. Dismutation
is a reaction where two identical molecules are transformed into different composites.
In the case of SOD, a superoxide ion oxidizes the other, generating O2
(normal) and oxygenated water (H2O2); this latter may
undergo further degradation through catalase or peroxidase (Yu,
Entamoeba and other protozoan are considered to be anaerobes because
they can be grown in vitro only under conditions of reduced oxygen tension,
being susceptible to the reactive oxygen species. However, these parasites have
been found to be aerotolerant, such data indicating the existence of efficient
mechanisms for detoxification (Mehlotra, 1996).
E. histolytica has been described as a microorganism of anaerobic metabolism.
Nevertheless, some works report that, even without mitochondria and Krebs cycle,
the parasite presents an incomplete respiratory chain with a complex (Fe-S)-protein
instead of cytochrome (Weinbach et al., 1980).
Another study identified on E. histolytica a mitochondrial homolog organelle
known as Crypton/Mitosome and suggested that it may share common ancestry with
mitochondria (Chan et al., 2005).
During the invasion of the host, E. histolytica trophozoites are exposed
to high quantities of reactive oxygen species, such as the superoxide radical.
The high toxicity of these molecules causes several injuries to the biologic
macromolecules, leading to metabolic damages. In order to survive in this environment,
the parasite must be able to inactivate free radicals (Murray
et al., 1981; Clark et al., 1986).
Chen et al. (1996) showed antioxidants mechanisms
in E. histolytica, found SOD and catalase enzymes. Lo
and Reeves (1980) described the purification of the enzyme NADPH:flavin
oxidoreductase in E. histolytica lysed cells. The parasite produces Fe-SOD
(SOD associated with iron), which is induced by the superoxide anion, as well
as H2O2, which can also be detoxified by NADPH:flavin
oxidoreductase (Bruchhaus et al., 1998).
Studies demonstrated a significant increase in the level of SOD and of the
surface enzyme EH29 (thiol-dependent peroxidase) when trophozoites are exposed
to high levels of oxygen, thus suggesting the role of said enzymes in the survival
of the parasite under oxidative stress (Ankri, 2002;
Akbar et al., 2004; Sen et
Wassmann et al. (1999) evaluated E. histolytica
resistance to metronidazole and detected that this data was related to enzymatic
changes, as SOD expression appeared to be increased at the resistant amoebae.
Classical studies involving amoebae and leukocytes, such as those performed
by Jarumilinta and Kradolfer (1964) and Guerrant
et al. (1981), demonstrated that virulent amoebae are lethal to leukocytes.
Salata et al. (1985) also demonstrated the capacity
of E. histolytica trophozoites (HM1) as killers of PMN and MN leukocytes.
Amoebae were able to kill leukocytes, even those found on previously immunized
Guerrant et al. (1981) described the interaction
between E. histolytica and PMNs. Both in vitro and in vivo
studies showed that trophozoites from less virulent E. histolytica strains
were surrounded, fragmented and ingested by PMNs. In contrast, contact with
trophozoites from more virulent amoebae strains caused loss of motility of these
leukocytes, which were phagocited and killed by them.
A study showed that, in the presence of IFN-γ, in vitro macrophages
presented an increased capacity of killing E. histolytica trophozoites.
Amoebicidal activity was determined by counting the number of dead trophozoites
in cultures containing macrophages and amoebic trophozoites, which were incubated
together for 4 h. The treatment with IFN-γ activated murine peritoneal
macrophages to kill amoebae, demonstrating that, upon activation, macrophages
are significantly more efficient (Ghadirian and Denis, 1992).
The study conducted by Sanchez-Guillen et al. (2002)
showed that invasive amoebiasis was related to IL-4 production, indicating a
Th2 profile and that, among asymptomatic carriers, the disease was correlated
with Th1 response, with high levels of IFN-γ.
Another study also evaluated the role of cytokines at the interactions and
showed that the immune response to LPPG is mediated by TLR2 and TLR4. The balance
between pro-inflammatory and anti-inflammatory cytokines produced by MNs regulates
the innate immune responses and an eventual unbalance is harmful to the host.
When used to challenge MNs, LPPG stimulated the production of anti-inflammatory
cytokines, such as IL-10, indicating that the parasite can modulate the host
response on its benefit (Maldonado-Bernal et al.,
A recent study showed that the lesions verified at amoebic colitis presented
a high concentration of neutrophils and lymphocytes, such data grounding the
authors theory that the interaction between PMNs and trophozoites contributes
to the pathogenicity (Dickson-Gonzalez et al., 2009).
Few data refer to the dose of superoxide in interactions between amoebae and
leukocytes and the results are still controversial. Lin
et al. (1993) investigated the effect of HM1 strain on the oxidative
burst of macrophages. The treatment of peritoneal macrophages with amoebic soluble
proteins increased a dose-dependent liberation of O2¯e de H2O2.
Ghadirian and Kongshavn (1988) studied the interaction
between MN and two E. histolytica strains, among which one was virulent
and verified that the levels of superoxide produced by macrophages were increased
in the presence of both of them, especially of the virulent one.
