Parasitology 2.

Dr. Llanera
January 20, 2014

AMOEBAE
OUTLINE
I. Subkingdom Protozoa
a. Class Lobosea
b. Structure
c. Life Cycle
d. Outbreaks
II. Entamoeba histolytica
a. Amoebiasis
b. Amoebic Colitis
III. Entamoeba hartmanii
IV. Entamoeba coli
V. Entamoeba polecki
VI. Endolimax nana
VII. Iodomoeba Butschii
VIII. Dientamoeba fragilis
IX. Entamoeba gingivalis
X. Naegleria fowleri
XI. Acanthamoeba sp.

SUBKINGDOM PROTOZOA
Phylum Sarcomastigophora
Subphylum Sarcodina

Class Lobosea
Subphylum Mastigophora

Class Zoomastigophora
Phylum Ciliophora
Class Kinetofragminophorea
Phylum Apicomplexa
Class Sporozoa

Table 1. Entamoeba histolytica VS Entamoeba coli

CLASS LOBOSEA

Intestinal Species
o Entamoeba histolytica
o Entamoeba hartmanni
o Entamoeba coli
o Entamoeba polecki
o Endolimax nana
o Iodamoeba butschlii
o Dientamoeba fragilis (now under Flagellate family)
o Entamoeba dispar
Other Species
o Entamoeba gingivalis
o Acanthamoeba sp.
o Naegleria fowleri

Entamoeba histolytica name comes from Histo tissue, lytic

destroy, meaning tissue destroying. Does not only involve
the invasion of colon but also has extra intestinal
involvement, the only member in its species pathogenic to
man
Entamoeba dispar morphologcally similar to histolytica but
genetically different because it is non-pathogenic
Entamoeba hartmanni, polecki and nana smallest ones in
its species, < 7 micra
E. hartmanni also called small race E. histolytica
Entamoeba coli larger than histolytica
Entamoeba polecki is rarely pathogenic in man, exposure
from pigs and monkeys
Similar to flagellates, it also has developmental stages:
trophozoites and cyst except for Dientamoeba fragilis and
Entamoeba gingivalis, they dont have cystic stage

STRUCTURE
Pseudopodia - with pseudopods or finger like structures which extends
for movement
o Lobose
o Crawling motion (not swimming motion)
o E. histolytica active progressive fast movement
o Entamoeba coli sluggish non progressive movement
Nucleus is compact or vesicular with a dark field structure called a
karyosome (endosome or nucleolus)
o In histolytica, the karyosome is smaller and centrally located with
an even peripheral chromatin
o In coli, the karyosome is larger and peripherally located with an
irregular peripheral chromatin
o Only genus entamoeba has peripheral chromatin
o Granules inside the periphery of karyosome is fine in E. histolytica
and coarse in Entamoeba coli
Nucleoplasm
Nuclear membrane
Endoplasm
o With mitochondria
o Food synthesis
o Food vacuoles stored in chromatoidal bodies;

Chromatoidal bodies in E. histolytica: blunt and round

Chromatoidal bodies in Entamoeba coli: sharp, splinter like

o Has golgi apparatus, endoplasmic reticulum and microsomes
Ectoplasm clear outer area
o Locomotor apparatus for procurement and ingestion of food
o Discharge of metabolic wastes and protection

Entamoeba Sp

Entamoeba
histolytica

Entamoeba coli

Karyosome Size

Smaller

Larger

Karyosome Locations

Centrally located

Peripherally
located

Peripheral Chromatin

Even

Uneven

Granules

Fine

Coarse

Chromatoidal Bodies

Blunt and round

Sharp, splinter like

Movement

Fast, active

Slow, sluggish

Trophozoite Content

RBC

Bacteria, debris

LIFE CYCLE
Person to person transfer (no cystic stage)
Encystation formation of cyst
o Protective - ciliates
o Reproductive flagellates & amoebae
Excystation when cyst becomes trophozites
No intermediate hosts, direct life cycles alternating trophozoites and
cyst
Mature cyst is the infective stage, for E. histolytica mature cyst will
have 4 nuclei and Entamoeba coli would have 8 nuclei

