27Chapter3Amoebation of contaminatedfood or waterwithLife Cycle (Fig.3.2)maturequadrinucleated cystsHost:E.histolytica completes its life cycle inSexual contact: Rare, either by anoge-singlehost,i.e.man.nitalororogenital contact.(especiallyinInfectiveform:Maturequadrinucleatedcystdeveloped countries among homosexualis theinfectiveform.Itcan resist chlorination,males)gastricacidityand desiccation and can survive.Vector:Veryrarely,flies andcockroachesinamoistenvironmentforseveralweeksmaymechanicallytransmitthecystsfromNote:Trophozoites and immature cysts can befeces,andcontaminatefoodandwater.passedin stool ofamoebicpatients,buttheycan'tDevelopmentinman(smallintestine)serveasinfectiveformastheyaredisintegratedin the environment or by gastric juice whenExcystation: In small intestine,the cystingested.wall gets lysed bytrypsin and a singleModeoftransmission:tetranucleated trophozoite(metacyst)is.Feco-oralroute(mostcommon):Byinges-liberated which eventually undergoesalungs abscessLiverabscessInfectiveform(Quadrinucleate cyst)SkinulcersBrainabscessPo nPuExtraintestinalamoebiasisEXCYSTATIONENCYSTATION(INSMALLINTESTINE)(INLARGEINTESTINE)TrophozoitePrecystOMetacystOUninucleated cyst8-BinucleatedcystMetacystictrophozoiteTetranucleated cystTrophozoiteIntestinalulcersCyst infecesTrophozoite in feces(seen in carriers and(seen inactivealso in active infection)infection)Fig.3.2:Life cycle of Entamoeba histolytica
Chapter 3 Amoeba 27 Life Cycle (Fig. 3.2) Host: E. histolytica completes its life cycle in single host, i.e. man. Infective form: Mature quadrinucleated cyst is the infective form. It can resist chlorination, gastric acidity and desiccation and can survive in a moist environment for several weeks. Note: Trophozoites and immature cysts can be passed in stool of amoebic patients, but they can’t serve as infective form as they are disintegrated in the environment or by gastric juice when ingested. Mode of transmission : z Feco-oral route(most common): By ingestion of contaminated food or water with mature quadrinucleated cysts z Sexual contact: Rare, either by anogenital or orogenital contact. (especially in developed countries among homosexual males) z Vector: Very rarely, flies and cockroaches may mechanically transmit the cysts from feces, and contaminate food and water. Development in man (small intestine) z Excystation: In small intestine, the cyst wall gets lysed by trypsin and a single tetranucleated trophozoite (metacyst) is liberated which eventually undergoes a Fig. 3.2: Life cycle of Entamoeba histolytica Chapter-03.indd 27 5/19/2014 12:47:33 PM
28Section2Protozoologyseries of nuclear and cytoplasmic divi-Mature quadrinucleated cysts released insions to produce eight small metacysticfeces can survive in the enviornment andtrophozoitesbecometheinfectiveform.Immature cystsMetacystic trophozoites are carried byand trophozoites are some times excreted,theperistalsistoileocecalregionoflargebutget disintegrated in the environement.intestine and multiplyby binaryfission, and(Fig. 3.2).then colonize on themucosal surfaces andPathogenesiscryptsofthelargeintestineAfter colonization,trophozoites showTrophozoite of E.histolytica is the majordifferent courses depending on variousinvasiveformand possessesmanyvirulencefactors like host susceptibility,age, sex,factors that playrole inthepathogenesis ofnutritionalstatus,hostimmunity,intestinalintestinalas well as extraintestinal amoebiasismotility,transittimeand intestinalflora(Table 3.2).Asymptomatic cyst passers:Inmajorityof individuals,trophozoites don't causePathogenesis of Intestinal Amoebiasisanylesion,transform intocystsand areTrophozoites invade the colonic mucosaexcretedinfecesproducing