Prof. Dr. Md. Akram Hossain March 09 - mmc.gov.bdmmc.gov.bd/downloadable file/Entamoeba akram.pdf · – E. coli, – E.gingivalis. 1/7/2014 4 Prof. Muahammad Akram, Entamoeba. Morphology

Entamoebahistolytica

Prof. Dr. Md. Akram Hossain

March 09

Introduction

Amoebiasis, the third most common cause ofparasitic death in the world is causedbyparasitic death in the world is causedbyEntamoeba histolytica protozoa withpseudopodia.

1/7/2014 2Prof. Muahammad Akram,

Entamoeba

Epidemiology

� Occurs worldwide. more common in tropics and subtropicsthan temperate zones, most in developing countries likeBangladesh, India, Pakistan and Nepal.

� About 500 million people are infected every year of which50 million (10%) suffers fromdisease and 100,000 dies.

� Common in adults, rare in children, and Amebic liverabscess is more common in male


Entamoeba

Taxonomy

�Phylum -Amoebozoa,

�Class –Entamoebea,– Order-Amoebida,

– Family - Entamoebidae– Family - Entamoebidae• Genus –Entamoeba,

• Species– E. histolytica,– E.dispar,

– E.hartmani,

– E. coli,

– E.gingivalis.


Entamoeba

Morphology

E. histolyticahas two stages:– trophozoite and– cyst with an intermediate– precyst.

The trophozoite is the activelyThe trophozoite is the activelymetabolizing, motile stage, and thecyst is dormant and environmentallyresistant. .


Entamoeba

Morphology

� Trophozoites vary remarkably in size-from 10 to 60µm or more in diameter, actively motile by pseudopodia.

� Amebas are anaerobic organisms and do not havemitochondria.

� Shape changes constantly due to pseudopodialmovement.

� Cytoplasm is divided into clear outerectoplasm andinner granularendoplasm.

� Endoplasm contains the nucleus andfoodvacuoles.

� The nucleus has a distinctive central karyosome and arim of finely beaded chromatin lining the nuclearmembrane.

� The food vacuoles contain may contain bacteria or redblood cells.


Entamoeba

Morphology of cyst

� The cyst is spherical,10-20 µm in diameter, with athin transparent wall.

� Fully mature cysts contain four nuclei. The nucleihave fine evenly distributed uniform granularperipheral chromatin, with small discrete centralkaryosome.karyosome.

� Chromatoidal bars, crystallizedribonucleoproteins, are present variably, and aremore common in immature cysts. They areelongated bars with bluntly rounded ends.

� Inclusions in the form of glycogen masses alsomay be present. Usually diffuse, concentratedmasses often present young cysts. They stainreddish brown with iodine

E.histolytica cyst : cystsmeasure 10-15 µm indiameter and are spherical.(Iodine stain)


Entamoeba

Morphology of E. histolytica at a glance

1. Has two stages - Trophozoite and Cyst2. Trphozoites are 20-60µm, motile with pseudopodia.3. Cytoplasmof trophozoite is divided into clear ectoplasm

and granular endoplasmoften contains food granules andRBC.RBC.

4. Cysts are non-motile, round, 10-20µm with thintransparent cyst wall.

5. 4 nuclei in mature cyst (immature cyst contains variablenumbers but less than 4)

6. Chromatoidal bars are characteristic but presence isvariable.


Entamoeba

Variation of E. histolytica

�Entamoeba Dispar– It is now established thatE. dispar (dispar =

different) (the name proposed by Brumpt in1925but recognizedonly in 1997by WHO) is1925but recognizedonly in 1997by WHO) isthe nonpathogenic species ofEntamoebawhichis morphologically identical withE. histolytica.

– E. dispar, is generally agreed to be about tentimes as common worldwide asE. histolytica.


