RESEARCHARTICLE Radiological evolution of porcine neurocysticercosis after combined antiparasitic treatment with praziquantel and albendazole CarlaCangalaya1,2*,JavierA.Bustos3,JuanCalcina4,AnaVargas-Calla4,JavierMamani5, DiegoSuarez6,GianfrancoArroyo3,ArmandoE.Gonzalez4,JuanChacaltana7, CristinaGuerra-Giraldez1,8,SiddharthaMahanty1,9,TheodoreE.Nash1,9,He´ctor a1111111111 H.Garc´ıa3,8,fortheCysticercosisWorkingGroupinPeru¶ a1111111111 1 LaboratoriodeInmunopatolog´ıaenNeurocisticercosis,FacultaddeCienciasyFilosof´ıa,Universidad a1111111111 PeruanaCayetanoHeredia,Lima,Peru,2 FacultaddeMedicinaHumana,UniversidadNacionalMayorde a1111111111 SanMarcos,Lima,Peru,3 UnidaddeCisticercosis,InstitutoNacionaldeCienciasNeurolo´gicas,Lima,Peru, a1111111111 4 FacultaddeMedicinaVeterinaria,UniversidadNacionalMayordeSanMarcos,Lima,Peru,5 Facultadde MedicinaVeterinariayZootecnia,UniversidadPeruanaCayetanoHeredia,Lima,Peru,6 Facultadde Medicina,UniversidadPeruanaCayetanoHeredia,Lima,Peru,7 DepartamentodeDiagno´sticopor ima´genes,InstitutoNacionaldeCienciasNeurolo´gicas,Lima,Peru,8 DepartamentodeCienciasCelularesy Moleculares,FacultaddeCienciasyFilosof´ıa,UniversidadPeruanaCayetanoHeredia,Lima,Peru, 9 LaboratoryofParasiticDiseases,NationalInstituteofAllergyandInfectiousDiseases,NationalInstitutesof OPENACCESS Health,Bethesda,Maryland,UnitedStatesofAmerica Citation:CangalayaC,BustosJA,CalcinaJ, ¶MembershipoftheCysticercosisWorkingGroupinPeruisprovidedintheAcknowledgments. Vargas-CallaA,MamaniJ,SuarezD,etal.(2017) *[email protected] Radiologicalevolutionofporcine neurocysticercosisaftercombinedantiparasitic treatmentwithpraziquantelandalbendazole.PLoS NeglTropDis11(6):e0005624.https://doi.org/ Abstract 10.1371/journal.pntd.0005624 Editor:CharlesD.Mackenzie,MichiganState University,UNITEDSTATES Background Received:November10,2016 Theonsetofanthelmintictreatmentofneurocysticercosis(NCC)provokesanacuteimmune responseofthehost,whichinhumancasesisassociatedwithexacerbationofneurological Accepted:May4,2017 symptoms.Thisinflammationcanoccuratthefirstdaysoftherapy.So,changesinthebrain Published:June2,2017 cystsappearancemaybedetectedbymedicalimaging.Weevaluatedradiologicalchanges Copyright:Thisisanopenaccessarticle,freeofall intheappearanceofbraincysts(enhancementandsize)ondaystwoandfiveafterthe copyright,andmaybefreelyreproduced, onsetofantiparasitictreatmentusingnaturallyinfectedpigsasamodelforhumanNCC. distributed,transmitted,modified,builtupon,or otherwiseusedbyanyoneforanylawfulpurpose. TheworkismadeavailableundertheCreative Methodsandresults CommonsCC0publicdomaindedication. ContrastT1-weightedmagneticresonanceimagingwithgadoliniumwasperformedbefore DataAvailabilityStatement:Allrelevantdataare andafterantiparasitictreatment.EightNCC-infectedpigsweretreatedwithpraziquantel withinthepaperanditsSupportingInformation files. plusalbendazoleandeuthanizedtwo(n=4)andfive(n=4)daysaftertreatment;another groupoffourinfectedpigsservedasuntreatedcontrols.Foreachlesion,gadolinium Funding:Thisworkwassupportedinpartbythe