2 PARA 3 - Amoebas

Parasitology 2.3

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.

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SUBKINGDOM PROTOZOA Phylum Sarcomastigophora Subphylum Sarcodina  Class Lobosea Subphylum Mastigophora  Class Zoomastigophora Phylum Ciliophora Class Kinetofragminophorea Phylum Apicomplexa Class Sporozoa

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Table 1. Entamoeba histolytica VS Entamoeba coli

CLASS LOBOSEA 

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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 

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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

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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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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

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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

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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

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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

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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 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

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

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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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 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   

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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

Figure 10. Entamoeba coli cyst 

ENTAMOEBA COLI TROPHOZOITES STAGE   

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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

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

Figure 11. Entamoeba polecki   

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

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

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

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Parasitology 2.3 o o

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Appearance: Coarse; Granular; Contains numerous vacuoles 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

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Figure 13. Endolimax nana cyst IODAMOEBA BUTSCHLII TROPHOZOITES STAGE   

“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

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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   

 Figure 12. Endolimax nana trophozoite   

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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

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

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

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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 Peripheral Chromatin: None o Karyosomal Chromatin: Fragmented into 4-8 segments Cytoplasm o Appearance: Vacuolated o Inclusions: Bacteria; Yeast; Starch granules

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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

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

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ACANTHAMOEBA

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Figure 16. Dientamoeba fragilis ENTAMOEBA GINGIVALIS      

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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

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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  

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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

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

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 

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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

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Pathogenic : 1 species Olfactory neuroepithelium Faster course

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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

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REFERENCES Dr. Llanera’s lecture & ppt Philippine Textbook of Medical Parasitology (Belizario)

Edited by: Gab Tan

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

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