Entamoeba

Comparison of E. histolytica& E. dispar

Criteria E. dispar E. histolytica

Microscopy No RBC seen within cytoplasm of trophozites

Ingested RBC may be seen & is diagnostic

In vitro culture Xenic (requires bacteria for growth Axenic (grows without In vitro culture Xenic (requires bacteria for growth in culture)

Axenic (grows without bacteria)

Cona agglutination Negative Positive

Complement Resistance

Negative Positive

Zymodemes (isoenzymes)

I & III II


Entamoeba

Life cycle


Entamoeba

Life cycle at a glance

Life cycle at a glance• Host :Man is the only host (Definitive)• Infective stage : Mature quadrinucleate cyst (Metacyst)• Route of infection : Fecal – oral • Route of infection : Fecal – oral • Site of lesion : Large intestine • Diagnostic stage : Cyst & trophozoite• Pathogenic stage : Trophozoite


Entamoeba

life cycle

� E. histolyticapasses its life cycle in single host, the man and� it has 2 stages, 1. trophozoites 2. cysts.� Mature Cysts containing four nuclei (metacyst) is the infective form, which

spread via the ingestion of faecally contaminated food or water.� When mature cysts are ingested via food and drinks their excystation occurs

within the lumen of the small intestine. During excystation, nuclear division isfollowed by cytoplasmic division, giving rise to eight young trophozoitesknown as amebula.

� Theseyoungtrophozoites,residein the lumenof thececumandlargeintestine,� Theseyoungtrophozoites,residein the lumenof thececumandlargeintestine,where they adhere to the colonic mucus and epithelial layers.

� Approximately 90% of individuals infected withE. histolytica areasymptomatically colonized. Re-encystation of the trophozoites occurs undercertain unfavorable conditions like dehydration within the lumen of the colon,resulting in the excretion of cysts in the faeces and continuation of the life cycle.

� Alternatively, the trophozoites can invade the colonic epithelium, causingamebic colitis (in ~10% of infected people).

� E. histolytica can spread in the bloodstream (hematogenously) after it haspenetrated the colonic epithelium and can establish persistent extraintestinalinfections, most commonly amebic liver abscess.


Entamoeba

Virulence factors1. lectin :Galactose andN-acetyl-D-galactosamine (Gal/GalNAc)

specific lectin by which the young trophozoites binds withcolonic mucin glycoproteins.

2. Apoptotic killing of host cells - Occurs by a novel pathway3. Pore forming protein(Amoebopore) - Kills endocytosed

bacteria and purified protein also capable of causing necroticdeathof hostcells.deathof hostcells.

4. Anti complement activity - Invasive E. histolytica resistdestruction by lectin mediated complement activation.

5. Motility - Invasion is probably also promoted by thecytoskeleton-induced motility

6. Enzymes- Invasive parasite secrete enzymes like proteases,Collagenases, elastase, hyaluronidase that degrade theextracellular matrix and antibody.


Entamoeba

Pathogenesis� Intestinal amoebiasis :

– The trophozoites are pathogenic form which colonize the intestine byadhering to colonic mucin glycoproteins, via a galactose and N-acetyl-D-galactosamine (Gal/GalNAc)–specificlectin.

– Host cells are killed via the induction of an apoptotic cascade.Apoptotic killing occurs by a novel pathway that is not blocked bybcl-2 and does not require fas, or the TNF-α receptor.

– Recentlythe amebashave beendemonstratedto activate“effector”– Recentlythe amebashave beendemonstratedto activate“effector”caspases immediately before destruction of the host cell. Inhibition ofthese human caspases blocks killing by the amebas.

– Trophozoites also contain apore-forming protein that is probablyinvolved in the destruction of endocytosed bacteria.

– The purified protein is also capable of inducing necrotic death ofeukaryotic cells.

– Motility and resistant to destruction by lectin mediated complmentactivation also play a role.

– Enzymes likeproteases destroy extracellular matrix.


Entamoeba

� Features of amoebic ulcer :– Trophozoites establish themselves in cecum and proximal colon, and

then invade tissues. This is frequently mild and self-limiting, withminimal symptoms. However, can cause extensive destruction of thecolonicmucosa,andinvadeotherorgans.colonicmucosa,andinvadeotherorgans.