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Parasitology 2.3

Colon would be the initial area of invasion by metacystic trophozoites,

especially the cecum. Because the gastric acidity and chime of intestine
is not conducive for reproductive action so they only become active
once they reach the cecum
They are usually present in the flexures, where the intestines bend like
splenic flexure, hepatic flexure, recto-sigmoid flexure
Trophozoites responsible for extra intestinal manifestations most
common site is the liver, especially the right lobe and then the lungs
Trophozoites (non-infective) and mature cyst (infective) are evacuated
in feces.
Trophozoites found in liquid, watery stools
Mature cyst found in formed stools

Table 3. Different Spectrums of Amoebic Infection

Classification
Characteristics
I. Asymptomatic
Intestinal Colonization
without
tissue
Infection
involvement
II. Symptomatic
Invasive infection
a. Amoebic dysentery
Fulminant
ulcerative
intestinal
disease
b. Nondysentery colitis
Ulcerative intestinal disease
c. Ameboma
Proliferative intestinal granuloma
d. Complicated
Intestinal Perforation, haemorrhage, fistula
Amebiasis
e. Postamebic colitis
Mechanism unknown
III. Extraintestinal Amebiasis
a. Nonspecific hepatomegaly No
demonstrable
invasion
accompanies intestinal infection
b. Acute
Nonspecific Amoeba in liver but without abscess
Infection
c. Amebic Abscess
Focal structural lesion
d. Amebic
Abscess Direct extension to pleura, lung,
Complicated
peritoneum, pericardium if there is
pericardial involvement left lobe of
liver more commonly involved
e. Amebiasis cutis
Direct extension to skin
f. Visceral amebiasis
Metastatic infection of lung, spleen
or brain

Figure 1. Life Cycle Mature cyst are ingested. Trophozoites then emerge
from the cyst and invade the colonic wall and reproduce. Mature cyst are
then released in feces
Table 2. Reasons for Amoebic Encystation and Excystation
Encystation
Excystation

Food supply high / low

Osmotic changes in medium

Excess of catabolic products of

Enzymatic action of the
the organism or associated
enclosed organism on the inner
bacteria
surface of the cyst wall

pH change (marked)

Among the parasitic protozoa,

favorable pH and enzymatic

Dessication of medium
action of the host tissues

O2 supply high / low

Overpopulation

OUTBREAKS
Single or multiple strain
Common in Mental institutions
Polymerase Chain Reaction (PCR) E. histolytica vs. E. dispar
Laredo strain (Laredo, Texas F. H. Connell in 1956)

Group Jobert| Members: Raph, Jobs, Dianelli, Paolo, Kinsley

ENTAMOEBA HISTOLYTICA
AMOEBIASIS: PATHOGENESIS & PATHOLOGY
Sites of colonization
o Intestinal lesion Colon (Most specific is in the cecum followed by
the rectosigmoid)
High requirement for iron
Primary sites of invasion in colon:
o Early flask shaped ulcer
o Late neutrophilic infiltrates
Secondary lesions other levels of the intestine / extraintestinal
VIRULENCE FACTORS
Susceptibility to agglutination by the lectin concanavalin A
Presence of an adhesion lectin that is inhibited by N-acetyl-Dgalactosamine
Ability to adhere to epithelial cells in vitro and to initiate cell-contactdependent cytolysis
Ability to phagocytize cells (E. histolytica demonstrate this thru
ingestion of RBCs erythrophagocytosis).
MOBILITY PATTERN BY STARCH GEL ELECTROPHORESIS
Performed on recent parasite isolates grown in the presence of
bacteria
Virulent strains of E. histolytica grown in axenic culture (w/o bacteria)
retain their zymodeme pattern (isoenzymes). Zymodeme is important
as one of the differentiating factor between E. histolytica and E. dispar.
E. dispar was recognized by Brumpt in 1925 as genetically distinct but
morphologically identical to E. histolytica. With regards to E.
hartmanni, it possesses all the features of E. histolytica but it is smaller
in size.
1913 WALKER AND SELLARDS
Human volunteers ingested cysts.
All became infected but only some developed acute dysentery.
As few as 10 cysts have been shown to produce infection.

