Chapter 2

Protozoan Infections

Intestinal Amebae
Pilarita T. Rivera, Windell L. Rivera, Juan Antonio A. Solon

S even species of amebae occur in humans.

These include the pathogenic Entamoeba
histolytica, and the commensals E. dispar, E.
reaction (PCR) restriction fragment length
polymorphism (RFLP), and typing with
monoclonal antibodies, these three species are
moshkovskii, E. hartmanni, E. coli, Endolimax now differentiated. E. hartmanni, formerly
nana, and Iodamoeba butschlii. Entamoeba referred to as “small race” of E. histolytica, is
polecki is an intestinal ameba of pigs and differentiated primarily on the basis of size.
monkeys that has been occasionally detected
Parasite Biology
in humans, and is a probable cause of diarrhea.
They are mainly differentiated on the basis of Entamoeba histolytica is a pseudopod-
structure and size. Trophozoites divide by binary forming non-flagellated protozoan parasite. It
fission. Most cyst-forming amebae go through is the most invasive of the Entamoeba parasites
nuclear division, and then divide again after (which includes E. dispar, E. moshkovskii, E.
excystation in a new host. hartmanni, E. polecki, E. coli, and E. gingivalis),
and the only member of the family to cause colitis
Entamoeba histolytica
and liver abscess. The life cycle of E. histolytica
Entamoeba histolytica is currently classified consists of two stages: an infective cyst (Plate
within the subphylum Sarcodina, superclass 2.1) and an invasive trophozoite form. No host
Rhizopoda, class Lobosea, order Amoebida, other than humans is implicated in the life cycle,
family Entamoebidae, and genus Entamoeba.
The members of this genus are characterized
by having a vesicular nucleus, a centrally (or near
central) located small karyosome, and varying
numbers of chromatin granules adhering to
the nuclear membrane. These nuclear and
other morphologic differences distinguish
the species of Entamoeba except E. histolytica,
E. dispar, and E. moshkovskii (previously
known as the Laredo strain). The three said
species are morphologically identical and of
the same size. It was only recently that this
E. histolytica species complex was resolved. Plate 2.1. Entamoeba histolytica cyst (Courtesy
Through isoenzyme analysis polymerase chain of the Department of Parasitology, UP-CPH)

20
 Chapter 2: Protozoan Infections 21

although natural infection of primates has been Infection with E. histolytica occurs when cysts
reported. The quadrinucleate cyst is resistant to are ingested from fecally-contaminated material
gastric acidity and desiccation, and can survive (Figure 2.1). Other modes of transmission
in a moist environment for several weeks. include venereal transmission through fecal-oral

Figure 2.1. Life cycle of Entamoeba histolytica

(Accessed from www.dpd.cdc.gov/dpdx)
22 Medical Parasitology in the Philippines

contact or direct colonic inoculation through

contaminated enema equipment. Excystation
occurs in the small or large bowel, where a cyst
undergoes nuclear followed by cytoplasmic
division to form eight trophozoites. The E.
histolytica trophozoites are highly motile and
possess pseudopodia (Plate 2.2). They vary in
size from 12 to 60 μm in diameter (about 20 μm
in average). Microscopic examination of fully-
passed stool specimens reveals the characteristic
progressive and directional movement of
trophozoites, with pseudopodia as locomotory
Plate 2.2. Entamoeba histolytica trophozoite
organelles. The hyaline pseudopodium is (From World Health Organization. Bench Aids for
formed when the clear, glasslike ectoplasm, the Diagnosis of Intestinal Parasites.
or outer layer is extruded, and the granular Geneva: World Health Organization; 1994)
endoplasm flows into it. Ingested red blood
cells are observed as pale, greenish, refractile
bodies in the cytoplasm of the ameba. Cysts are
usually spherical, and the size may vary from 10
to 20 μm. They are characterized by a highly
refractile hyaline cyst wall, one to four nuclei,
and rod-shaped (or cigar-shaped) chromatoidal
bars. Trophozoites have the ability to colonize
and/or invade the large bowel, while cysts are
never found within invaded tissues. E. histolytica
trophozoites multiply by binary fission. They
encyst producing uninucleate cysts, which
then undergo two successive nuclear divisions Plate 2.3. Entamoeba histolytica quadrinucleate
to form the characteristic quadrinucleate cysts cyst (From World Health Organization. Bench Aids
(Plate 2.3). for the Diagnosis of Intestinal Parasites. Geneva:
E. histolytica is a eukaryotic organism but World Health Organization; 1994)
has several unusual features, including the lack
of organelles that morphologically resemble lack of glutathione metabolism, the use of
mitochondria. Because nuclear-encoded pyrophosphate instead of ATP at several steps
mitochondrial genes such as pyridine nucleotide in glycolysis, and the inability to synthesize
transhydrogenase and hsp60 are present, E. purine nucleotides de novo. Glucose is actively
histolytica, at one time may have contained transported into the cytoplasm, where the
mitochondria. There is no rough endoplasmic end products of carbohydrate metabolism are
reticulum or Golgi apparatus, although cell ethanol, carbon dioxide, and under aerobic
surface and secreted proteins contain signal conditions, acetate.
sequences, and tunicamycin inhibits protein Pathogenesis and Clinical Manifestations
glycosylation. Ribosomes form aggregated
crystalline arrays in the cytoplasm of the The proposed mechanisms for virulence
trophozoite. Some differences in biochemical are: production of enzymes or other cytotoxic
pathways from higher eukaryotes include the substances, contact-dependent cell killing,
 Chapter 2: Protozoan Infections 23