characteristic ulcerative lesionsAmoebic dysentery: Trophozoites ofand profusebloodydiarrhea(amoebicE.histolytica secrete proteolytic enzymesdysentery).Males and females are affectedthat cause destruction and necrosis ofequallywith a ratio of 1:1.tissue,and producesflask shapedulcers onthe intestinal mucosa.At this stage, largeAmoebiculcernumbersoftrophozoites areliberatedalongThe classical ulcer is flask-shaped (broadwith blood and mucus in stoolproducingbase with a narrow neck).amoebic dysentery.Trophozoites usually.Itmaybelocalizedtoileocecalregion(mostdegeneratewithin minutesAmoebic liverabscess:In few cases,common site)or sigmoidorectal region ormay be generalized involving the wholeerosion and necrosis of small intestinelengthof the large intestineare soextensive thatthetrophozoites gainentrance into theradicals ofportal veinsUlcersare usuallyscatteredwith interveningandarecarriedawaytotheliverwheretheynormalmucosamultiplycausingamoebicliverabscessItmaybesuperficial (confinedtomuscularismucosaandheal without scar)ordeepDevelopment in man (large intestine)ulcer(beyondmuscularismucosa andhealsEncystation:After some days,when thewithscarformation)intestinal lesion starts healing and patientSizerangingfrompinhead to inchesimproves,thetrophozoitestransform intoShape round to ovalprecysts then into quadrinucleated cysts.Marginraggedand underminedwhichareliberated infecesBaseisformed onmusclecoat...Encystation occurs only in the largeComplications of intestinal amoebiasisgut.Cysts are never formed once the(Fig. 3.3)trophozoites are excreted in stool·Factors that induce cystformation includeTherearefollowingtypesofcomplications:food deprivation, overcrowding,desic-.Fulminantamoebiccolitis:Resultingfromcation,accumulation ofwasteproducts,generalized necrotic involvementofentireand cold temperatureslarge intestine,occurs morecommonly
28 Section 2 Protozoology series of nuclear and cytoplasmic divisions to produce eight small metacystic trophozoites z Metacystic trophozoites are carried by the peristalsis to ileocecal region of large intestine and multiply by binary fission, and then colonize on the mucosal surfaces and crypts of the large intestine z After colonization, trophozoites show different courses depending on various factors like host susceptibility, age, sex, nutritional status, host immunity, intestinal motility, transit time and intestinal flora z Asymptomatic cyst passers: In majority of individuals, trophozoites don’t cause any lesion, transform into cysts and are excreted in feces z Amoebic dysentery: Trophozoites of E. histolytica secrete proteolytic enzymes that cause destruction and necrosis of tissue, and produces flask shaped ulcers on the intestinal mucosa. At this stage, large numbers of trophozoites are liberated along with blood and mucus in stool producing amoebic dysentery. Trophozoites usually degenerate within minutes z Amoebic liver abscess: In few cases, erosion and necrosis of small intestine are so extensive that the trophozoites gain entrance into the radicals of portal veins and are carried away to the liver where they multiply causing amoebic liver abscess. Development in man (large intestine) z Encystation: After some days, when the intestinal lesion starts healing and patient improves, the trophozoites transform into precysts then into quadrinucleated cysts which are liberated in feces z Encystation occurs only in the large gut. Cysts are never formed once the trophozoites are excreted in stool z Factors that induce cyst formation include food deprivation, overcrowding, desiccation, accumulation of waste products, and cold temperatures z Mature quadrinucleated cysts released in feces can survive in the