Amoeba Part 2
Metacystic trophozoite/
Pathogenic Amoeba
- E. histolytica
E. histolytica
- Tissue invading amoeba
- Pathogenic amoeba of man
Clinical manifestations
- Amoebiasis
- Amoebic dysentery
- Amoebic hepatitis (liver abscess)
Synonyms
- Amoeba coli
- Amoeba dysenteriae
- Entamoeba dysenteriae
- Entamoeba tetragena
- Entamoeba dispar
- Endamoeba histolytica
- Endamoeba dysenteriae
Geographic Distribution
- Same as non-pathogenic amoeba
- Worldwide with high incidence in the
tropics and subtropics
Morphology, Biology, Life Cycle
- 5 Stages similar to E. coli
1. Cyst
2. Metacyst
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3.
like/broadly rounded pseudopodal
amoebulae extensions of the ectoplasm
4. Trophozoite o Shares motility of E.
5. Precyst gingivalis, but differ in
direction where the E.
Trophozoite
histolytica can go anywhere
- Invasive, growing, feeding stage of since it is unidirectional
the parasite o Direction changes rapidly in
- Invasive means the parasite is capable response to conditions of their
of invading tissues inside the stomach microclimate
- Invades the tissues wherein it will try - Cytoplasm is described as having a
to feed there ground glass appearance and is
o Parasites are commensal; they differentiated into a clearer outer
need a host to survive and feed ectoplasm and inner, finely granular
off the host endoplasm in which food vacuoles
- I&H Stain of containing ingested RBCs may be
Histolytica observed
o Same with E. coli except
trophozoite
different cytoplasm
appearance, where E.
histolytica has ground glass
appearance while E. coli is
I. Living Trophozoite honeycomb shaped (dirty
looking full of vacuoles of
General Characteristics & Structure
ingested bacteria, not RBCs)
- Size: 10-60 micrometer in diameter - Nucleus can be occasionally seen in
- Motility: Exhibits remarkable the center of the endoplasm
locomotion which can be observed in - (I&H Preparation): Nucleus is
freshly passed dysenteric or diarrheic spherical; contains a small distinct
stools dotlike central karyosome surrounded
o Only the trophozoite stage is by an unstained halo and anchored by
motile numerous delicate, radiating
- Movement: Progressive achromatic fibrils to the inner surface
unidirectional movement resulting of the nuclear membrane
from long, fingerlike/tongue-
 o The achromatic fibrils give is now able to penetrate the small B. Young Cyst
the nucleus the appearance of intestine.
- 1-2 nuclei
a spokes of a wheel or bull’s
A. Mature Cyst - Glycogen mass with hazy margins
eye
- Chromatoidal bodies, which are long
- 4 nuclei with centrally located
or short rods with rounded ends (cigar
karyosome, rarely 8 nuclei
or sausage-shaped) under the
o Characteristic of E. histolytica
microscope in a freshly-prepared
to have centrally located
slide
karyosome
II. Cyst - No cytoplasmic inclusions, making it
clear in appearance
General Characteristics & Structures - Nuclear structure: can be
uninucleated or quadrinucleated
o Uninucleated: contains
chromatid body and a large
glycogen mass/glycogen
vacuole
o Quadrinucleated: mature
cysts present only in the gut Recent evidence shows that there are 2
lumen, they NEVER invade significantly different sizes for strains:
the intestinal wall.
▪ Only the trophozoite 1. Large race
stage is invasive stage - Average cyst diameter is > 10
- Size: 10-20 micrometer, (average of micrometer
12-13 micrometer) - Generally virulent
- Spherical, may be subspherical or 2. Small race
ovoidal - Cyst
- Covered with smooth chitinous layer, o Size: 5-10 micrometer
making itself resistant to gastric acid o Commensal, non-pathogenic,
and other adverse conditions do not normally invade tissues
- Location of Cyst: inside the stomach; o Considered as a separate
with the chitinous layer, this parasite species: E. hartmanni
is capable of surviving the harsh o Life cycle, general
environment inside the stomach, and morphology, and overall
appearance are almost
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 identical to E. histolytica, with
the exception of size (closely
resembles E. nana)
- Immature cyst
o Contains glycogen mass
o Several to numerous
chromatoidals that are short
with tapered ends (rice grain-
shaped) or thin and bar-like
structure
- Trophozoite
o Size: 12-15 micrometer
o Do not ingest RBC
o Motility is less vigorous
(more sluggish) than E.
