ØØFascioliasisFascioliasis isis anan importantimportant parasiticparasiticdiseasedisease causedcaused byby FasciolaFasciola hepaticahepatica andand FF..giganticagigantica..

ØØTheThe diseasedisease hashas 22 clinicalclinical phasesphases withwithdifferentdifferent signssigns andand symptoms,symptoms, thethe hepatichepatic(acute)(acute) phasephase andand biliarybiliary (chronic)(chronic) phasephase(acute)(acute) phasephase andand biliarybiliary (chronic)(chronic) phasephase

ØØInIn manymany endemicendemic areas,areas, childrenchildren andandfemalesfemales areare mostmost severelyseverely affectedaffected..Therefore,Therefore, thethe capacitycapacity toto correctlycorrectlydiagnosediagnose humanhuman fascioliasisfascioliasis becomesbecomescrucialcrucial..

Endoscopic diagnosisEgg detection Imaging features

Methods of diagnosis

Serological diagnosis

Polymerase chainreaction

Surgical diagnosis

ØØEggsEggs ofof FasciolaFasciola areare operculatedoperculated,, largelarge,,lightlight yellowishyellowish--brownbrown,, broadlybroadly ellipsoidalellipsoidalandand nonnon--embryonatedembryonated whenwhen excretedexcreted ininfaeces,faeces, andand theirtheir lengthlength andand widthwidth arearegenerallygenerally withinwithin aa specificspecific rangerange(considered(considered toto bebe 150150 μmμm inin lengthlength andand

((11) ) Fasciola Fasciola egg detection:egg detection:

(considered(considered toto bebe 150150 μmμm inin lengthlength andand9090 μmμm inin width)width)..

ØØ AtAt thethe abopercularabopercular endend ofof thethe shell,shell, therethere isisoftenoften aa roughenedroughened oror irregularirregular areaarea thatthat isisnotnot seenseen inin FasciolopsisFasciolopsis eggs,eggs, however,however, itit isisfoundfound toto bebe populationpopulation--dependentdependent..

ØØShapeShape andand sizesize ofof thethe flukefluke eggseggs arearecrucialcrucial diagnosticdiagnostic featuresfeatures forfor identificationidentificationofof speciesspecies.. Therefore,Therefore, studyingstudying theirtheirmorphometricmorphometric traitstraits (length(length ((ELEL),), widthwidth ((EWEW),),EL/EWEL/EW usingusing microscopymicroscopy oror perimeterperimeter ((EPEP),),areaarea ((EAEA)) andand roundnessroundness ((ERER)) usingusing aaareaarea ((EAEA)) andand roundnessroundness ((ERER)) usingusing aacomputercomputer imageimage analysisanalysis system)system) waswasconsideredconsidered toto detectdetect thethe influenceinfluence ofof bothboththethe geographicalgeographical locationlocation andand ofof thethe hosthost(human(human andand livestock)livestock) onon eggegg sizesize..

Shape and size variability of fasciolid eggs shed in human stools: (A–G) Fasciolahepatica of the Northern Altiplano, Bolivia; (H–P) F. hepatica of the Cajamarca Valley,Peru; (Q–S) F. hepatica of the Kutaisi region, Georgia; (T–V) fasciolid eggs (mainly F.hepatica phenotypes) of the Nile Delta region, Egypt; (W–Y) F. gigantica of the QuyNhon area, Vietnam. Note the presence of lateral irregularities on the egg shell surfacein I, J and R, presence of such irregularities on the abopercular end of the shell in C, E–G, N–P and X, and their absence on the same abopercular shell end in M and Y.

Factor map corresponding to Fasciola eggs shed by humans from Bolivia, Peru, Georgia, Egypt and Vietnam.Georgia, Egypt and Vietnam.

Factor map corresponding to Fasciola eggs from humans and cattle from the same endemic area in Vietnam.