Gandhi et al. (1987) detected that PMN cells
from patients showing severe forms of amoebiasis presented high levels of superoxide,
in contrast to the data observed in cells from patients affected by non-invasive
forms of the disease.
However, Arbo et al. (1990) reported that the
oxidative response of neutrophils appeared to be reduced in the presence of
amoebae. Manrique et al. (2002) found no increase
in the superoxide production by PMN cells in the presence of antigens from E.
histolytica pathogenic strains.
During invasion, trophozoites are exposed to elevated quantities of reactive
oxygen species, such as the superoxide radical. According to Ramos-Martinez
et al. (2009), the highly virulent E. histolytica phenotype
is related to its great skill to reduce superoxide.
Entamoeba histolytica produces SOD with iron and this enzyme is induced
by the superoxide anion, leading to the production of H2O2.
Similarly to SOD, NADPH:flavin oxidoredutase (Eh34) also plays an antioxidant
role by converting oxygen into H2O2, which, on its turn,
may be eliminated by the peroxiredoxin enzyme (Bruchhaus
et al., 1998).
Studies showed that both SOD and EH29 surface enzyme this latter acting as
a free radical blocker, are significantly increased when trophozoites are exposed
to high levels of oxygen, suggesting that both of them are involved in the survival
of the parasite under oxidative stress (Akbar et al.,
2004; Sen et al., 2007).
Studies confirmed the existence of superoxide in amoebae. Akbar
et al. (2004) reported the presence of free radicals in trophozoites
that encountered high-oxygen environment. Munoz-Sanchez
et al. (1997) ascertained that amoebae in cultures produce free
radicals from oxygen, although these radicals are likely to cause biological
damages on them. Crisostomo-Vazquez et al. (2002)
evaluated the correlation between free radicals produced by E. histolytica
and proteases (azocasein and azoalbumin), suggesting that free radicals contribute
to the proteases action.
Guerrant et al. (1981) studied the interaction
between E. histolytica and polymorphonuclear (PMN) phagocytes. Both in
vitro and in vivo studies showed that trophozoites of less virulent
E. histolytica strains were surrounded by PMNs which fragmented and ingested
them, while virulent amoebae were able to internalize and kill PMNs.
Vinayak et al. (1990) evaluated the interaction
between peritoneal macrophages and trophozoites of a virulent E. histolytica
strain (NIH:200) and noted that, in the presence of antibodies, these latter
were destroyed by macrophages when opsonized.
Ghadirian and Denis (1992) analyzed the role of IFN-γ
at the activation of in vitro macrophages to kill E. histolytica
trophozoites. Amoebicidal activity was determined by counting the number of
dead trophozoites in cultures containing macrophages and trophozoites of a E.
histolytica virulent strain, which were incubated together for four hours.
After treatment with IFN-γ, a significant increase in the number of killed
amoebae was verified.
Franca-Botelho et al. (2010) showed interactions
between leukocytes and amoebae in vitro, as well as the correlated oxidative
stress. In the presence of the E. histolytica HM1-IMSS and ICB-32 strains,
associated with PMN leukocytes, the levels of superoxide were increased. Surprisingly,
such augmentation was still more significant in relation to the ICB-32 strain,
which may be possibly related to the differences at the stock of detoxification
substances. The levels of SOD were higher for this strain, what could justify
a lower level of superoxide at the incubations of PMN with HM1-IMSS, when compared
to ICB-32. As to the E. dispar ICB-ADO strain, O2- was
found in its lowest levels. Nevertheless, contrarily to what happened to HM1-IMSS,
SOD was also disclosed in low levels, such data giving rise to the following
possibilities: leukocytes may have used non-oxidative mechanisms to destroy
these amoebae, or this particular strain is so susceptible to O2-
that it was incapable to resist even to the contact with low levels of
Studies showed an important role also played by nitric oxide at the macrophage-mediated
death of amoebae, since the survival of this parasite depends on its ability
to evade such immune mechanism. This skill is described by works that report
the amoeba capacity to inhibit the nitric oxide synthesis and to catabolize
this substance (Lin et al., 1992; Seydel
et al., 2000; Ankri, 2002).
Epidemiological data estimate that about 90% of amoebiasis cases are asymptomatic
and that, among the symptomatic forms, the intestinal amoebiasis is the most
frequent one (Stanley, 2003).
Patients with non-dysenteric colitis present abdominal colics and intervals
of diarrhoea and asymptomatic periods of normal intestinal activity. Some cases
involve a dysenteric colitis, characterized by exacerbated dyspeptic symptoms
(pain, eructation, burning sensation and nausea), abdominal distension, flatulence,
more than ten daily muco-bloody evacuations and constant sensation of evacuation
need. Submucosa is then filled with ulcers, that may cause hydroelectrolytic
disturbs and energetic-proteic malnutrition (Melo et
In extraintestinal amoebiasis, trophozoites can migrate through the superior
mesenteric vessel and reach the liver, where they cause inflammation, cellular
degeneration and liquefative necrosis, thus forming the amoebic abscess, generally
located at the right lobe. Patients present fever, intense pain at the right
hypochondrium, as well as typical irradiations of biliary colic and painful
hepatomegaly at palpation, which does not use to be icteric (Thompson
et al., 1985).