– Initially produces small, discrete erosions or minute crypt lesions.These then extend through the mucosa, into the submucosa, andexpand laterally to produce flask-shaped lesions. These may merge andcause denudation of overlying mucosa, producing a minute hole ofcommunication. These flasks shaped ulcer usually have raggedmargins, and bases due to digesting effect of proteases.

– The floor remains full of altered blood from oozing of the sidewalls.Base of the ulcer usually remains limited to muscularis mucosa, rarelyreach to submucosa, and up to muscularis externa.


Entamoeba

Extra intestinal amoebiasis

� E. histolyticacan metastasize in any organ which hepatic amoebiasis isthe commonest.

� Hepatic amoebiasis – Ameba can reach the liver via the portal veinfrom which they extend progressively in all directions, producing livercell necrosis and liquefaction, to form an amebic liver abscess, whichwill extend eventually into adjacent structures. They staysmall orcontinue to grow – center of abscess is full of necrotic fluid, outer wallfull of trophozoites. If abscessruptures,organismsareavailableto eatfull of trophozoites. If abscessruptures,organismsareavailableto eatother organs. The necrotic fluid is well comparable to ancovy sauce asper its colour and consistency. Microscopically, this fluid contains nil tovery few pus cells, trophozoites can be found but never cysts.

� Pulmonary amoebiasis -develop generally when liver abscess rupturesthrough diaphragm.

� Other ectopic sites includebrain, skin, penis (possibly acquiredsexually). Rare sites includekidneys, spleen, male and femalegenitaliaand pericardium.


Entamoeba

Pathogenesis of intestinal amoebiasis

• Young trophozoites released fromcyst binds with colonicmucosa by lectin (Gal/GalNAc)

• Colonization follows binding and then destruction by contactdependentcytolysis.dependentcytolysis.

• Invasion is facilitated by many factors like – pore formingproteins, proteases, motility and phagocytosis.


Entamoeba

Immunity

� Immunity to infection withE. histolyticais associated witha mucosal IgA response against the carbohydraterecognition domain of the Gal/GalNAc lectin.

� These IgA molecules act as the receptor blockingantibodies, and interfere attachmentof trophozoite onantibodies, and interfere attachmentof trophozoite onmucosal epithelial surface.

� Over a one-year period, children with this response had 86percent fewer newinfections than children without thisresponse. Cell-mediated responses have been described in-patients with amebic liver abscess, characterized bylymphocyte proliferation and lymphokine secretion that isamoebicidal in vitro.


Entamoeba

Clinical Features� Individuals who are infected withE. histolyticacan either remain asymptomatic

or present clinically with dysentery or extraintestinal disease.

� Asymptomatic : Asymptomatic infection withE. histolytica is defined as thepresence ofE. histolytica in stool in the absence of colitis or extraintestinalinfection. Colonization with the morphologically identical parasiteE. dispar ismore common in developing countries.E. histolyticacolonization is frequentlyobservedin high-risk settings. Colonizationwith E. histolyticacarrieda low butobservedin high-risk settings. Colonizationwith E. histolyticacarrieda low butdefinite risk of development of invasive amoebiasis.

� Amebic colitis : Amebic dysentery occurs gradually, with symptoms (such asabdominal pain and tenderness, painful sudden bowel evacuation (tenesmus), andintermittent diarrhoea, vomiting and general malaise) developing over a period ofone to several weeks. Amebic diarrhea is marked by 4-6 loose stools per day.Fever occurs in only a minority of patients with amebic colitis, and onlymicroscopically detectable blood is present in a majority.Because of thechronicity of the illness, weight loss is common.


Entamoeba

Clinical FeaturesAmebic liver abscess :

Liver abscess is overwhelmingly the most common manifestationof amoebiasis occurring outside the intestine (extraintestinal).

This complication is 10 times more common in adult men than inadultwomen. Childrenrarelydevelopamebicliver abscess.adultwomen. Childrenrarelydevelopamebicliver abscess.