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Parasitology 2.3

PATHOGENIC ACTIVITIES OF E. HISTOLYTICA DEPEND ON:

Host innate resistance
Virulence and invasiveness of the strain
Conditions of the GI tract

Amoebic granuloma (ameboma) in colonic wall sequel to an amoebic

ulcer
-Remember the oldest/primary lesion is in the cecum, thats why the pain is
similar to appendicitis.
EXTRAINTESTINAL LESIONS
Liver

Right lobe (Amoebic hepatitis)

o Amoebae caught in the occlusion produce lytic necrosis of the wall
of the vessels enter periportal sinusoids and digest pathways
into the lobules.
o 3 zones (gross / LPO)

Necrotic center filled with thick fluid

Median zone with coarse stroma

Outer zone of nearly normal tissue being invaded by

amoebae

Figure 2. Stages and manifestations of Enatamoeba histolytica infection

Figure 3. Gross image showing erosion of the intestine with minute

hemorrhages caused by E. histolytica

Figure 5. Aspiration of amoebic abscess aspiration of the liver showing

anchovy-like, dark-brown, or sardines-like aspirate.

If FNAB (Fine Needle Aspiration Biopsy) is done, it is usually CT scan or

ultrasound guided.

Lungs

Extension of a hepatic abscess by rupture through the diaphragm

(hepatobronchial fistula) liver-colored sputum

Independent of the liver from the intestine

Pericardial

Left lobe of liver mostly affected

Figure 4. Histology of the intestine showing flask-shaped ulcer. Amoeba is

able to penetrate the tunica mucosa by way of the crypts of Lieberkuhn
towards the muscularis mucosa, and the subserosa will be the one to contain
the infection to stop it causing the appearance of a flask-shape ulcer, a
pathognomonic histopathologic sign
Note: In organs without submucosa like the gallbladder, when they stretch
the epithelium becomes denuded and disappear in some areas. In effect the
muscle layer increases and leads to deepening of the epithelium forming
rokitansky-aschoff sinuses.

SECONDARY LESIONS
Lower colonic segments (rectosigmoid) by regurgitation

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Skin

Amebiasis Cutis
o Perianal extension of acute amoebic colitis
o Abdominal wall through rupture or open drainage of a colonic,
appendiceal, or hepatic lesion
o Penis
o Vulvar amoebiasis is less common.
Brain Hematogenous / Arises from or concomitant with liver or lungs
Spleen
Adrenals
Renal System Kidneys, Ureters, Urinary bladders, Urethras
Clitoris
Nasal polyp
Eye

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Parasitology 2.3

AMOEBIC COLITIS: PATHOLOGIC ANATOMY

The earliest, oldest and most advanced erosive ulceration is seen in the
cecal region (cecum, ileocecal valve, appendix, ascending colon)
nd
2 in frequency and intensity: sigmoid, rectum
rd
3 : splenic and hepatic flexures
SYMPTOMATOLOGY
Incubation period
o Biological incubation: 2 5 days or more
o Clinical incubation: 4 days 1 year
o Expected incubation: 1 4 months
Onset
o Gradual development of symptoms
o Diarrhea, abdominal cramps, or may be asymptomatic
Amoebic Colitis is acute if <1 month
Bacterial complications
Extraintestinal manifestations

DYSENTERIC STAGE OF AMOEBIASIS AND SHIGELLOSIS

Table 4. Amoebiasis VS Shigellosis
Amoebiasis
Shigellosis
Epidemiology
Typically endemic; long Typically
epidemic;
incubation period
short incubation period
Pathology
Stool w/ blood, mucus, Numerous pus cells, no
necrotic tissue cells; Charcot
Leyden
few WBC; usually with crystals; leukocytosis
Charcot Leyden crystals
Symptomatology
No fever, moderate Fever usually present,
tenesmus;
localized severe
tenesmus,
abdominal discomfort
generalized abdominal
tenderness
Complications
Severe hemorrhage
Polyarthritis
Diagnostic Tests
Stool examination
Stool culture

Buffered methylene blue view within 30 minutes (for fresh,

unpreserved; if with amoebic trophozoites on saline) - not for
amoebic cysts, flagellates

DETECTION
Specimen: feces, saline-purged or enema specimen, aspirate, surgical
biopsy, necropsy material
Artifacts and confusers WBCs or other parasites
Seroimmunologic diagnosis
o Craig 1928-1931 Complement fixing abilities
o Goldman 1962 1964 Fluorescent antibody
o Indirect hemagglutination Hepatic abscess
o Latex agglutination
o ELISA
o Agar or cellulose acetate diffusion
o Moan hemagglutination test

Figure 6. Entamoeba histolytica trophozoite. Nucleus with centrally located

small karyosome and peripheral chromatin.