Direct coprological analysis of fasciolid Direct coprological analysis of fasciolid eggs continues to be the most appropriate eggs continues to be the most appropriate

diagnostic strategydiagnostic strategy

In spite of

•The recognized lower sensitivity of egg•The recognized lower sensitivity of eggdetection in faecal samples,

•Its uselessness for the diagnosis of patients inthe acute phase, and•The lack of an accurate relationship betweenegg counts per gram of faeces and fluke burden

The most common imaging findings of The most common imaging findings of fascioliasis are fascioliasis are multiple small nodularmultiple small nodular andandbranching linear lesionsbranching linear lesions, frequently in the , frequently in the subcapsular areassubcapsular areas of the liver parenchymaof the liver parenchyma

that appear that appear hypoechoichypoechoic onon USUS,,

(2) Imaging features of fascioliasis

ØØ hypoechoichypoechoic onon USUS,,ØØ hypodensehypodense onon CTCT,,ØØ isointenseisointense oror hypointensehypointense lesionslesions onon TT11--

weightedweighted MRIMRI imagesimages andandØØ isointenseisointense oror hyperintensehyperintense lesionslesions withwith

surroundingsurrounding hyperintensityhyperintensity onon TT22--weightedweighted MRIMRIimagesimages..

In the biliary phaseIn the biliary phase, ductal changes are , ductal changes are observed mainly in the observed mainly in the central bile ducts:central bile ducts:

ØØDuctDuct wallwall thickeningthickening andand periductalperiductalfibrosisfibrosis..ØØThickeningThickening ofof thethe gallbladdergallbladder wallwall..ØØ BileBile ductduct dilationdilation maymay bebe seenseen duedue totoobstructionobstruction..ØØAA largelarge cavitarycavitary lesionlesion maymay bebe seenseen duedue totoØØAA largelarge cavitarycavitary lesionlesion maymay bebe seenseen duedue totowormworm reinvasionreinvasion ofof thethe hepatichepatic parenchymaparenchymaandand rupturerupture ofof thethe bilebile ductduct..ØØ AcuteAcute hemobiliahemobilia relatedrelated toto thethe bleedingbleedingulcerulcer inin thethe CBDCBD maymay bebe seenseen..ØØSometimes,Sometimes, migrationmigration ofof thethe parasiteparasite fromfromthethe liverliver parenchymaparenchyma toto thethe biliarybiliary ductduct cancanresultresult inin subcapsularsubcapsular hemorrhagehemorrhage inin thetheliverliver parenchymaparenchyma..

Abdominal US. Dilated biliary tree and diffuse and irregular thickening of the CBD wall, which is 5 mm wide.

US

CTCT

thethe mostmost usefuluseful imagingimaging methodmethod ininhepatichepatic phasephase andand helpfulhelpful inin followfollow--upuptoto evaluateevaluate thethe efficacyefficacy ofof therapytherapy

moremore usefuluseful inin biliarybiliary stagestage ofof thetheCTCT

MRI

moremore usefuluseful inin biliarybiliary stagestage ofof thethediseasedisease andand inin followfollow--upup toto evaluateevaluatethethe efficacyefficacy ofof medicalmedical therapytherapy

helpfulhelpful inin determiningdetermining thethe phasephase andandactivityactivity andand demonstratesdemonstrates variousvariouschangeschanges associatedassociated withwith traumatictraumatichepatitishepatitis causedcaused byby wormworm migrationmigration

EndoscopyEndoscopy isis aa usefuluseful diagnosticdiagnostic tool,tool, whetherwhetherbyby itsits directdirect visualizationvisualization ofof thethe wormworm oror bybyobservationobservation ofof itsits shadowshadow inin ERCPERCP imagesimages..