IntramuralResearchProgramoftheNational enhancementintensity(GEI)andcystvolumeweremeasuredatbaselineandafterantipar- InstituteofAllergyandInfectiousDiseases,andby asitictreatment.VolumeandGEIquantificationratios(post/pre-treatmentmeasures)were FIC-NIHtraininggrantTW001140,NIH usedtoappraisetheeffectoftreatment.Cystsfromuntreatedpigsshowedlittlevariations 1R01AI116456,andBecaAnualdeMedicina betweentheirbasalandposttreatmentmeasures.Atdays2and5thereweresignificant “FranciscoTejadaySem´ıramisRea´tegui”2014 UniversidadPeruanaCayetanoHeredia.HHGis increasesinGEIratiocomparedwiththeuntreatedgroup(1.32and1.47vs1.01,p=0.021 PLOSNeglectedTropicalDiseases|https://doi.org/10.1371/journal.pntd.0005624 June2,2017 1/14 Radiologicalevolutionofneurocysticercosisaftertreatmentwithpraziquantelandalbendazole supportedbyaWellcomeTrustSenior andp=0.021).Cystvolumeratiosweresignificantlyloweratdays2and5comparedwith InternationalResearchFellowshipinPublicHealth theuntreatedgroup(0.60and0.22vs0.95,p=0.04andp=0.02).Cystswithlowercystvol- andTropicalMedicine.Thefundershadnorolein umeratiosshowedmoremarkedpost-treatmentinflammation,lossofvesicularfluidand studydesign,datacollectionandanalysis,decision topublish,orpreparationofthemanuscript. cystwallwrinkling. Competinginterests:Theauthorshavedeclared thatnocompetinginterestsexist. Conclusion/Significance Asignificantanddrasticreductionofcystsizeandincreasedpericysticenhancementoccur intheinitialdaysafterantiparasitictreatmentasaneffectofacuteperilesionalimmune response.Thesesignificantchangesshowedthatearlyanthelminticefficacy(daytwo)can bedetectedusingmagneticresonanceimaging. Authorsummary Neurocysticercosis(NCC)isafrequentparasiticinfectionofthehumanbrainandthe mostcommoncauseofadultonsetepilepsyindevelopingcountries.Acuteinflammatory responseinNCCplaysanimportantroleinthepathogenesisofsymptomsbyanthelmini- tictherapies.TheanthelminticrecommendedtherapyforNCChasdrawbacksasthe exacerbationofinflammationarounddegeneratingcystsprovokestheappearanceof symptomsatthefirstdaysoftreatment.Radiologicalchangesintheappearanceofcysts usuallyareseenaftermonthsoftherapy.Toevaluateifsignificantradiologicalchanges (enhancementandsize)occurinthefirstdaysoftherapy,weusedaporcineNCCmodel andmagneticresonanceimaging(MRI)withcontrastsolution.Themajorradiological changesobservedaftertreatmentwithalbendazoleandpraziquantelwereanincreasein enhancementandthesignificantreductionincystsizebyday2andmoreevidentonday 5.Cystswithgreaterchangesalsoexperiencedexacerbatedinflammation,lossofvesicular fluidandwrinklingofthecystwall.Theseresultsshowanearlytherapeuticeffectandthe possibleutilityofrepeatMRIimagingwithinafewdaysafterstartingtreatment.Finally, thesefindingscontributetoourunderstandingofthetreatmentinducedearlyexacerba- tionofsymptoms. Introduction Neurocysticercosis(NCC)isaneurologicalparasiticdiseasecausedbytheinfectionofthe brainbythelarvalstageofTaenia solium[1].NCCrepresentsaseriousandpersistingpublic