2 PARA 3 - Amoebas

Group Jobert | Members: Sayoc, Sebastian, Segovia, Segovia, Serencio

Page 1 of 8

AMOEBAE

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 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 don’t 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

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 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

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

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

Page 2 of 8

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

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  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 tissue 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

Amebiasis

Perforation, haemorrhage, fistula e. Postamebic colitis Mechanism unknown

III. Extraintestinal Amebiasis

a. Nonspecific hepatomegaly No demonstrable invasion accompanies intestinal infection b. Acute Nonspecific

Infection

Amoeba in liver but without abscess c. Amebic Abscess Focal structural lesion

d. Amebic Abscess Complicated

Direct extension to pleura, lung, 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

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-D-galactosamine

 Ability to adhere to epithelial cells in vitro and to initiate cell-contact-dependent 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.

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

Page 3 of 8

PATHOGENIC ACTIVITIES OF E. HISTOLYTICA DEPEND ON:

 Host innate resistance

 Virulence and invasiveness of the strain  Conditions of the GI tract

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 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

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

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

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

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

Page 4 of 8

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 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

incubation period

Typically epidemic; short incubation period

Pathology Stool w/ blood, mucus, necrotic tissue cells; few WBC; usually with Charcot Leyden crystals

Numerous pus cells, no Charcot Leyden crystals; leukocytosis

Symptomatology No fever, moderate tenesmus; localized abdominal discomfort

Fever usually present, severe tenesmus, generalized abdominal tenderness

Complications Severe hemorrhage Polyarthritis

Diagnostic Tests Stool examination Stool culture

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 (Lugol’s)/ Iron Hematoxylin

Table 5. Detection & Cultivation of Amoeba Consistency Protozoan

stage most likely to be found

Saline 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 (Lugol’s)

 Glycogen, nuclei of cysts

o 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.

Figure 7. A) Precystic stage; B) Cyst (has 4 nuclei and a well-defined wall)

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

Page 5 of 8

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 STAGE

 Size: 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 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

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; 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

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

Page 6 of 8

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 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 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 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

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 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 CYSTIC STAGE

 Size: 5-20 μ

 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

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

Page 7 of 8

 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

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 prior; CSF; 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 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 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.

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

Page 8 of 8

Figure 19. Life Cycle of Naegleria fowleri & Acanthamoeba 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

APPENDIX

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. Llanera’s lecture & ppt

 Philippine Textbook of Medical Parasitology (Belizario)

Edited by: Gab Tan

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

MORPHOLOGY Naegleria Acanthamoeba

Trophozoites

Broad pseudopods Filamentous pseudopods (acanthopodia)

Motility Active Sluggish

Flagellate stage +

Does not form this stage

Cysts

Thin

walled Double walled

Pores in cyst wall None May have pores or osteioles

Encystment in