(3) Endoscopic diagnosis

ERCP imagesERCP images

Filling defect Linear defect Disappearing stone

ØØ FillingFilling defectsdefects withwith aa characteristiccharacteristicperipheralperipheral radiolucencyradiolucency asas aa resultresult ofof thetheedgeedge ofof thethe wormworm foldingfolding onon itselfitself asas itit movesmoves..ØØWellWell--defineddefined linearlinear defectsdefects inin closeclose relationrelationtoto thethe wallwall ofof thethe bilebile ductsducts.. TheThe shapeshape ofofwhichwhich dependsdepends onon whetherwhether thethe wormworm isis seenseenfaceface onon oror fromfrom thethe sideside..ØØDisappearingDisappearing stonestone isis theirtheir inconsistentinconsistentØØDisappearingDisappearing stonestone isis theirtheir inconsistentinconsistentappearance,appearance, whichwhich confusesconfuses thethe endoscopistendoscopistwithwith regardregard toto whetherwhether aa fillingfilling defectdefect isis presentpresent..ThisThis phenomenonphenomenon isis mostmost probablyprobably thethe resultresult ofofthethe temporarytemporary movementmovement ofof thethe lowerlower partpart ofofthethe worm’sworm’s bodybody awayaway fromfrom thethe bilebile ductduct wallwallwhilewhile remainingremaining attachedattached toto thethe wallwall byby itsitsventralventral suckersucker..

ØØ Although,Although, inin biliarybiliary fascioliasisfascioliasis ERCPERCP andandsphincterotomysphincterotomy havehave beenbeen usedused forfordiagnosisdiagnosis andand extractionextraction ofof thethe parasitesparasitesfromfrom thethe biliarybiliary treetree byby balloonballoon oror basketbasket ororeveneven continuouscontinuous suctionsuction andand thatthat thetheinfectioninfection isis sometimessometimes diagnoseddiagnosedinfectioninfection isis sometimessometimes diagnoseddiagnosedunexpectedlyunexpectedly byby ERCPERCP inin patientspatients suspectedsuspectedofof havinghaving choledocholithiasis,choledocholithiasis, howeverhoweverinadequateinadequate incisionincision ofof papillapapilla maymay resultresult inincholangitischolangitis asas wellwell asas stonesstones inin thethe CBDCBD

ØØ VariousVarious serologicalserological teststests havehave beenbeen usedused forforFasciolaFasciola diagnosisdiagnosis asas indirectindirect haemaglutinationhaemaglutination(I(IHAHA),), indirectindirect fluorescencefluorescence antibodyantibody testtest ((IFATIFAT),),immunoperoxydaseimmunoperoxydase ((IPIP),), counterelectrophoresiscounterelectrophoresis

(4) Serological diagnosis

immunoperoxydaseimmunoperoxydase ((IPIP),), counterelectrophoresiscounterelectrophoresis((CEPCEP),), enzymeenzyme--linkedlinked immunosorbentimmunosorbent assayassay((ELISAELISA)) andand DotDot--ELISAELISA..

ØØ AlmostAlmost allall ofof thesethese techniquestechniques concernconcern withwith thethedetectiondetection ofof circulatingcirculating antibodiesantibodies andand onlyonly aa fewfewareare designeddesigned toto detectdetect circulatingcirculating antigensantigens andandimmuneimmune complexescomplexes

ØØ thethe advantagesadvantages ofofserologicalserological teststestsareare beingbeingapplicableapplicable duringduringallall stagesstages ofof thethedisease,disease, butbutespeciallyespecially duringduring

ØØ Unfortunately, there Unfortunately, there is some problems as: is some problems as: (i) useless for (i) useless for intensity estimation; intensity estimation; (ii) tests are generally (ii) tests are generally expensive and expensive and require a minimum of require a minimum of laboratory laboratory especiallyespecially duringduring

thethe invasiveinvasive ororacuteacute phase,phase, andand ininotherother situationssituations ininwhichwhich coprologicalcoprologicaltechniquestechniques areareinappropriateinappropriate..

laboratory laboratory infrastructure; and infrastructure; and (iii) Tests have so far (iii) Tests have so far never proved to be never proved to be able to differentiate able to differentiate between infection by between infection by F. hepaticaF. hepatica and and F. F. giganticagigantica

ELISAELISAIHAIHA

9898%,%,8686%.%.the specificitythe specificity

detected from detected from the second the second week.week.

detected from detected from third week after third week after infectioninfection

Antibodies Antibodies directed directed againstagainst F.F.hepaticahepatica

ØØ FasciolaFasciola wormsworms releaserelease substantialsubstantial amountsamountsofof excretoryexcretory––secretorysecretory productsproducts (ESP)(ESP)