healthproblembecauseitisthemostfrequentcauseoflate-onsetseizuresindevelopingcoun- tries[1,2]. Treatmentwithanthelminticdrugssuchaspraziquanteland/oralbendazolehasbeenasso- ciatedwithincreasedseverityofsymptomswithinthefirstdaysoftherapy[3–7].Eventhough praziquantelandalbendazolehavedifferentmechanismsofaction[8,9],bothdrugscausethe destructionofcystsandsubsequentreleaseofantigens,triggeringthehostimmuneresponse [7,10–13].UsingtheporcineNCCmodelandtheantihelminticdrugpraziquantel,thisacute post-treatmentinflammatoryresponsewasassociatedwithpericysticinflammation[14] accompaniedbyanincreaseofvascularpermeability,pro-inflammatoryandregulatorycyto- kineprofiles[15]duringthesecondandfifthday.Usingthesamemodel,radiologicalchanges intheappearanceofbraincystshavebeenreportedaftertwoweeksofpraziquanteltreatment PLOSNeglectedTropicalDiseases|https://doi.org/10.1371/journal.pntd.0005624 June2,2017 2/14 Radiologicalevolutionofneurocysticercosisaftertreatmentwithpraziquantelandalbendazole [16–18].Similarly,theuseofalbendazoleintheporcinemodelresultedinanincreaseofpro- inflammatorycytokines[14]. Medicalimaginghasbeenausefultoolinthediagnosisandmedicalfollow-upofNCC patients[7].Cystappearance,size,perilesionalenhancementandedemaareimagingcriteria todeterminetheradiologicalresolutionofNCCaftertreatment[19].Theearliestradiological changesrelatedtothesizeandappearancesofbraincystsafterconventionalanthelmintic treatmenthasbeenreportedduringthefirstweekoftreatmentinhumans[20,21]andinpigs aftertwoweeks[16–18].However,theradiologicalevolutionofbraincystsduringthefirst daysoftreatment,whenperilesionalinflammationestablishesandsymptomsincreasein treatedpatients,hasbeenscarcelyexplored. Inthepresentstudy,weevaluatedtheearlyradiologicalchangesonMRIfollowingthe onsetofantiparasitictreatment(daystwoandfive)inpigsnaturallyinfectedwithT.soliumas amodelforhumanNCCandconfirmedtheradiologicalfindingswithanex-vivohistopatho- logicalexamination. Materialsandmethods Studydesignandanimals AtotaloftwelvepigsnaturallyinfectedwithTaenia soliumcysticercosiswereobtainedin endemicvillages,transportedtoourfacilitiesinLima,andrandomlydividedinthreegroups, controloruntreated,PZQ+ABZ2dandPZQ+ABZ5d,asfollows:Fourpigsremained untreatedasacontrolgroupand8pigsweretreatedwiththesamecombinationofanthelmin- ticdrugsandsacrificedattwo(n=4)andfive(n=4)daysaftertreatment.Thetreatmentcon- sistedofcombinedtherapywithpraziquantel(Helmiben,Farmindustria,Peru)givenforonly thefirstdayat75mg/kg/day,dividedintothreedosesof25mg/kgadministeredeverytwo hours[10],andalbendazole(Zentel,GlaxoSmithKline,Peru)givendailyuntilsacrificeat15 mg/kg/day[22]. Interventions Allpigshadpreandpost-contrastMRIbeforetreatment(Pre-treatmentMRI)andontheday ofsacrifice(Post-treatmentMRI).Twohoursbeforesacrifice,anEvansbluesolutionwas infusedaspreviouslyreported[15].Forallinterventions,pigswereanesthetizedwithanintra- muscularinjectionofamixtureofketamine(Ket-A-10050mg/kg,AgrovetMarketSA,Peru) andxylazine(Dormi-Xyl2mg/kg,AgrovetMarketSA,Peru)[15]. Imageacquisitionprotocol. Pre-andpost-treatmentMRIswereperformedona3-Tesla