Laboratory Diagnosis: Detection and Cultivation

Stool examination with Charcot Leyden

Trophozoites seen in unstained (but black and white); more seen with
Buffered Methylene blue

Cysts more stained by Iodine (Lugols)/ Iron Hematoxylin

Figure 7. A) Precystic stage; B) Cyst (has 4 nuclei and a well-defined wall)
Table 5. Detection & Cultivation of Amoeba
Consistency
Protozoan
Saline
stage most
likely to be
found

Iodine

Buffered
methylene
blue
(if
trophozoites
are seen)

Cysts

Cysts
(occasionally
trophozoites)
Trophozoites

Trophozoites

MICROSCOPIC EXAMINATION OF WET MOUNTS

Directly from fecal smear (DFS) or from concentrated specimens
Types / Procedures:
o Saline

Ova, larvae, trophozoites and cysts; RBC, pus

o Iodine (Lugols)

Glycogen, nuclei of cysts

Group Jobert| Members: Raph, Jobs, Dianelli, Paolo, Kinsley

Figure 8. Entamoeba histolytica Life Cycle

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Parasitology 2.3

ANTIAMOEBICS
Metronidazole kills trophozoites; for E. histolytica, Giardia,
Trichomonas; has metallic taste; do not use with alcohol (disulfiram-like
reactions)
Iodoquinol luminal amoebicide
Diloxanide Furoate luminal, if asymptomatic flatulence, nausea,
rash
Paromomycin Sulfate luminal
Emetine & Dehydroemetine toxic
TREATMENT
Metronidazole 750 mg tid x 5-10 days only used for confirmed cases
of E. histolytica and prophylaxis prior to abdominal surgery
Iodoquinol
Emetine HCl (6% soln) SQ/IM 1 mg/kg BW daily x 5 days (max daily
dose of 60 mg) relieves symptoms > eradicate infection
Dehydroemetine & Emetine with toxic effects on myocardium &
peripheral nerves
DO NOT give the following: Loperamide HCl, Diphenoxylate HCl,
Thephenamil HCl (may produce toxic megacolon in acute ulcerative
colitis).
ENTAMOEBA HARTMANII
small race of E. histolytica

Figure 9. Entamoeba coli trophozoite

TROPHOZOITES STAGE
Size: 5-12
Motility: Usually nonprogressive
Nucleus
o Number: Not visible in unstained preparations
o Peripheral Chromatin: Fine granules; Evenly distributed;
Uniform in size
o Karyosomal Chromatin: Small; Discrete; Eccentrically located
Cytoplasm
o Appearance: Finely granular
o Inclusions: Bacteria
CYSTIC STAGE
Size: 5-10
Shape: Spherical
Nucleus
o Number: 4 in mature cyst; 1-2 in immature cyst
o Peripheral Chromatin: Fine uniform granules; Evenly
distributed
o Karyosomal Chromatin: Small; Discrete; Centrally located
Cytoplasm
o Chromatid Bodies: Elongated bars with bluntly rounded ends
o Glycogen: Diffuse; Stains reddish-brown with iodine

Figure 10. Entamoeba coli cyst

ENTAMOEBA COLI
TROPHOZOITES STAGE

Size: 15-50
Motility: Sluggish; Non-progressive with blunt pseudopods
Nucleus
o Number: Often visible in unstained preparations
o Peripheral Chromatin: Coarse granules; Irregular in size and
distribution
o Karyosomal Chromatin: Large; Discrete; Eccentrically located
Cytoplasm
o Appearance: Coarse often vacuolated
o Inclusions: Bacteria, Yeasts, etc