enviornment and become the infective form. Immature cysts and trophozoites are some times excreted, but get disintegrated in the environement. (Fig. 3.2). Pathogenesis Trophozoite of E. histolytica is the major invasive form and possesses many virulence factors that play role in the pathogenesis of intestinal as well as extraintestinal amoebiasis (Table 3.2). Pathogenesis of Intestinal amoebiasis Trophozoites invade the colonic mucosa producing characteristic ulcerative lesions and profuse bloody diarrhea (amoebic dysentery). Males and females are affected equally with a ratio of 1:1. Amoebic ulcer The classical ulcer is flask-shaped (broad base with a narrow neck). z It may be localized to ileocecal region (most common site) or sigmoidorectal region or may be generalized involving the whole length of the large intestine z Ulcers are usually scattered with intervening normal mucosa z It may be superficial (confined to muscularis mucosa and heal without scar) or deep ulcer (beyond muscularis mucosa and heals with scar formation) z Size ranging from pin head to inches z Shape round to oval z Margin ragged and undermined z Base is formed on muscle coat. Complications of intestinal amoebiasis (fig. 3.3) There are following types of complications: z Fulminant amoebic colitis: Resulting from generalized necrotic involvement of entire large intestine, occurs more commonly Chapter-03.indd 28 5/19/2014 12:47:33 PM
29Chapter3AmoebaTable3.2:Virulencefactors of EntamoebahistolyticaAmoebiclectinantigen:Itisa260kDagalactoseandN-acetylgalactosamine inhabitablesurfaceprotein (Gal/NAG lectin)It has two subunits-heavy (170 kDa) and-light (35 kDa) subunits linked by disulfde bridge.The 170 kDasubunithasacytoplasmicandtoxicmembranedomainLectin antigen istheprinciplevirulencefactor,presentonthesurfaceoftrophozoitesofpathogenicE.histolyticabutnotonnonpathogenicE.dispar.Itsvariouspathogenicmechanismsare:Adhesion:Bybindingtoglycoproteinreceptorson intestinalandhepaticsurfacesCytotoxicity:Bycontactdependentcytolysis ofthetargetcellsbyincreasingthecalciumlevelComplementresistance:170kDasubunitresemblesCD59(ahumancomplementblocker)thatpreventC5b-9complexformationAmoebapore(5kDaAmoebicporeformingprotein):It insertsionchannelsinthetargetcell membranecausingleakageofions.ItsequivalentfoundinE.dispariscalledasdisparporesCysteineproteasesTheydegradeextracellularmatrix,responsibleforinvasion,secretedonlybytrophozoitesofpathogenicE.histolytica.Examplesincludehistolysin,amoebapain andcathepsinBlikeproteasesHydrolyticenzymesSuchas RNAse,neutralproteaseandphosphatases-help inthedestructionofthetargettissueNeuraminidaseandmetallocollagenaseHelpininvasioninimmunocompromisedpatientsandin(segmentof intestine invaginates into theadjoiningintestinal lumen,causingbowelpregnancyAmoebic appendicitis:Results when theobstruction)Perianalskinulcers:Bydirectextension ofinfectioninvolvesappendix·Intestinal perforation and amoebic peri-ulcerstoperianal skintonitis: Occurs when the ulcerprogressesAmoeboma(amoebicgranuloma):Abeyondtheserosadiffusepseudotumorlikemassofgranulo.Toxic megacolon and intussusceptionmatoustissuefoundinrectosigmoidregionDeepextensionofAmoebomaulcerbeyondInvasion ofFlask-shapedSinusformationmuscularismucosaePerforationblood vesselslcerQ0008100071MucosaLMuscularismucosSubmucosaCircularmusclesLongitudinalmusclesSerosaPeritoneal liningFig.3.3:Complications ofintestinalamoebiasis (crosssectionof intestinalwall)
