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Parasitology 2.3

AMOEBAEDr. Llanera

January 20, 2014

UTLINE

I. Subkingdom Protozoa

a. Class Lobosea

b. Structure

c. Life Cycle

d. Outbreaks

II. Entamoeba histolytica

a. Amoebiasis

b. Amoebic ColitisIII. 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

CLASS LOBOSEA

Intestinal Species

o Entamoeba histolytica

o Entamoeba hartmanni

o Entamoeba coli

o

Entamoeba poleckio 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 don’t have cystic stage

STRUCTURE

Pseudopodia - with pseudopods or finger like structures which e

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 ca

karyosome (endosome or nucleolus)o In histolytica, the karyosome is smaller and centrally locate

an even peripheral chromatin

o In coli, the karyosome is larger and peripherally located w

irregular peripheral chromatin

o Only genus entamoeba has peripheral chromatin

o Granules inside the periphery of karyosome is fine in E. hist

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

o Has golgi apparatus, endoplasmic reticulum and microsome

Ectoplasm – clear outer area

o Locomotor apparatus for procurement and ingestion of food

o Discharge of metabolic wastes and protection

Table 1. Entamoeba histolytica VS Entamoeba coli

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 trophozoite

cyst

Mature cyst is the infective stage, for E. histolytica mature cy

have 4 nuclei and Entamoeba coli would have 8 nuclei

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 li

Movement Fast, active Slow, sluggish

Trophozoite Content RBC Bacteria, debris

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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

gure 1. Life Cycle – Mature cyst are ingested. Trophozoites then emerge

om the cyst and invade the colonic wall and reproduce. Mature cyst are

en released in feces

ble 2. Reasons for Amoebic Encystation and Excystation

cystation Excystation

Food supply – high / low

Excess of catabolic products of

the organism or associated

bacteria

pH change (marked)Dessication of medium

O2 supply – high / low

Overpopulation

Osmotic changes in medium

Enzymatic action of the

enclosed organism on the inner

surface of the cyst wall

Among the parasitic protozoa,favorable pH and enzymatic

action of the host tissues

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)

Table 3. Different Spectrums of Amoebic Infection

Classification Characteristics

I.

Asymptomatic Intestinal

Infection

Colonization without

involvement

II. Symptomatic Invasive infection

a. Amoebic dysentery Fulminant ulcerative int

disease

b. Nondysentery colitis Ulcerative intestinal disease

c.

Ameboma Proliferative intestinal granulo

d. Complicated Intestinal

Amebiasis

Perforation, haemorrhage, fist

e. Postamebic colitis Mechanism unknown

III. Extraintestinal Amebiasis

a. Nonspecific hepatomegaly No demonstrable in

accompanies intestinal infectio

b.

Acute Nonspecific

Infection

Amoeba in liver but without ab

c. Amebic Abscess Focal structural lesion

d. Amebic Abscess

Complicated

Direct extension to pleura,

peritoneum, pericardium – if t

pericardial involvement left lo

liver more commonly involvede. Amebiasis cutis Direct extension to skin

f. Visceral amebiasis Metastatic infection of lung,

or brain

ENTAMOEBA HISTOLYTICA

AMOEBIASIS: PATHOGENESIS & PATHOLOGY

Sites of colonization

o Intestinal lesion – Colon (Most specific is in the cecum follow

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-ac

galactosamine

Ability to adhere to epithelial cells in vitro and to initiate cell-co

dependent cytolysis

Ability to phagocytize cells (E. histolytica demonstrate this

ingestion of RBCs – “ erythrophagocytosis” ).