histolytica
Various reports indicate that there are 2
strains of E. histolytica differing in
pathogenicity
E. histolytica has now been divided into 2
species, the first species E. histolytica, the
other one being the noninvasive E. dispar
- Although E. dispar is noninvasive, it
is evidently capable of producing
intestinal lesions in experimental
animals (E. dispar is pathogenic in
zoonotic cases so far)
Differentiation of the 2 species is not possible
by light microscopy
- Only differentiated by Polymerase
Chain Reaction (PCR), Enzyme-
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linked Immunosorbent Assay
(ELISA), and Isoeznyme analysis (or
research techniques)
E. histolytica Life Cycle
Infective stage: Quadrinucleated mature cyst
of E. histolytica.
Ingestion
The first part of the life cycle is humans get
infected by the ingestion of quadrinucleated
cyst from the contaminated food and water.
This mature cyst reaches the stomach once
ingested, and there, they resist the gastric acid
because of the chitinous wall of the mature
cyst of E. histolytica. Later on, it will pass
through the small intestine.
In the small intestine, the quadrinucleated
cyst will now be hydrolyzed by the enzyme
trypsin inside the small intestine, dissolving
the chitinous layer/wall of the cyst. This
dissolution of the wall releases the
quadrinucleated trophozoites.
The quadrinucleated trophozoites will further
divide into 8 metacystic trophozoites and will
propagate in the small intestine environment.
Once they mature in the small intestine, they
will now pass through the colon.
Multiplication
Metacystic trophozoites are carried by
peristaltic movements to the ileo-cecal region
and divide by binary fission.
There, they colonize the mucosal surfaces
and crypts of the large intestine.
Encystation
Encystation occurs in the small intestine and
is where the metacystic trophozoites are
formed.
Encystation occurs when there is food
deprivation, crowding, desiccation, and
accumulation of waste products.
Later on will exit the excreta and get mixed
with the food and water then the life cycle
goes on.
Contamination of food and water is only
possible when there is no proper sanitation.
When the community doesn’t have comfort
rooms with septic tanks, E. histolytica will
most likely propagate.
Habitat
- Cecum; make contact with mucosa or
become lodged in the glandular crypts
Modes of Transmission
- Ingestion of food and drinks
contaminated with feces containing
the quadrinucleated cysts (infective
stage)
- Polluted water supply: cysts may
remain viable in:
o Damp soil = at least 8 days
o In other moist cool conditions
= for over 12 days
o Water (normal temperature) =
9-30 days
o Water at 4 degrees Celsius = 3
months
- Unclean handling of infected
individuals (hands, clothing, fomites)
o Fomites like flies may carry E.
histolytica and land on your
food
- Droppings of flies and other insects
o Cysts are unchanged in the
intestine of flies and
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cockroaches and remain
viable in feces and vomitus
for 48 hours
o Note: Filth flies (Musca
domestica) and cockroaches
are important mechanical
vectors of cysts. Their sticky,
bristly appendages can easily
carry cysts from fresh stool to
the dinner table; their habit of
vomiting and defecating while
feeding is an important means
of transmission
▪ Even a second or
millisecond when the
fly touches the food
renders the entire food
contaminated with the
parasite
- Use of human excreta in vegetable
gardens
- Gross carelessness in personal
hygiene in children’s asylums, mental
hospitals, prisons, and other places
- Sexually transmitted disease
- Human carriers or cyst passers
Pathogenicity
When E. histolytica is capable of invading
tissue, it is now called Intestinal Amebiasis
- In the intestines
- May remain localized in the colon
(where they colonize, feed, and grow)
and multiply in the crypts
- Attachment is mediated by an
amoebal galactose or N-acetyl-d-
galactosamine adherence lectins
(Gal/GalNac lectin)
- Inside the intestine, the parasites
ingest foodstuffs such as starch
granules (rice) that most humans are
eating
- Probably utilize mucous secretions as
food
- Metabolize anaerobically with certain
enteric bacteria, since they do not
need O2 but more CO2
- Invasion of tissue
o Initiated when the trophozoite
is able to penetrate the mucus
layer covering the colonic
epithelium
o Facilitated by the expression
of virulence factors which
 have been extensively studied
and characterized up to the
molecular level. These are:
▪ Gal/GalNac lectin;
▪ Amebapores; and
▪ Cysteine proteinases
o They no longer depend on
bacteria but obtain their
nourishment through the
absorption of dissolved
tissue juices Diagnosis
▪ They lyse mucosal
1.