and cytophagocytosis. In vitro, amebic killing study involving 206 patients with probable
of target cultivated mammalian cells involve ALA as diagnosed by ultrasound, the two
receptor-mediated adherence of ameba to most frequent manifestations were fever in
target cells, amebic cytolysis of target cells, 77% and RUQ pain in 83%. Pain is either
and amebic phagocytosis of killed or viable localized in or referred to the right shoulder.
target cells. E. histolytica trophozoites adhere The liver is tender, especially in acute cases,
to the colonic mucosa through a galactose- and hepatomegaly is present in 50% of cases.
inhibitable adherence lectin (Gal lectin). Then, Chronic disease (>2 weeks duration) is found
the amebae kill mucosal cells by activation of in older patients and it involves wasting with
their caspase-3, leading to their apoptotic death significant weight loss rather than fever. Only
engulfment. 30% of ALA cases have concurrent diarrhea.
Recent studies have shown that susceptibility However, daily stool cultures revealed that 72%
of humans to E. histolytica infection is associated harbored trophozoites even in asymptomatic
with specific alleles of the HLA complex. infections. Mortality in uncomplicated ALA is
Majority of cases present as asymptomatic less than 1%.
infections with cysts being passed out in The onset of amebic colitis may be sudden
the stools (cyst carrier state). The recent after an incubation period of 8 to 10 days, or
differentiation of E. dispar and E. histolytica after a long period of asymptomatic cyst carrier
by PCR has confirmed the high prevalence state. ALA may have all acute presentation of
of non-pathogenic E. dispar compared to the less than 2 weeks duration or a chronic one of
pathogenic E. histolytica. However, studies also more than 2 weeks duration. The recurrence
revealed that most E. histolytica infections in rate was found to be 0.29% in a five-year study
endemic communities are asymptomatic. of ALA in Mexico.
Amebic colitis clinically presents as gradual The most serious complication of amebic
onset of abdominal pain and diarrhea with or colitis is perforation and secondary bacterial
without blood and mucus in the stools. Fever peritonitis. Colonic perforation occurs in 60%
is not common and it occurs only in one third of fulminant colitis cases.
of patients. Although some patients may only In ALA, the most serious complications are
have intermittent diarrhea alternating with rupture into the pericardium with a mortality
constipation, children may develop fulminant rate of 70%, rupture into the pleura with
colitis with severe bloody diarrhea, fever, and mortality of 15 to 30%, and super infection.
abdominal pain. Intraperitoneal rupture, which occurs in 2 to
Ameboma occurs in less than 1% of 7.5% of cases, is the second most common
intestinal infections. It clinically presents as complication. However, it is not as serious as
a mass-like lesion with abdominal pain and colonic perforation because ALA is sterile.
a history of dysentery. It can be mistaken for Secondary amebic meningoencephalitis
carcinoma. Asymptomatic ameboma may also occurs in 1 to 2%, and it should be considered
occur. in cases of amebiasis with abnormal mental
Amebic liver abscess (ALA) is the most status. Renal involvement caused by extension
common extra-intestinal form of amebiasis. of ALA or retroperitoneal colonic perforation is
The cardinal manifestations of ALA are fever rare. Genital involvement is caused by fistulae
and right upper quadrant (RUQ) pain. Several from ALA and colitis or primary infection
studies have shown these two as the most through sexual transmission.
frequent complaints, particularly in acute Natural or innate immunity to E. histolytica
cases (<2 weeks duration). In a Philippine in the intestines involves mucin inhibition of
24 Medical Parasitology in the Philippines