MRIscanner(PhilipsAchieva,Best,TheNetherlands).Sequencesincludedcoronal,sagittal andaxialTFE(Turbofieldecho)T1-weightedgradient-echoimagesunderconditionsofrepe- titiontime(TR)=7miliseconds(ms),echotime(TE)=4ms,flipangle=8˚,pixelbandwidth= 270kHz,sectionthickness=0.5–4mm,matrix=256–480pixels,beforeandafterinjectionofa contrastsolutionofgadoliniumdiethylenetriaminopentaaceticacid(Gd-DTPA,0.1mmol/kg) throughanearIVcatheter. Collectionofspecimens Aftereuthanasia,thepigbrainswereplacedondryiceslabsandcutin1-cmsections.Cysts withpericysticcapsuleswerecollectedfrombothhemispheresforhistopathologyandRNA studies.Specimensfromtherighthemispherewerefixedin10%neutralbufferedformalin, embeddedinparaffinandthensectionedcoronallyat4μmthickness.Conventionalhematox- ylin-eosinwasperformedoneveryslideandtwosectionswereexaminedwithconventional PLOSNeglectedTropicalDiseases|https://doi.org/10.1371/journal.pntd.0005624 June2,2017 3/14 Radiologicalevolutionofneurocysticercosisaftertreatmentwithpraziquantelandalbendazole lightmicroscopy.Microphotographsweretakenat15XmagnificationwithaCarlZeissstereo- scopewithAxioVisionsoftwaretoobtainasinglelargeimage(“cystmap”)[22]. Radiologicalmeasures Gadoliniumenhancementintensity(GEI). Quantitativemeasurementsofgadolinium enhancementintensity(GEI)wereobtainedfrompre-andpost-treatmentscanimages,indi- vidually.Theopenaccessimage-processingFIJIprogram(ImageJ,http://imagej.nih.gov/ij/) wasusedtoadjustimagebrightnessandcontrasttobetterdefineanddelineateindividualperi- cysticlesionareas(selectionoftheregionofinterest).Thegrayvaluesineachpixelwithin theseselectionswerenormalizedaspercentagesofacontinuousscalefrom0(black)to256 (brightest)andanaverageGEIvaluefromallpixelswithintheselectionofeachimage/slice wascalculated.ThetheoreticalmaximumvalueforGEIwas100%.Finally,usingsoftwareR version3.2.2,theaverageGEIfromallsectionsliceswasobtainedasasinglevalueforeach cyst[22].Thesenumericalvalueswererangedbetween0and100.Inaddition,eachcysthad twovaluesofGEIfrompre-andpost-treatmentscanimages(preGEIandpostGEI,respec- tively).Additionally,aratiobetweenpre(preGEI)andposttreatmentGEI(postGEI)wascal- culatedtomeasureGEIincreaseaftertreatmentforeachcyst(postGEI/preGEI). Cystvolume. Individualcystvolumeswerecalculatedusingcoronalscanimagesbasedon sphericalcap([(1/6)(cid:3)π(cid:3)height(cid:3)(3(cid:3)area2+height2)])andsegment([(1/6)(cid:3)π(cid:3)height(cid:3)(3(cid:3)(area /π) 1 +3(cid:3)(area /π)+height2)])formulaswhencystshadthreeormoreslicerepresentations(S1Fig). 2 ForcystsimagedinonlyoneortwoMRIslices,theellipsoidvolumeformula(4/3(cid:3)π(cid:3)Longaxis ofsagittal(cid:3)axial(cid:3)coronal)wasused.