ENTAMOEBA POLECKI
Usually seen in hogs and monkeys; rarely diagnosed in man

Figure 11. Entamoeba polecki

Group Jobert| Members: Raph, Jobs, Dianelli, Paolo, Kinsley

CYSTIC STAGE
Size: 10-35
Shape: Spherical; Occasionally oval, triangular or another shape
Nucleus
o Number: 8 in mature cyst but there are supernucleate cysts
with 16; 2 in immature cysts
o Peripheral Chromatin: Coarse; Irregularly shaped granules;
Irregularly distributed
o Karyosomal Chromatin: Large; Discrete; Usually eccentrically
located; Occasionally centrally located
Cytoplasm
o Chromatid Bodies: Less in number than E. histolytica;
Splinter-like with pointed ends
o Glycogen: Diffuse; May be a well-defined mass in immature
cysts; Stains reddish-brown with iodine

TROPHOZOITES STAGE
Size: 10-25
Motility: Sluggish; May be progressive in diarrheic stool
Nucleus
o Number: Slightly visible in unstained preparations; May be
distorted by pressure from vacuoles in cytoplasm
o Peripheral Chromatin: Fine granules; Evenly distributed;
Occasionally irregularly arranged; in plaques or crescents
o Karyosomal Chromatin: Small; Discrete; Eccentrically located;
Occasionally large, diffuse or irregular
Cytoplasm

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Parasitology 2.3
o
o

Appearance: Coarse; Granular; Contains numerous vacuoles

Inclusions: Bacteria; Yeast

CYSTIC STAGE
Size: 9-18
Shape: Spherical or Oval
Nucleus
o Number: 1 to 2
o Peripheral Chromatin: Fine granules evenly distributed
o Karyosomal Chromatin: Small; Eccentrically located
Cytoplasm
o Chromatid Bodies: Many small bodies with angular or pointed
ends; May be oval or rodlike
o Glycogen: Small diffuse masses; Stains reddish-brown with
iodine; A dark area called Incusion Mass is often present;
Inclusion Mass doesnt stain with iodine
ENDOLIMAX NANA

Figure 13. Endolimax nana cyst

IODAMOEBA BUTSCHLII
TROPHOZOITES STAGE

Dwarf Internal Slug

TROPHOZOITES STAGE
Size: 6-12
Motility: Sluggish; Usually nonprogressive with blunt pseudopods
Nucleus
o Number: Occasionally visible in unstained preparations
o Peripheral Chromatin: None
o Karyosomal Chromatin: Large; Irregularly shaped (blot-like)
Cytoplasm
o Appearance: Granular, vacuolated
o Inclusions: Bacteria

Size: 8-20
Motility: Sluggish; Nonprogressive
Nucleus
o Number: Not usually visible in unstained preparations
o Peripheral Chromatin: None
o Karyosomal Chromatin: Large; Centrally located; Surrounded
by refractive achromatic granules
Cytoplasm
o Appearance: Coarse, granular, Vacuolated
o Inclusions: Bacteria; Yeasts; Etc.

Figure 14. Iodamoeba butschlii trophozoite

Figure 12. Endolimax nana trophozoite

CYSTIC STAGE
Size: 5-10
Shape: Spherical, Ovoid or Ellipsoidal
Nucleus
o Number: 4 in mature cysts; Less than 4 in immature cysts
(rarely seen)
o Peripheral Chromatin: None
o Karyosomal Chromatin: Large; Blot-like; Centrally located
Cytoplasm
o Chromatid Bodies: Granules or small oval masses
o Glycogen: Diffuse; Concentrated mass may be seen in young
cysts; Stains reddish-brown with iodine

Group Jobert| Members: Raph, Jobs, Dianelli, Paolo, Kinsley

CYSTIC STAGE
Size: 5-20
Shape: Ovoid, Ellipsoidal, Triangular or of another shape
Nucleus
o Number: 1 in mature cyst
o Peripheral Chromatin: None
o Karyosomal Chromatin: Large; Eccentrically located; Refractile
achromatic granules on one side; Indistinct in iodine
preparations
Cytoplasm
o Chromatid Bodies: Granular
o Glycogen: Compact well-defined mass; Stains dark brown
with iodine

Figure 15. Iodamoeba butschlii cyst

DIENTAMOEBA FRAGILIS

Trophozoite only
Non-invasive

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Parasitology 2.3

Size: 6-12
Motility: Single pseudopodia are multiple leaflike hyaline structures;
motion is active and progressive
Nucleus
o Number: Usually 1 but may be 2
o Rosette-shaped nuclei (Belizario)
o Peripheral Chromatin: None
o Karyosomal Chromatin: Fragmented into 4-8 segments
Cytoplasm
o Appearance: Vacuolated
o Inclusions: Bacteria; Yeast; Starch granules