amebic attachment to the underlying mucosal Acute amebic colitis should be differentiated
cells. In the systemic circulation, the mechanism from bacillary dysentery of the following
is that of complement-mediated killing of etiology: Shigella, Salmonella, Campylobacter,
trophozoites. Acquired immunity primarily Yersinia, and enteroinvasive Escherichia coli
involves cell-mediated responses, although (Table 2.1). Although stools may be grossly
humoral responses may also contribute to bloody or heme-positive in both conditions,
anti-amebic immunity. Activated T-cells kill fever and significantly elevated leukocyte count
E. histolytica by: a) directly lysing trophozoites are less common in amebic colitis. Another
in a contact-dependent process; b) producing differential is inflammatory bowel disease.
cytokines which activate macrophages and other Amebic colitis should be ruled out before
effector cells (neutrophils and eosinophils); and steroid therapy for inflammatory bowel disease
c) providing helper effect for B-cell antibody is started because of the risk of developing toxic
production. In vitro studies using activated megacolon.
murine and human T-cells demonstrated The differential diagnoses of ALA include
significant killing of trophozoites in a contact- pyogenic liver abscess, tuberculosis of the liver,
dependent and antibody independent manner. and hepatic carcinoma. On the other hand,
Cytokine studies revealed that interferon (IFN) genital amebiasis should be differentiated
and interleukin (IL-2) may have a role in from carcinoma, tuberculosis, chancroid, and
activating macrophages for amebicidal activity. lymphogranuloma venereum.
More recent studies demonstrated that activated
macrophages produce nitric oxide (NO) which Table 2.1. Comparison of bacillary and amebic
was lethal to trophozoites. Tumor necrosis factor dysentery
(TNF) was shown to stimulate NO production.
Bacillary Dysentery Amebic Dysentery
Although it is known that antibodies are
May be epidemic Seldom epidemic
produced against amebic antigens, there has
Acute onset Gradual onset
been no direct evidence of T-cell help for
Prodromal fever and No prodromal features
B-cells. Studies have revealed that the principal malaise common
antibody-dependent cell cytotoxicity (ADCC) Vomiting common No vomiting
did not work against amebae. Antibodies which Patient prostrate Patient usually ambulant
were detected by seroepidemiologic studies and
Watery, bloody diarrhea Bloody diarrhea
secretory IgA isolated in the gut may merely
Odorless stool Fishy odor stool
be an indicator of current or recent invasive
Stool microscopy:
amebiasis. numerous bacilli, pus
Amebic modulation of host immune cells,
responses exists. For instance, infected human macrophages, red cells, Stool microscopy: few
no Charcot-Leyden bacilli, red cells,
subjects and animals have been shown to be in crystals trophozoites with
a state of immunosuppression during the acute ingested red blood
cells, Charcot-Leyden
stage of amebiasis. This state, characterized crystals
by T-cell hyporesponsiveness, suppressed Abdominal cramps Mild abdominal cramps
proliferation and cytokine production, depressed common and severe
delayed-type hypersensitivity (DTH), and Tenesmus common Tenesmus uncommon
macrophage suppression, is favorable for amebic Natural history: Natural history: lasts for
survival. It is the reversal of these modulatory spontaneous recovery weeks; dysentery
in a few days, weeks or returns after remission;
effects, which is the key in controlling amebiasis. more; no relapse infection persists for
years
 Chapter 2: Protozoan Infections 25