(DetailsofthesecalculationsareprovidedinS1Fig).Finally, wecalculatedtheratiobetweenpre-andpost-treatmentcystvolumestoevaluateindividualcyst volumereductionaftertreatment(postCystVolume/preCystVolume). Ex-vivoexamination Inflammatoryscore–composite(ISC). Theinflammationaroundthecyst(capsule)was measuredusingasemiquantitativehistologicalscore[14,23,24],whichisdescribedina graphicalmannerinotherstudies[14].Usingthissystem,inflammatorystages(IS)arecatego- rizedasIS1(alayerofcollagenwithscarceornoimmunecells),IS2(athickerlayerofcollagen withanincreasednumberofnon-organizedimmunecells),IS3(atypicalgranulomatousreac- tionwithabundantorganizedimmunecellsdistributedinlayers[anepithelioid-richcelllayer nexttothecystwall,andincreaseofeosinophils]containingafewmultinucleatedgiantcells [Langerhanscells])orIS4(withIS3featuresplusadditionalabundanteosinophilsdistributed inalayeradjacenttothecystwallandabundantmultinucleatedgiantcellsaddedtoaseverely damagedparasitestructure).DifferentISsectorsmayappeararoundthecystperimeter,sowe calculatedthepercentageofthecystforeachISusingapanoramicimageofeachcyst.The compositescoreisaweightedmeanofthepercentageofeachinflammatorystagealongthe perimeterofacyst,multipliedbythenumericalvalueoftheIS.[ISC=(%ofIS1(cid:3)1)+(%of IS2(cid:3)2)+(%ofIS3(cid:3)3)+(%ofIS4(cid:3)4)].ThereforetheISCresultsinanumericalvariablewith valuesbetween100and400. Cystdamagescore–composite(CDSC). SimilartoISC,weusedapreviouslydescribed scoringsystemtomeasurethecystdamage[14].Cystdamagewascategorizedintofourstages, CD0(nodamage),CD1(fewalterationsofthecystvesicularwall[tegumentandsubtegument layers]withoverallpreservationofthecystarchitecture),CD2(moderatealterationswithloss ofmicrotrichesormicrovilliontheoutertegumentallayer,distendedtegumentandsubtegu- ment,andhyperchromophilicanddilatedcanalicularsystemsinthesubtegumentlayer),or CD3(severealterationsincludinglossofarchitectureofthevesicularwallandlossofdefinition PLOSNeglectedTropicalDiseases|https://doi.org/10.1371/journal.pntd.0005624 June2,2017 4/14 Radiologicalevolutionofneurocysticercosisaftertreatmentwithpraziquantelandalbendazole intheinternalregionofthecyst)[14].Thecompositedamagescoreisaweightedmeanofthe extensionsofeachcystdamagestagealongtheperimeterofacyst,multipliedbythenumerical valueoftheCD[CDSC=(%ofCD0(cid:3)0)+(%ofCD1(cid:3)1)+(%ofCD2(cid:3)2)+(%ofCD3(cid:3)3)]. ValuesoftheCDSCvarybetween0and300[14]. Statisticalanalysis Pre-andpost-treatmentGEI,pre-andpost-treatmentcystvolume,cystvolumeratio,GEI ratio,InflammatoryScoreComposite(ISC)andcystdamagescorecomposite(CDSC)wereall continuousparameters.Treatmentsgroupswereusedasacategoricalvariable(untreated, beentreatedat2dand5d).MannWhitneytestwasusedtocomparepre-GEIandpreCystvol- umebetweenthedifferenttreatmentgroups.Pre-posttreatmentdifferencesforGEIandcyst volumewereanalyzedbytheWilcoxontestineachtreatmentgroup,individually.Toevaluate ifthemeanchangeinGEIandcystvolumefrompretopost-treatmentmeasuresdifferedin thethreegroups,weusedageneralizedestimatingequation(GEE)analysis.Toverifythose post-treatmentdifferences(cystvolumeandGEI)trulyresultfromtreatmentratherthanfrom left-overeffectsof(usuallyrandom)pre-testdifferencesbetweengroups,weusedananalysis ofcovariance(ANCOVA)withpre-treatmentmeasuresascovariates.Finally,weusedthe Mann-WhitneyUtesttocompareratios(changesbetweenpre-andpost-treatmentmeasures) ofGEIandcystvolumebetweentreatmentgroups.Sinceratioanalysisresultswerehighlycor- relatedwithunstandardizedgroupanalyses,weusedratiosforthecorrelationswithhistopa- thology.Spearmancorrelationwasusedtoassesstherelationbetweeneachradiological(GEI