Chapter 3 Amoeba 29 Fig. 3.3: Complications of intestinal amoebiasis (cross section of intestinal wall) in immunocompromised patients and in pregnancy z Amoebic appendicitis: Results when the infection involves appendix z Intestinal perforation and amoebic peritonitis: Occurs when the ulcer progresses beyond the serosa z Toxic megacolon and intussusception (segment of intestine invaginates into the adjoining intestinal lumen, causing bowel obstruction) z Perianal skin ulcers: By direct extension of ulcers to perianal skin z Amoeboma (amoebic granuloma): A diff use pseudotumor like mass of granulomatous tissue found in rectosigmoid region table 3.2: Virulence factors of Entamoeba histolytica amoebic lectin antigen • It is a 260 kDa galactose and N-acetylgalactosamine inhabitable surface protein (Gal/NAG lectin) • It has two subunits—heavy (170 kDa) and—light (35 kDa) subunits linked by disulfi de bridge. The 170 kDa subunit has a cytoplasmic and toxic membrane domain • Lectin antigen is the principle virulence factor, present on the surface of trophozoites of pathogenic E. histolytica but not on nonpathogenic E. dispar. Its various pathogenic mechanisms are: − Adhesion: By binding to glycoprotein receptors on intestinal and hepatic surfaces − Cytotoxicity: By contact dependent cytolysis of the target cells by increasing the calcium level − Complement resistance: 170kDa subunit resembles CD59 (a human complement blocker) that prevent C5b-9 complex formation amoebapore (5 kDa Amoebic pore forming protein) : It inserts ion channels in the target cell membrane causing leakage of ions. Its equivalent found in E. dispar is called as dispar pores Cysteine proteases They degrade extracellular matrix, responsible for invasion, secreted only by trophozoites of pathogenic E. histolytica. Examples include histolysin, amoebapain and cathepsin B like proteases Hydrolytic enzymes Such as RNAse, neutral protease and phosphatases—help in the destruction of the target tissue Neuraminidase and metallocollagenase Help in invasion Chapter-03.indd 29 5/19/2014 12:47:35 PM
30Section2 ProtozoologyChronic amoebiasis: Itischaracterizedby>Outerzone:comprised ofhealthythickening,fibrosis,strictureformationwithhepatocytes invaded with amoebicscarring and amoeboma formation.trophozoitesAnchovy saucepus:Liver abscess pus isPathogenesisof Extraintestinalthickchocolatebrownincolor.ThefluidAmoebiasisis acidic and pH5.2-6.7 and is comprisedFollowing1-3monthsofintestinal amoebiasis,of necrotic hepatocytes without anyabout5-10%ofpatientsdevelopextraintestinapus cells and occasional amoebic tropho-amoebiasis.Liveris the most common sitezoites (mainly found in last few drops of(because of the carriage of trophozoitespus as amoebaemultiplyin thewall ofthrough the portal vein) followed by lungs,abscess).brain,genitourinarytractand spleen.ComplicationsofamoebicliverabscessAmoebicliverabscessWithcontinuoushepatic necrosis,abscessThemost common group affected:Adultmay grow in various direction of liver dis-males (male and femaleratio is 9:1).charging the contents into the neighboringThemostcommonaffectedsiteistheorgans (Fig.3.4).posterior-superior surface of the right lobe.Rightsided liverabscess mayruptureexter-of liver. Abscess is usually single or rarelynally to skin causing granuloma cutis ormultiple (Fig.3.4).rupture intolungs (pulmonaryamoebiasisAmoebictrophozoites occludethehepaticwith trophozoites in sputum)orintothevenules; which leadsto anoxicnecrosis ofrightpleura (amoebicpleuritis)the hepatocytes.Inflammatory responseRuptureof liver abscess belowthediapsurroundingthehepatocytesleadstothehragm leads to subphrenic abscess andformationofabscessesgeneralizedperitonitis.Microscopicallytheabscess wall is com-Left sided liverabscess mayrupture intoprised of:stomachorleftpleuraorpericardial cavityInner central zone of necrotichepato-(amoebicpericarditis)cyteswithoutamoebaHematogenous spread can occur from>Middle zone of