Diagnosis following morphologic structures are noted:

size of the cyst, number of nuclei, location and
The standard method of parasitologic
appearance of the karyosome, the characteristic
diagnosis is microscopic detection of the
appearance of chromatoid bodies, and presence
trophozoites and cysts in stool specimens.
of cytoplasmic structures such as glycogen
Ideally, a minimum of three stool specimens
vacuole. E. histolytica can, thus, be differentiated
collected on different days should be examined.
from the non-pathogenic species, E. hartmanni,
For detection of trophozoites, fresh stool
E. coli, E. nana, and Iodameba bütschlii. Stool
specimens should be examined within 30
culture using Robinson’s and Inoki medium is
minutes from defecation. Using the direct fecal
more sensitive than stool microscopy, but is not
smear (DFS) with saline solution alone, the
routinely available.
microscopist can observe trophozoite motility.
Differentiation between E. histolytica and
Unidirectional movement is characteristic
E. dispar is not possible by microscopy. This
of E. histolytica. Using saline and methylene
can only be done by PCR, enzyme-linked
blue, Entamoeba species will stain blue, thus,
immunosorbent assay (ELISA), and isoenzyme
differentiating them from white blood cells.
analysis. The last is primarily a research
Using saline and iodine, the nucleus and
technique. On the other hand, an ELISA-based
karyosome can be observed to differentiate E.
assay for stool is now commercially available
histolytica from the non-pathogenic amebae
and studies have demonstrated a sensitivity of
(E. hartmanni, E. coli, Endolimax nana).
80% and specificity of 99%. The use of PCR
The detection of E. histolytica trophozoites
is limited by the requirement of sophisticated
with ingested red blood cells is diagnostic of
equipment. A Philippine study (n=497 stool
amebiasis. Charcot-Leyden crystals (Plate 2.4)
samples) looked into the reliability of stool
can also be seen in the stool.
ELISA with PCR as gold standard (Plate 2.5).
Sensitivity and specificity were 91% and 97%,
respectively.
Detection of antibodies in the serum is
still the key in the diagnosis of ALA. It must
be noted that in ALA, microscopic detection
cannot be done because aspiration is an invasive
procedure, and trophozoites are missed because
they are located in the periphery of the abscess.

Plate 2.4. Charcot-Leyden crystal observed

in stool specimen of a patient suffering from
amebiasis (Courtesy of the Department of
Parasitology, UP-CPH)

Concentration methods such as Formalin

Ether/Ethyl Acetate Concentration Test
Plate 2.5. Agarose gel showing the 100bp PCR
(FECT) and Merthiolate Iodine Formalin products of Entamoeba histolytica-positive
Concentration Test (MIFC) are more sensitive stool specimens (lanes 2-15)
than the DFS for detection of cysts. The (Courtesy of Dr. Windell Rivera)
26 Medical Parasitology in the Philippines