cells by secreting
lysosomal enzymes
forming flask-shaped
ulcers (teardrop-
shaped) and
trophozoites are found
in the wall of the
abscess
▪ Primary ulcers are
found in the cecum,
appendix, or
adjacent portion of
the ascending colon
▪ Ulceration of the
intestinal wall may
give rise to amoebic
dysentery
o Encystation does not occur in
the tissue or outside the
intestinal lumen, therefore,
specimens taken outside the
lumen will contain the
trophozoite stage only
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▪The invasive form of
E. histolytica is the
trophozoite, so it will
not encyst or form
back to cyst form
o May affect other organs such
as the liver, brain, lungs,
spleen
Intestinal Amebiasis
- Stool Examination (S/E) by direct
smears and stained mounts
o 3 or more specimens at 3- or
4-day intervals give more
positive (+) results than
examination of stool by
successive days because of the
random irregularity in the
excretion of cyst
- In diarrheic/dysenteric/watery stool,
trophozoites are the parasite stage
only seen, accompanied by blood and
mucus
o Scrape a small portion of the
sample where the blood and
mucus is located since that is
where the cyst is most likely
located
o Should be examined as soon
as possibly preferably within
30 minutes after voiding; if
examined after 30 minutes a
false negative result may
appear, so fixate the sample as
needed
o Wet mounts using Normal
Saline Solution (NSS)
o Preserve parasites if one
cannot examine directly or
immediately
▪ Fix with MIF
(Merthiolate Iodine
Formaldehyde
fixative); or
▪ PVA (Polyvinyl
Alcohol); or
▪ Schaudinn’s fixative
for permanent staining
Note: in lab practice, fixating stool samples
are uncommon because the fixatives are
expensive, and stool examinations cost only
P90-P250, so it is recommended to examine
stool samples immediately after collection
and no longer fixate the specimen.
- In solid/formed stool, only cysts are
the parasite stage seen (usually seen
in carriers or chronic patients,
amebiasis is worse)
o Any part of the feces can be
scraped but should include a
portion or any fleck of
mucous adherent to feces or
blood, get a sample where
there is mucous or blood like
in formed stool
 o Wet mounts using Normal o May be expressed from ulcers • Uncommon
Saline Solution (NSS) or by means of gentle pressure technique
Iodine Preparation (I2) from long-handled curette or - Culture
o If cysts are too few to be seen, loop o Commonly conducted in
do concentration technique o Obtaining specimens from the microbiology
(zinc sulfate [ZnSO4] tissues o Used in the study of its
centrifugal flotation method, o 1/3 of lesions are in the metabolism and
Formalin Ether Concentration sigmoidorectal area pathogenicity, and in the
Test [FECT] and the ▪ Insert instrument in production of antigens for
Merthiolate Iodine Formalin the anus serodiagnosis
Concentration [MIFC] Test) o Look for typical lesions (or o Trophozoite or cyst from
- Saline-purged specimens samad or lu-as) feces, material aspirated by
o Will provide material for o Scrapings or aspirates from sigmoidoscopy
positive (+) diagnosis when suspected sites of amebic ▪ After conducting
routine fecal examination has ulceration may be obtained or sigmoidoscopy,
been unrewarding/yield no punch biopsies secured conduct culture
result ▪ If previous test fails, immediately
o Sodium sulfate (Na2SO4 or aspiration can be used - Culture medium
Glauber salts) or phosphosoda ▪ Aspirated material o Dibasic medium of Boeck and
is preferred ingredient for this should be examined Drbohlav (egg slant base with
test microscopically for isotonic overlay = LES-Locke
o Following saline purgation, motile trophozoites egg serum)
earlier fecal evacuations are immediately (using o Diamond’s medium or TYI-
discarded. Sedimented Normal Saline S-33
elements of mucus and tissue Solution suspension) ▪ May reveal the
cells from the 2nd or 3rd bowel ▪ Punch biopsy – more presence of E.