degenerative hepato-liver affecting brain, lungs, spleen andcytes,RBC,fewleucocytesandoccasio-genitourinary organs.nallyamoebictrophozoitesClinicalManifestationsofAmoebiasisAsymptomaticamoebiasisAbout90%ofinfectedpersonsareasymptomatic carriers and excrete cysts in theirfeces.Nowit is confirmed that many of thesecarriers harbor E. dispar.Theremaining10%ofpeople(whoaretrulyinfectedbypathogenicE.histolytica)producesa spectrumofdiseasesvaryingfromintestinalamoebiasistoamoebicliverabscess.Fig.3.4: Cross section of liver showingIntestinalamoebiasisamoebicliverabscess (rightside)Incubation period varies from oneto fourCourtesy:HOD,Department ofPathologyweeks.Intestinal amoebiasisischaracterizedMeenakshi medical college, Chennai
30 Section 2 Protozoology z Chronic amoebiasis: It is characterized by thickening, fibrosis, stricture formation with scarring and amoeboma formation. Pathogenesis of Extraintestinal amoebiasis Following 1–3 months of intestinal amoebiasis, about 5–10% of patients develop extraintestinal amoebiasis. Liver is the most common site (because of the carriage of trophozoites through the portal vein) followed by lungs, brain, genitourinary tract and spleen. Amoebic liver abscess The most common group affected: Adult males (male and female ratio is 9:1). The most common affected site is the posteriorsuperior surface of the right lobe of liver. Abscess is usually single or rarely multiple (Fig. 3.4). z Amoebic trophozoites occlude the hepatic venules; which leads to anoxic necrosis of the hepatocytes. Inflammatory response surrounding the hepatocytes leads to the formation of abscesses z Microscopically the abscess wall is comprised of: h Inner central zone of necrotic hepatocytes without amoeba h Middle zone of degenerative hepatocytes, RBC, few leucocytes and occasionally amoebic trophozoites h Outer zone: comprised of healthy hepatocytes invaded with amoebic trophozoites z Anchovy sauce pus: Liver abscess pus is thick chocolate brown in color. The fluid is acidic and pH 5.2–6.7 and is comprised of necrotic hepatocytes without any pus cells and occasional amoebic trophozoites (mainly found in last few drops of pus as amoebae multiply in the wall of abscess). Complications of amoebic liver abscess With continuous hepatic necrosis, abscess may grow in various direction of liver discharging the contents into the neighboring organs (Fig. 3.4). z Right sided liver abscess may rupture externally to skin causing granuloma cutis or rupture into lungs (pulmonary amoebiasis with trophozoites in sputum) or into the right pleura (amoe bic pleuritis) z Rupture of liver abscess below the diaphragm leads to subphrenic abscess and generalized peritonitis z Left sided liver abscess may rupture into stomach or left pleura or pericardial cavity (amoebic pericarditis) z Hematogenous spread can occur from liver affecting brain, lungs, spleen and genitourinary organs. Clinical manifestations of amoebiasis Asymptomatic amoebiasis About 90% of infected persons are asymptomatic carriers and excrete cysts in their feces. Now it is confirmed that many of these carriers harbor E. dispar. The remaining 10% of people (who are truly infected by pathogenic E. histolytica) produces a spectrum of diseases varying from intestinal amoebiasis to amoebic liver abscess. Intestinal amoebiasis Incubation period varies from one to four weeks. Intestinal amoebiasis is characterized Fig. 3.4: Cross section of liver showing amoebic liver abscess (right side) Courtesy: HOD, Department of Pathology, Meenakshi medical college, Chennai Chapter-03.indd 30 5/19/2014 12:47:35 PM