MOBILITY PATTERN BY STARCH GEL ELECTROPHORESIS

Performed on recent parasite isolates grown in the presen

bacteria

Virulent strains of E. histolytica grown in axenic culture (w/o ba

retain their zymodeme pattern (isoenzymes). Zymodeme is imp

as one of the differentiating factor between E. histolytica and E. di

E. dispar was recognized by Brumpt in 1925 as genetically distin

morphologically identical to E. histolytica. With regards

hartmanni, it possesses all the features of E. histolytica but it is s

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

gure 2. Stages and manifestations of Enatamoeba histolytica infection

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

morrhages caused by E. histolytica

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

ble to penetrate the tunica mucosa by way of the crypts of Lieberkuhn

wards the muscularis mucosa, and the subserosa will be the one to contain

e infection to stop it causing the appearance of a flask-shape ulcer, a

athognomonic histopathologic sign

ote: In organs without submucosa like the gallbladder, when they stretch

e epithelium becomes denuded and disappear in some areas. In effect the

uscle layer increases and leads to deepening of the epithelium forming

kitansky-aschoff sinuses.

SECONDARY LESIONS

Lower colonic segments (rectosigmoid) by regurgitation

Amoebic granuloma (ameboma) in colonic wall – sequel to an am

ulcer

-Remember the oldest/primary lesion is in the cecum, that’s why the

similar to appendicitis.

EXTRAINTESTINAL LESIONS

Liver

Right lobe (Amoebic hepatitis)

o Amoebae caught in the occlusion produce lytic necrosis of th

of the vessels → enter periportal sinusoids and digest patinto 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 invad

amoebae

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

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

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

ultrasound guided.

Lungs

Extension of a hepatic abscess by rupture through the diap

(hepatobronchial fistula) – liver-colored sputum

Independent of the liver from the intestine

Pericardial

Left lobe of liver mostly affected

Skin

Amebiasis Cutis

o

Perianal extension of acute amoebic colitiso Abdominal wall through rupture or open drainage of a c

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)

2nd

in frequency and intensity: sigmoid, rectum

3rd

: splenic and hepatic flexures

SYMPTOMATOLOGY

Incubation period

o Biological incubation: 2 – 5 days or more

o

Clinical incubation: 4 days – 1 yearo Expected incubation: 1 – 4 months

Onset

o Gradual development of symptoms

o Diarrhea, abdominal cramps, or may be asymptomatic

Amoebic Colitis is acute if

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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

TROPHOZOITES STAGE

Size: 5-12μ

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 μ

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

ENTAMOEBA COLI

TROPHOZOITES STAGESize: 15-50 μ

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

Figure 9. Entamoeba coli trophozoite

CYSTIC STAGE

Size: 10-35 μ

Shape: Spherical; Occasionally oval, triangular or another shape

Nucleus

o Number: 8 in mature cyst but there are supernucleate

with 16; 2 in immature cysts

o Peripheral Chromatin: Coarse; Irregularly shaped gra

Irregularly distributed

o Karyosomal Chromatin: Large; Discrete; Usually eccent

located; Occasionally centrally located

Cytoplasm

o

Chromatid Bodies: Less in number than E. histoSplinter-like with pointed ends

o Glycogen: Diffuse; May be a well-defined mass in imm

cysts; Stains reddish-brown with iodine

Figure 10. Entamoeba coli cyst

ENTAMOEBA POLECKI

Usually seen in hogs and monkeys; rarely diagnosed in man

Figure 11. Entamoeba polecki

TROPHOZOITES STAGE

Size: 10-25 μ

Motility: Sluggish; May be progressive in diarrheic stool

Nucleus

o Number: Slightly visible in unstained preparations; M

distorted by pressure from vacuoles in cytoplasm

o Peripheral Chromatin: Fine granules; Evenly distri

Occasionally irregularly arranged; in plaques or crescent

o Karyosomal Chromatin: Small; Discrete; Eccentrically lo

Occasionally large, diffuse or irregular

Cytoplasm

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Parasitology 2.3

o Appearance: Coarse; Granular; Contains numerous vacuoles

o Inclusions: Bacteria; Yeast

CYSTIC STAGE

Size: 9-18 μ

Shape: Spherical or Oval

Nucleus

o Number: 1 to 2

o Peripheral Chromatin: Fine granules evenly distributed

o

Karyosomal Chromatin: Small; Eccentrically locatedCytoplasm

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 doesn’t stain with iodine

ENDOLIMAX NANA

“Dwarf Internal Slug”