movements are pipetted onto satisfactory to fix, histolytica when
the slide, placed in a cover section, and stain microscopic
slip, and examined for specimen before examination has
trophozoites (a complex attempting to examine failed, but they should
procedure, but this test is only it NEVER be used as a
conducted if the fecal exam • Conduct if S/E substitute for
has failed). and/or microscopic
- Sigmoidoscopy material aspiration fails examination

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 ▪ This type of test is
more expensive
compared to S/E and
TAT for this test is
around 3 days for
culture purposes
2. Hepatic amoebiasis and other
extraintestinal lesions
- Results when trophozoites enter the
mesenteric venules and travel to the
liver through the hepato-port system.
This is where the amoeba is spreading
to the different parts of the body. The
center of the abscess is filled with
necrotic fluid, a median zone
consisting of liver stroma and the
outer zone consisting of liver tissue
being attacked by amoebae,
although it is bacterially sterile.
- Determine or establish the presence
of intestinal amoebiasis
o Clinical manifestations
include increased WBC and
liver function test (elevated
BSP and ALP results)
o Aspiration of abscess – punch
or needle biopsy, similar to
sigmoidoscopy
▪ Characteristic
chocolate colored
(anchovy sauce)
content of abscess
▪ Mixture of sloughed
liver tissue and blood
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or degenerated liver Note: Most common serologic tests
cells, RBC, occasional performed for amebiasis
leukocytes are found
▪ Trophozoite recovered
in about 1/3 of cases, Table 1: Amebic vs Bacillary Dysentery
best obtained from the
wall of the abscess
- X-Ray
o Cannot diagnose or identify a
particular amoeba
o Exhibit the extent of the
damage in the colon, or small
intestine, or liver (if the
infection is systemic)
- Seroimmunologic tests
o When direct microscopic
examination fails to reveal the
presence of E. histolytica –
Antibody or Antigen
detection
o Indirect hemagglutination test
(IHAT)
▪ Detect Antibodies of Free-living Amoebae
past infection
▪ Not very useful since Free-living: because they don’t need a host in
it only detects past order for them to survive or live.
infection Taxonomical Classification
o Agar gel Diffusion (AGD)
o Counter-current Order: Schizopyrenida
Immunoelectrophoresis (CIE) Family: Valkamphidae
o Enzyme-linked
Immunosorbent Assay Genus: Naegleria
(ELISA)
o Indirect Fluorescent Antibody
Test (IFAT) Naegleria fowleri = Naegleria aerobia
 - Cause of Primary Amebic - Polymerase Chain Reaction (PCR) differentiated ectoplasm and a granular
Meningoencephalitis (PAM) and Enzyme-linked endoplasm.
- Amoebae proliferate rapidly in water Immunosorbent Assay (ELISA) are
as temperature rises now used as specific N. fowleri tests
Parts of N. fowleri
1. Amoeboid Locally occurring species has been named
- Elongate with a broad anterior and Naegleria philippinensis
tapered posterior end
- Usually has a lobose
monopseudopodium Life Cycle of Naegleria fowleri
2. Flagellate
- 2 long flagella at the tip of a pear-
shaped body
o Amoeba to flagellate
transformation can take place
within a period of 2-3 hours or
up to 3-4 days
3. Nucleus
- Very prominent with a large centrally It starts with the penetration of the holes of
located karyosome the human (nose, ears, mouth, eyes).
o Might be confused with E.
histolytica The uninucleated cyst will pass through any
4. Conspicuous contractile vacuoles of the facial holes and will form several
5. Food vacuoles trophozoites, and these trophozoites are able
- Contain bacteria when free-living to multiply by binary fission, then several
- Host cell debris when parasitic trophozoites are produced and produce
6. Uninucleated cyst flagella to become motile, then excystation
- The free-living amoeba cyst form of occurs.
this parasite is uninucleated The pictures at the left side are the
uninucleated cyst, and the pictures at the right
side are the trophozoite form.
Diagnosis
In the stained picture (top right), the
trophozoite form of N. fowleri has a well-
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