The most common extraintestinal manifestation, which occurs at the liver, was
considered to be invariably fatal in the past. However, since the introduction
of more efficient methods of diagnostic and treatment, mortality rates have
fallen to 1-3% (Thompson et al., 1985; Barnes
et al., 1987).
Amoebiasis may be aggravated by bacterial secondary infections, which are likely
to cause an abscess rupture to abdomen, to the lung, to the pleura or the pericardium.
The hematogenic dissemination of trophozoites can injure the lung, the skin,
the pericardium, the genitourinary system and the brain (Stanley,
A study conducted in Vietnam reported 21 cases of hepatic amoebiasis in each
group of one hundred thousand persons living in the city of Hue (Blessmann
et al., 2002). Hepatic lesions seem to be more incident among HIV
(Human Immunodeficiency Virus) patients, as described by a study with patients
from the National Hospital of Seul, which found that 32% of patients with amoebic
hepatic lesions were HIV positive (Muzaffar et al.,
Papavramidis et al. (2008) reported a case of
acute abdomen due to the rupture of a gigantic amoebic liver abscess, which
reflects a severe form of the disease with high mortality rates. The authors
emphasized that prompt diagnosis and treatment are fundamental to preserve the
Fulminant amoebic colitis is a severe form of the disease, mostly identified
among pregnant women and immunocompromised patients and is usually reported
in association with diabetes mellitus and chronic alcoholism (Takahashi
et al., 1997; Stanley, 2003; Suarez-Artacho
et al., 2006).
DIAGNOSIS AND TREATMENT
Intestinal amoebiasis is traditionally diagnosed through laboratorial tests
to detect the parasite on the feces. Cystic forms use to be found on consistent
feces, while the trophozoitic ones are present on a diarrheic or pasty fecal
material. However, the lack of technical experience, the intermittent elimination
of E. histolytica/E. dispar cysts (Walsh, 1986)
and the non-differentiation from other intestinal amoebae, cells and artefacts
may jeopardize the microscopic diagnosis (Bruckner, 1992).
At the immunodiagnostic, the reaction of indirect immunofluorescence (RIFI)
for the research of parasite-specific antibodies at the serum of patients, as
well as the immunoenzymatic assay (ELISA) for detection of coproantigens at
the feces have been used as diagnostic alternatives. Both techniques may be
recommended for diagnosis of isolated cases or for epidemiological studies (Haque
et al., 1998), showing higher specificity and sensitiveness if compared
to microscopy (Katzwinkel- Wladarsch et al., 1994).
The Polymerase Chain Reaction (PCR) is reputed as the most specific method for
the identification of E. histolytica infections, thus offering new perspectives
for future use at the laboratorial routine. However, this method still requires
optimization to become more practical and less expensive (Silva
and Gomes, 2005).
In an effort to improve the diagnosis of intestinal amoebiasis, Gutierrez-Cisneros
et al. (2010) showed that real-time PCR has been used for the detection
and differentiation of E. histolytica and E. dispar infections.
Fecal samples from 130 individuals with positive microscopic examination were
analyzed by real-time PCR, which detected E. histolytica DNA in materials
from only 10 (7.7%), while E. dispar DNA was found in samples from 117
As to the therapeutic perspectives, metronidazole is the most used amoebicidal
drug worldwide. Being well tolerated, it is nowadays the drug recommended for
both intestinal and hepatic amoebiasis, at the doses of 500-800 mg, three times
a day, during five to ten days. If such a treatment does not reach satisfactory
results, it is recommendable to associate metronidazole to antibiotics. Apart
from drug therapy, fecal contamination of water and food should be avoided by
fundamental prophylactic policies such as, for instance, installation of basic
sanitary conditions, as well as a sanitary education and a strict control of
individuals who manipulate food and may be, sometimes, asymptomatic carriers
of amoebiasis (Silva and Gomes, 2005).
CONCLUSION AND FINAL CONSIDERATIONS
Amoebiasis is reputed by the World Health Organization as one of the leading health problems in developing countries, constituting important cause of death. E. histolytica is capable of causing devastating dysentery, colitis and liver abscess.
Although, being broadly spread, the disease tends to reach the highest prevalence rates in developing countries, where the investments in basic sanitary conditions are usually insufficient to meet the necessary requirements, thus allowing the expansion of amoebiasis through orofecal transmission. In the lack of significant improvements and progress in the public health area, which would strongly contribute to the reduction of cases of amoebiasis, it is mandatory to advance on the study of the parasite biology and its pathogenicity, in an attempt to develop more efficient methods for the treatment and prevention of the disease.
Many works focused on such aspects have been developed worldwide, but there are still unclear features that require new researches. A better understanding on the relationship between parasite and the host immune response is crucial for the development of vaccines and for the improvement of therapeutic alternatives to amoebiasis.
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