ØØ FF.. hepaticahepatica releaserelease approximatelyapproximately 6060proteins,proteins, ofof whichwhich 2929 consistconsist essentiallyessentially ofofcathepsincathepsin LL proteases,proteases, superoxidesuperoxidedismutase,dismutase, thioredoxinthioredoxin peroxidase,peroxidase,glutathioneglutathione SS--transferasestransferases andand fattyfatty acidacidbindingbinding proteinsproteins..glutathioneglutathione SS--transferasestransferases andand fattyfatty acidacidbindingbinding proteinsproteins..

ØØ FF.. giganticagigantica ESPESP areare ofof 6666,, 6464,, 5858,, 5454,, 2828 andand2626 kDakDa.. TheThe formerformer fourfour moleculesmolecules derivedderivedfromfrom thethe tegument,tegument, whilewhile thethe 2828 andand 2626 kDakDareleasedreleased fromfrom cellscells lininglining thethe gutgut

ØØ CysteineCysteine proteinasesproteinases areare probablyprobably thethe mostmostabundantabundant proteinsproteins amongamong E/SE/S productsproducts fromfromfasciolidsfasciolids

ØØ SeveralSeveral cysteinecysteine proteinasesproteinases offeroffer highlyhighlysensitivesensitive andand specificspecific markersmarkers forfor humanhumanfascioliasisfascioliasis serodiagnosisserodiagnosis asas::

1.1. secretedsecreted byby thethe adultadult andand juvenilejuvenile formsforms2.2. areare highlyhighly antigenicantigenic inin animalsanimals andand humanshumans3.3. aa veryvery valuablevaluable sourcesource ofof antigensantigens forfor3.3. aa veryvery valuablevaluable sourcesource ofof antigensantigens forfor

diagnosisdiagnosis

ØØ FasciolaFasciola hepaticahepatica recombinantrecombinant cysteinecysteineproteinasesproteinases producedproduced inin yeastyeast oror ininEscherichiaEscherichia colicoli havehave beenbeen usedused inin ELISAELISAmethodsmethods forfor humanhuman infectioninfection diagnosis,diagnosis,providingproviding resultsresults similarsimilar toto nativenative antigensantigens..

ØØ TheThe usefulnessusefulness ofof molecularmolecular geneticgenetictechniquestechniques (as(as PCR)PCR) inin fascioliasisfascioliasisdiagnosisdiagnosis waswas emphasizedemphasized withwith choosingchoosing ofofsequencessequences notnot repeatedrepeated multiplemultiple timestimes inin thethegenomegenome inin orderorder toto limitlimit sensitivitysensitivity..

PCR in fasciola differentiation

genomegenome inin orderorder toto limitlimit sensitivitysensitivity..ØØ variousvarious molecularmolecular methodsmethods andand DNADNA

markersmarkers havehave beenbeen used,used, asas comparisonscomparisons ofofnucleotidenucleotide sequencessequences ofof mitochondrialmitochondrialNADHNADH dehydrogenasedehydrogenase 11 (nad(nad11)) andandcytochromecytochrome cc oxidaseoxidase subunitsubunit 11 (cox(cox11)) genesgenes

ØØ Recently,Recently, toto distinguishdistinguish betweenbetween FF.. hepaticahepaticaandand FF.. giganticagigantica,, aa simplesimple andand rapidrapid PCRPCR--restrictionrestriction fragmentfragment lengthlength polymorphismpolymorphism(RFLP)(RFLP) assay,assay, usingusing differentdifferent enzymesenzymes asas::

common restriction enzymes common restriction enzymes AvaAva II and II and DraDra IIII

restriction endonucleases restriction endonucleases HspHsp92 92 II and II and RcaRca II

internal transcribed spacer internal transcribed spacer 1 1 and and 2 2 (ITS(ITS11, ITS, ITS22))

Restriction fragment length polymorphism (RFLP) patterns of PCRproducts of liver flukes after digestion with AvaII and DraIIrestriction enzymes: Lanes M (left and right), 100 bp DNA sizerestriction enzymes: Lanes M (left and right), 100 bp DNA sizemarker; Lanes 1 and 2, 618-bp-long PCR products of Fasciolahepatica and F. gigantica, respectively; Lanes 3 and 5, PCRproducts of F. hepatica after digestion with AvaII and DraII,respectively; Lanes 4 and 6, PCR products of F. gigantica afterdigestion with AvaII and DraII, respectively.