To date, serological tests for amebic disease Treatment and Prognosis

include indirect hemagglutination (IHAT),
The treatment of amebiasis has two
counter immunoelectrophoresis (CIE), agar gel
objectives: a) to cure invasive disease at both
diffusion (AGD), indirect fluorescent antibody
intestinal and extraintestinal sites; and b) to
test (IFAT), and ELISA. The IHAT can detect
eliminate the passage of cysts from the intestinal
antibodies of a past infection even as long as 10
lumen. Metronidazole is the drug of choice
years ago. In contrast, the antibodies detected by
for the treatment of invasive amebiasis. Other
ELISA, AGD, and CIE are of short duration,
5-nitroimidazole derivatives such as tinidazole
lasting for a few months. Antibodies have
and secnidazole are also effective. Diloxanide
been demonstrated in asymptomatic intestinal
furoate is the drug of choice for asymptomatic
infections so that serology can be used in the
cyst passers. It is also given after a course of
monitoring of a cyst carrier.
metronidazole for invasive amebiasis.
Ultrasound, computerized tomography
Percutaneous drainage of liver abscess is
(CT scan), and magnetic resonance imaging
indicated for patients who do not respond
(MRI) are non-invasive and sensitive methods
to metronidazole and who need prompt
in early detection of ALA. Ultrasound (Plate
symptomatic relief of severe pain. It is also done
2.6) typically shows a round or oval hypoechoic
for those who have left lobe abscess that may
area with wall echoes. In 80% of cases, this
rupture into the pericardium, large abscesses in
finding is seen in the right lobe of the liver.
danger of rupture, and multiple abscesses with
Multiple lesions occur in 50% of acute cases,
a probable associated pyogenic etiology.
and aspiration may be required to differentiate
amebic from pyogenic abscess. Using serological Epidemiology
methods (IHAT and IFAT) as gold standard, a
For a long time, the species-complex
Philippine study has shown that the sensitivity
referred to as E. histolytica was believed to
and specificity of ultrasound were 95% and
infect 500 million people, or 10% of the
40%, respectively. However, as the results of
world’s population. However, with the recent
the study still revealed some limitations in the
redescription into three different species: the
use of ultrasound in the diagnosis of ALA,
pathogenic E. histolytica, and the commensals, E.
additional diagnostic ultrasound findings have
dispar and E. moshkovskii, the true prevalence of
yet to be identified.
amebiasis is approximately 1 to 5% worldwide.
There are 50 million E. histolytica infection
cases, and 40,000 to 100,000 deaths due to
amebiasis in the world per year. Thus, amebiasis
is the third most important parasitic disease,
after malaria and schistosomiasis, and second
to malaria as the top cause of mortality among
parasitic protozoans.
Humans are the major reservoirs of
infection with E. histolytica. Ingestion of food
and drink contaminated with E. histolytica
cysts from human feces, and direct fecal-
oral contact are the most common means of
Plate 2.6. Ultrasound showing a solitary infection. Amebic infection is prevalent in the
hypoechoic mass at the right lobe of the liver Indian subcontinent, Africa, East Asia, and
suggesting ALA (Courtesy of Dr. Pilarita Rivera) South and Central America. In developing
 Chapter 2: Protozoan Infections 27

countries, prevalence depends on the level of cases should be done. Food handlers should be
sanitation, crowding, socio-economic status, screened for cyst carriage, and asymptomatic
cultural habits, and age. In developed countries, cyst carriers should be treated.
infection is usually caused by E. dispar, and Vaccines can be a cost-effective and
is prevalent in certain groups: immigrants, potent strategy for amebiasis prevention
travelers from endemic countries, homosexual and eradication. Unlike in other protozoan
males (men having sex with men), HIV patients, infections, amebic vaccine development has
and institutionalized people. fewer problems. The ameba life cycle is simple,
A microscopic study of diarrheic stools in and no intermediate hosts are involved. Amebae
Australia (n=5,921) revealed 177 (3%) positive are extracellularly located, and do not undergo
samples. PCR detected 5 E. histolytica, 63 E. antigenic variation. All these characteristics are
dispar, and 55 E. moshkovskii infections. The supportive of an achievable amebic vaccine.
latter two species, which are both commensals, Studies have also demonstrated the
are 10 times more prevalent than E. histolytica. acquisition of protective immunity to amebae,
A stool survey done in Iran (n=16,592) showed particularly that of mucosal immune response.
226 positive samples. Only 101 isolates were Trials with recombinant amebic antigens as
successfully cultured in Robinson’s medium. vaccines have proven to be more advantageous
Of these isolates, 93 (92.1%) were E. dispar, than inactivated/attenuated amebae. The
and only 8 (7.9%) were E. histolytica or mixed candidate vaccine molecules which have been
infections by PCR- RFLP. most intensely studied are the serine-rich E.
A field study in Northern Philippines histolytica protein (SREHP), the adherence
(n=1,872) showed 137 (7.3%) E. dispar, and lectin (Gal/GalNAc lectin), and the 29 kDa
18 (0.96%) E. histolytica by PCR. A study in a cysteine-rich amebic antigen. However, most
mental institution (n=113) showed E. histolytica of these studies have utilized animal models
or E. dispar in 43 subjects (38.1%), while PCR and artificial infection during challenge.
detected 74 (65.5%) E. histolytica-positive Testing these candidate vaccines in humans
samples, and 6 (5.3%) E. dispar/E. histolytica and developing them as food-based vaccines
mixed samples. will be in the forefront of future directions of
amebiasis control.
Prevention and Control
References
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education and promotion. The proper use of (Emended Walker, 1911) separating it
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