Most of the patients, who present clinically with an amebic liverabscess, do not have coexistent dysentery, although a past historyof dysentery is common.Multiple cysts or cysts >10 cmin size usually occur in superiorright liver lobe involvement Spontaneous resolution by 6 monthsin 66% and Persist >1 year in 10%.


Entamoeba

Isoenzyme patterns � Isoenzyme patterns are known for four amebic enzymes which helps in

differentiating pathogenic strains from non pathogenic strains and alsohas epidemiological importance.

� The isoenzymes patterns are as follows :– glucose phosphate isomerase (GPI),– hexokinase (HK),– malate:NADP+ oxidoreductase (ME), and– phosphoglucomutase(PGM).– phosphoglucomutase(PGM).

� The isoenzyme patterns of three of these, GPI, HK, and PGM, can beused to define >20 zymodemes ofE histolytica. The enzyme markersassociated with pathogenicity are the presence of a b band and theabsence of an a band for PGM.

� Zymodemes II, VI, VII, XI, XII, XIII, XIV, XIX, and XX are pathogenic.� Zymodemes II and XI are responsible for liver abscesses. Zymodeme

patterns are of epidemiologic and research interest but their limitedavailability makes them less useful clinically.


Entamoeba

Laboratory Diagnosis

�Principle :– Laboratory diagnosis of intestinal amoebiasis is based on

microscopic demonstration of hematophagous trophozoiteor cyst of E. histolyticaand antigen detection fromstool.Nucleicacidstechniquesarealsohelpful. CulturefollowedNucleicacidstechniquesarealsohelpful. Culturefollowedby isoenzyme analysis is the ‘gold standard’ though it istime consuming and highly technical.

– Laboratory diagnosis of hepatic amoebiasis is practicallydone by antigen detection fromblood, saliva and pus fromliver.


Entamoeba

Laboratory Diagnosis Stool ME

� Diagnosis in most laboratories is still by microscopic examination of fresh,warm, liquid faeces for hematophagous trophozoites (i.e. trophozoites thathave ingested red blood cells).

� Sensitivity is only 30-50%. This may be improved by merthiolate iodineformalin concentration and staining of multiple stool specimens bymodified Ritchie formalin-ether concentration and examination of wetmounts and preparationsstainedby iron haematoxylinor trichrome formounts and preparationsstainedby iron haematoxylinor trichrome fortrophozoites and cysts, or by examining sigmoidoscopic swabs andscrpaings from large bowel ulcers and biopsies of rectal mucosa.

� Pathogenic strains of E. histolytica can only be differentiated by indirectimmunofluorescence with monoclonal antibodies.

� Delays in the processing of stool samples affect the sensitivity of lightmicroscopy, which under the best of circumstances is only 60% of that ofthe stool culture method. T

� he young cyst has a single nucleus but this rapidly divides twice to formfirstly a two- and then four-nucleus stage


Entamoeba

Laboratory Diagnosis M/E Stool

� In semiformed stool it is possible to find precysts and cystswith 1-4 nucleibut quadrinucleate (4 nuclei) cysts, the metacyst, is most common informed stool.

� Culture of stool samples followed by isoenzyme analysis canaccuratelydistinguish E. histolytica from E. dispar, and is considered to be the 'goldstandard'for diagnosis.standard'for diagnosis.

� However, one to several weeks required for this test and alsorequiresspecial laboratory facilities, making it impractical for use in the developingworld.


Entamoeba

Lab diagnosis� Antigen detection by ELISAon stool is the most sensitive

and specific method. The sensitivity of this method is >85%,and its specificity is >90%.

� Serological methods for antibody detection can also be usedto distinguish accurately between prior infection withE.histolyticaandE. dispar.

� Levels of anti-amebic antibodies remain elevated in the� Levels of anti-amebic antibodies remain elevated in theserumfor years, so detection of immunoglobulin M (IgM)antibodies prove to be useful for the diagnosis of acutedisease.