TROPHOZOITES STAGE

Size: 6-12 μ

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

Figure 12. Endolimax nana trophozoite

CYSTIC STAGE

Size: 5-10 μ Shape: Spherical, Ovoid or Ellipsoidal

Nucleus

o Number: 4 in mature cysts; Less than 4 in immature cysts

(rarely seen)


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

Figure 13. Endolimax nana cyst

IODAMOEBA BUTSCHLII

TROPHOZOITES STAGE

Size: 8-20 μ

Motility: Sluggish; Nonprogressive

Nucleus

o Number: Not usually visible in unstained preparations


o Karyosomal Chromatin: Large; Centrally located; Surro

by refractive achromatic granules

Cytoplasm

o

Appearance: Coarse, granular, Vacuolatedo Inclusions: Bacteria; Yeasts; Etc.

Figure 14. Iodamoeba butschlii trophozoite

CYSTIC STAGE

Size: 5-20 μ

Shape: Ovoid, Ellipsoidal, Triangular or of another shape

Nucleus

o Number: 1 in mature cyst


o Karyosomal Chromatin: Large; Eccentrically located; Ref

achromatic granules on one side; Indistinct in

preparations

Cytoplasm

o Chromatid Bodies: Granular

o Glycogen: Compact well-defined mass; Stains dark

with iodine

Figure 15. Iodamoeba butschlii cyst

DIENTAMOEBA FRAGILIS

Trophozoite only

Non-invasive

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Parasitology 2.3

Size: 6-12 μ

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 Karyosomal Chromatin: Fragmented into 4-8 segments

Cytoplasm

o

Appearance: Vacuolatedo Inclusions: Bacteria; Yeast; Starch granules

Figure 16. Dientamoeba fragilis

ENTAMOEBA GINGIVALIS

Trophozoite only

First amoeba to be described

Present only in the mouth

Size: 10-20 μ

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

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

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 prio

histopath

Prognosis: fatal within a week

Treatment: none; Amphotericin B and Sulfadiazine

ACANTHAMOEBA

A. culbertsoni

A. polyphaga

A. castellanii

A. stronyxis

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 hemorrh

abscesses. (Belizario)

Most affected areas of the brain: posterior fossa, diencep

thalamus, brainstem

On the affected areas, the leptomeninges are opaque with pu

exudates & vascular congestion. (Belizario)

Figure 18. Pathogenesis of Acanthamoeba. The route of invasion

penetration into the CNS is via the circulatory system, while the prim

sites of infection are either the skin or lungs.

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Parasitology 2.3

Figure 19. Life Cycle of Naegleria fowleri & Acanthamoeba

DIAGNOSIS

Amoebae in CSF, scrapings from lesions in cases of corneal orcutaneous infections; cultures of material from those sources; stained

vaginal smears; purulent discharge from infected ear

TREATMENT

Amphotericin B and sulfadiazine

NAEGLERIA VS ACANTHAMOEBA

APPENDIX

Throphozoites (top) and Cysts (bottom): From left to right: (A) E. histo

(B) E. hartmanii, (C) E. coli, (D) E. polecki, (E) Endolimax nana, (F) Iodam

butschii, (G) Dientamoeba fragilis

REFERENCES

Dr. Llanera’s lecture & ppt

Philippine Textbook of Medical Parasitology (Belizario)

Edited by: Gab Tan

aegleria 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

MORPHOLOGY Naegleria Acanthamoeba

Trophozoites

Broad pseudopods Filamentous pseudopo

(acanthopodia)

Motility Active Sluggish

Flagellate stage

+

Does not form this stage

Cysts

Thin

walled

Double walled

Pores in cyst wall NoneMay have pores

osteioles

Encystment in

tissue No May encyst in tissue

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