FasciolagiganticaFasciolahepaticaRestrictionenzyme322, 269, 27529, 62, 27AvaII618529, 89DraII

ØØ Recently,Recently, PCRPCR isis usedused asas aa sensitivesensitive andandspecificspecific methodmethod forfor detectiondetection ofof FasciolaFasciola ininfieldfield--collectedcollected LymnaeaLymnaea spsp.. snailssnails.. AA primerprimerpairpair waswas designeddesigned toto amplifyamplify aa 405405 bpbpfragmentfragment ofof thethe cytochromecytochrome cc oxidaseoxidasesubunitsubunit 11 genegene ofof FF.. hepaticahepatica.. TheThe techniquetechniqueshowedshowed toto bebe effectiveeffective forfor detectingdetecting specificspecificshowedshowed toto bebe effectiveeffective forfor detectingdetecting specificspecificFF.. hepaticahepatica infectionsinfections ofof lowlow intensityintensity ((1010 pgpgofof genomicgenomic FF.. hepaticahepatica DNA)DNA) inin thetheintermediateintermediate host,host, andand hencehence itit couldcould bebeusedused toto studystudy thethe epidemiologicalepidemiological situationsituation

ØØ TheThe wormworm oror thethe eggegg inin itsits normalnormal habitathabitatmaymay bebe detecteddetected accidentallyaccidentally duringduring surgerysurgerydonedone forfor otherother diseasesdiseases inin thethe liverliver oror biliarybiliarytracttract..

ØØ AA strangestrange manifestationmanifestation ofof

(6) Surgical diagnosis

AA strangestrange manifestationmanifestation ofoffascioliasisfascioliasis isis thethe diseasedisease ininsitessites otherother thanthan thethe liverliver((ectopicectopic fascioliasisfascioliasis))ØØ TheThe juvenilejuvenile flukesflukes dodo notnot reachreach maturitymaturity inin

thesethese sitessites.. TheyThey produceproduce granulomatousgranulomatousnodulesnodules andand maymay bebe calcifiedcalcified

ØØ ExcisionExcision biopsybiopsy ofof ectopicectopic parasiticparasiticnodulesnodules isis thethe bestbest methodmethod forfordiagnosisdiagnosis whichwhich revealsreveals eosinophiliceosinophilicgranulomatousgranulomatous lesionslesions withwith eithereither focifociofof vasculitisvasculitis,, necrosisnecrosis oror immatureimmatureofof vasculitisvasculitis,, necrosisnecrosis oror immatureimmaturewormworm oror eggegg inin thethe centercenter.. LaparotomyLaparotomyoror endoscopicendoscopic surgerysurgery isis carriedcarried outout totodiagnosediagnose andand treattreat intraabdominalintraabdominalnodulesnodules

Macroscopic appearance of Fasciola hepatica granulomas on colon wall

Appearance of Fasciola hepatica egg in Fasciola hepatica granulomas

ØØ ChengCheng etet alal.. ((20072007)) illustratedillustrated anan unusualunusual sitesiteofof ectopicectopic fascioliasisfascioliasis whichwhich isis thethe ocularoculartissuetissue inin 66--yearsyears oldold childchild presentedpresented withwithfascialfascial swellingswelling andand monocularmonocular blindnessblindness..DuringDuring surgery,surgery, FasciolaFasciola withwith itsits cephaliccephalicconecone waswas demonstrateddemonstrated withinwithin thethe leftleftconecone waswas demonstrateddemonstrated withinwithin thethe leftleftcorneacornea..