31Chapter3Amoebaby four clinical forms:LaboratoryDiagnosisIntestinalamoebiasisAmoebicdysentery:Symptomsinclude1.bloodydiarrhea with mucus and pus cells,oStoolmicroscopybywetmount,permanentcolicky abdominal pain,fever, prostration,stains,etc-detectscystsandtrophozoitesand weight loss. Amoebic dysentery.Stoolcultureshouldbe differentiated frombacillary>Polyxenicandaxenicculturedysentery (Table 3.3)Stool antigen detection (copro-antigen)2.Amoebic appendicitis:Presented withCIEP,EISA,ICTacuterightlowerabdominalpain.Serology3.Amoeboma:It present as palpable abdo->Amoebicantigen-ELISAminalmass>Amoeboicantibody-IHA,ELISAandIFA4.Fulminantcolitis:PresentsasintenseoIsoenzyme(zymodene)analysiscolickypain,rectaltenesmus,morethanMolecular diagnosis20motions/day,fever,nausea,anorexia>NestedmultiplexPCRandrealtimePCRand hypotension.Table3.3:Differences in stool characters between amoebic dysentery and bacillarydysenteryCharacterAmoebicdysenteryBacillarydysenteryPathologyUlcerDeepShallowMarginUniformRagged and underminedNormalInflamedIntervening mucosaNecrosistypePyknosis (pyknoticbodies)Karyolysis (ghost cells)Cellular responseMononuclear cellsPolymorphonuclear cellsStoolmacroscopicfeature 6-8/day>10/dayNumberofmotionAmountCopious amountSmall quantityColorDark redBright redOdorOffensiveOdorlessAcidicReactionAlkalineConsistencyNot adherentto the containerAdherent to the containerStoolmicroscopicfeatureRed blood cells (RBCs)InclumpsDiscrete or in rouleauxPus cellsFewNumerousFewMacrophagesNumerous,maycontain RBCs,socanbemistakenasE.histolyticaEosinophilsPresentAbsentorrarePresentCharcot Leyden crystalAbsentPresentAbsentPyknotic body(nuclear remains of tissue cellsand leukocytes)Ghost cellAbsentPresentOrganism detectedE.histolyticacystortrophozoiteBacteria (Shigella)
Chapter 3 Amoeba 31 table 3.3: Diff erences in stool characters between amoebic dysentery and bacillary dysentery Character amoebic dysentery Bacillary dysentery Pathology Ulcer Deep Shallow Margin Ragged and undermined Uniform Intervening mucosa Normal Infl amed Necrosis type Pyknosis (pyknotic bodies) Karyolysis (ghost cells) Cellular response Mononuclear cells Polymorphonuclear cells Stool macroscopic feature Number of motion 6–8/day > 10/day Amount Copious amount Small quantity Color Dark red Bright red Odor Off ensive Odorless Reaction Acidic Alkaline Consistency Not adherent to the container Adherent to the container Stool microscopic feature Red blood cells (RBCs) In clumps Discrete or in rouleaux Pus cells Few Numerous Macrophages Few Numerous, may contain RBCs, so can be mistaken as E. histolytica Eosinophils Present Absent or rare Charcot Leyden crystal Present Absent Pyknotic body (nuclear remains of tissue cells and leukocytes) Present Absent Ghost cell Absent Present Organism detected E. histolytica cyst or trophozoite Bacteria (Shigella) by four clinical forms: 1. Amoebic dysentery: Symptoms include bloody diarrhea with mucus and pus cells, colicky abdominal pain, fever, prostration, and weight loss. Amoebic dysentery should be differentiated from bacillary dysen tery (Table 3.3) 2. Amoebic appendicitis: Presented with acute right lower abdominal pain 3. Amoeboma: It present as palpable abdominal mass 4. Fulminant colitis: Presents as intense colicky pain, rectal tenesmus, more than 20 motions/day, fever, nausea, anorexia and hypotension. Laboratory Diagnosis Intestinal amoebiasis z Stool microscopy by wet mount, permanent stains, etc—detects cysts and trophozoites z Stool culture h Polyxenic and axenic culture z Stool antigen detection (copro-antigen)— CIEP, ELISA, ICT z Serology h Amoebic antigen—ELISA h Amoeboic antibody—IHA, ELISA and IFA z Isoenzyme (zymodene) analysis z Molecular diagnosis h Nested multiplex PCR and real time PCR Chapter-03.indd 31 5/19/2014 12:47:36 PM