� PCR is helpful for the differentiation ofE. histolyticafromE. dispar from faeces, pus and culture. The sensitivity andspecificity of PCR-based methods for the diagnosis ofE.histolytica infection is similar to stool culture followed byisoenzyme analysis, and is more sensitive than antigendetection tests.


Entamoeba

�Hepatic amoebiasis is best diagnosed by ELISAdetection of lectin antigen. The test on serumhassensitivity of 96% and specificity of 75-100%,while that on salivahasspecificity of 70%. Whenwhile that on salivahasspecificity of 70%. Whenperformed on abscess fluid or pus, the specificityis 100%.

�Other extraintestinal complications include splenicabscess, empyema and pericarditis. Diagnosis inthese cases is similar to that for liver abscess


Entamoeba

Treatment

� Intestinal amoebiasis– Metronidazole is the drug of choice for treatment of symptomatic

intestinal amoebiasis 750 mg t.i.d., 5-10 days or 50 mg/kg, singledose for adults, cure rate 90%. OrTinidazole can be used 50 mg/kgq.d. 3 days, cure rate 86%.

�Amoebic liver abscess– Metronidazole 750 mg t.i.d., 5-10 days or 2.4 g q.d., 1-2 days, cure

rate is 90%.OrTinidazole / Ornidazole 2 g orally single dose.

�Asymptomatic cyst passers– Diloxanide furoate, 500 mg t.i.d., 10 days, cure rate 87-96%. Or

Iodoquinol (Diiodohydroxyquin) 650 mg t.i.d., 20 days, cure rate95%. OrParomomycin 500 mg t.i.d, 10 days or 30 mg/kg/day in 3doses, 5-10 days, cure rate85-90%.


Entamoeba

Prevention

� Non-Pathogenic Ameba

1. Entamoeba coli2. Entamoebahartmanni :2. Entamoebahartmanni :

3. Endolimax nana4. Iodamoeba bütschlii5. Entamoeba dispar


Entamoeba

Summary1) Entamoeba histolytica causes amoebiasis, the third most common cause of

parasitic death in the world, which kills 100000 people every year.

2) E. histolytica has two stages in its life cycle, Cyst and Trophozoites with anintermediate Precystic stage of which cysts are the infective stageandtrophozoites are the pathogenic stage and both the stages found in the stool ofpatient with amoebiasis. It is transmitted by fecal oral route. It passes its lifecycle in a single host, the man.

3) E. histolytica causes intestinal and extraintestinal amoebiasis. Intestinalamoebiasis is manifested by dysentery. Clinical manifestation of extraintestinalamoebiasis varies with site of involvement. Hepatic amoebiasis is commonestextraintestinal amoebiasis.

4) Diagnosis of intestinal amoebiasis is based on identification of cysts ortrophozoites of E. histolytica by microscopic examination of stool and also bydetection of antigen from stool. Direct fluorescent antibody test, EIA and PCRare also helpful. Extraintestinal amoebiasis is diagnosed by microscopy ofaspirated pus from the site of lesion and detection of antigen and antibody.

5) Amoebiasis can be effectively treated by metronidazole or tinidazole.


Entamoeba

Study Questions

1. What is the mortality and morbidity of amoebiasis? What is its clinical significance?

2. Describe morphology of Entamoeba histolytica. 3. Write about the laboratory diagnosis of amoebiasis? Which

test is most sensitive? test is most sensitive? 4. Write about the pathogenesis of amoebiasis.5. What are the virulence factors of Entamoeba histolytica. 6. What is Entamoeba dispar? Why it is so called? How it can

be differentiated from E. histolytica.7. What are the non-pathogenic intestinal amebas? How

E.histolytica can be differentiated from Entamoeba coli.8. Enumerate important non-pathogenic amebas? What is

their clinical significance?1/7/2014 31

Prof. Muahammad Akram, Entamoeba

Documents

Prof. Dr. Md. Akram Hossain March 09 - mmc.gov.bdmmc.gov.bd/downloadable file/Entamoeba akram.pdf · – E. coli, – E.gingivalis. 1/7/2014 4 Prof. Muahammad Akram, Entamoeba. Morphology