PATHOGENESIS, PATHOLOGY & SYMPTOMATOLOGY

Findings:
o Like fulminant bacterial meningitis
o Amoebae in exudates
Primary Amebic Meningoencephalitis
Diagnosis: swimming in thermal/stagnant water 3 to 6 days prior; CSF;
histopath
Prognosis: fatal within a week
Treatment: none; Amphotericin B and Sulfadiazine

A. culbertsoni
A. polyphaga
A. castellanii
A. stronyxis

ACANTHAMOEBA

Figure 16. Dientamoeba fragilis

ENTAMOEBA GINGIVALIS

Trophozoite only
First amoeba to be described
Present only in the mouth
Size: 10-20
Motility: Pseudopodia are usually blunt; moderately active and
progressive motility
Nucleus
o Number: One spheroid nucleus
o Peripheral Chromatin: Fine; Evenly distributed
o Karyosomal Chromatin: Coarse
Cytoplasm
o Appearance: Vacuolated
o Inclusions: Food, debris, bacteria

ACANTHAMOEBA CULBERTSONI
Amoebic meningoencephalitis, uveitis and ulceration of cornea
Active trophic forms
o No flagellate form
Resistant cysts resistant to chlorine and can withstand drying
Slow movement of acanthopodia
PATHOGENESIS, PATHOLOGY & SYMPTOMATOLOGY
Purulent leptomeningitis, brain edema, foci of necrosis
Olfactory nerves and lobes not affected
Cerebral hemispheres may be edematous and soft with hemorrhages &
abscesses. (Belizario)
Most affected areas of the brain: posterior fossa, diencephalon,
thalamus, brainstem
On the affected areas, the leptomeninges are opaque with purulent
exudates & vascular congestion. (Belizario)

Figure 17. E. gingivalis

NAEGLERIA FOWLERI
Free-living amoebo-flagellate
Motile trophozoites
o Amoeboid
o Flagellate (w/ 2 flagella) shed flagella then resume amoeboid
motility and reproduction
Non motile resistant cysts
Flagellate stage enters nasal cavity; where it reverts to amoeboid form
before invading olfactory tissues and the brain
Cysts instilled intranasally are not infective in experimental animals

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Figure 18. Pathogenesis of Acanthamoeba. The route of invasion &

penetration into the CNS is via the circulatory system, while the primary
sites of infection are either the skin or lungs.

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Parasitology 2.3

MORPHOLOGY

Naegleria

Acanthamoeba

Broad pseudopods

Filamentous pseudopods
(acanthopodia)

Active

Sluggish

Trophozoites

Motility

Flagellate stage

Does not form this stage

+
Thin
walled

Double walled

None

May have
osteioles

No

May encyst in tissue

Cysts

Pores in cyst wall

Encystment
tissue

in

pores

or

Figure 19. Life Cycle of Naegleria fowleri & Acanthamoeba

APPENDIX

DIAGNOSIS
Amoebae in CSF, scrapings from lesions in cases of corneal or
cutaneous infections; cultures of material from those sources; stained
vaginal smears; purulent discharge from infected ear
TREATMENT
Amphotericin B and sulfadiazine
NAEGLERIA VS ACANTHAMOEBA

Naegleria

Acanthamoeba

Pathogenic : 1 species
Olfactory neuroepithelium
Faster course

Pathogenic: 4 species
Broken or ulcerated skin or eye;
lungs or genitourinary tract
Slow tissue invasion
Granuloma formation
Gradual onset; prolonged chronic
course
Chronically
ill
/
immunosuppressed

Throphozoites (top) and Cysts (bottom): From left to right: (A) E. histolytica,
(B) E. hartmanii, (C) E. coli, (D) E. polecki, (E) Endolimax nana, (F) Iodamoeba
butschii, (G) Dientamoeba fragilis

REFERENCES
Dr. Llaneras lecture & ppt
Philippine Textbook of Medical Parasitology (Belizario)

Edited by: Gab Tan

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