andcystvolume)andhistopathological(ISCandCDSC)parameters.Allstatisticalanalyses wereperformedusingsoftwareRprogramforWindows,version3.2.2.Graphswereper- formedusingtheggplot2package[25].Valuesofpunder0.05wereconsideredtobestatisti- callysignificant. Ethicalstatement ThestudywasconductedinaccordancewiththeNationalInstitutesofHealth/AALCguide- lines,andwasreviewedandapprovedbytheInstitutionalEthicsCommitteeforAnimalUseat UniversidadPeruanaCayetanoHeredia(assurancenumberA5146-0). Results Experimentalresults Thestudyanimalsweresevenmaleandfivefemalepigs.Theirweightrangewasvariable (mean:69.8kg;range:15–120kg).Atotalof328braincystswereobtainedfromthe12pigs. Theparasitecystburdenineachpigbrainwasalsovariable(mean:27.3;range:1–152) (Table1)[22]. Gadoliniumenhancementintensity(GEI) AnumberofestimatesofGEIshowedincreasesaroundcystsintreatedpigscomparedtocysts incontroluntreatedpigs.Atbaseline(beforeantiparasitictreatment),cystsinpigsfromthe ControlandPZQ+ABZ2dgroupshadhigherGEIthancystsfromthePZQ+ABZ5dgroup (30.22and28.32vs.24.1,p<0.05).Post-treatmentGEIvalueswerehigherinbothtreated groupscomparedwithcontrolpigs(PZQ+ABZ2d:36.04andPZQ+ABZ5d:35.8vs.Control: 33.31,p<0.001).Whencomparingpre-andpost-treatmentGEIineachgroup,therewere marginaldifferencesincystsfromcontrolanimals(30.22vs.33.31,p=0.048),whileGEI aroundcystsintreatedgroupsincreasedmarkedly(PZQ+ABZ2d:28.32vs.36.04,p<0.001 PLOSNeglectedTropicalDiseases|https://doi.org/10.1371/journal.pntd.0005624 June2,2017 5/14 Radiologicalevolutionofneurocysticercosisaftertreatmentwithpraziquantelandalbendazole Table1. Generalcharacteristicsofstudyanimals. Pigcharacteristics Treatmentconditions Control PZQ+ABZ2d PZQ+ABZ5d Numberofpigs(male/female) 4(2/2) 4(3/1) 4(2/2) Weight(kg) 120,111,78,42 70,45,45,15 117,90,79,25 Totalbraincysts(bypig) 73(1,11,16,45) 63(10,11,13,29) 192(4,13,23,152) https://doi.org/10.1371/journal.pntd.0005624.t001 andPZQ+ABZ5d:24.1vs.35.8,p<0.001)(S1Table).GEEanalysisconfirmedthattheeffect oftreatmentinincreasingtheenhancementaroundcystschangedfrombasaltodays2and5 (RCforinteractiontermbetweenpre-postGEImeasuresandgroups:4.996,<0.001)(S2 Table). Additionally,afteradjustingforpre-treatmentdifferences,GEIincreasedsignificantlyin bothtreatedgroups(PZQ+ABZ2d:7.324,p-value=0.001andPZQ+ABZ5d:9.442,p- value<0.001)comparedwiththecontrolgroup(S2Table). Ratioanalysiswasalsousedtoassesstheincreasesinenhancementbetweengroups(across time).IndividualcystGEIratio(post-/pre-treatmentGEI)demonstratedasimilareffect (meanratioswere1.01forcystsofcontrolpigs,1.32forcystsofpigsinPZQ+ABZ2dgroup, and1.47inPZQ+ABZ5dgroup;p=0.021betweengroupsandp=0.387comparingboth treatmentgroups)(Table2,Fig1). Cystvolume OnbaselineMRI(beforeantiparasitictreatment),cystsfromthecontrolandPZQ+ABZ5d groupshadlargervolumes(106.16mm3and114.18mm3,respectively)thanthosefromPZQ +ABZ2dpigs(74.56mm3)(p<0.05).Onpost-treatmentMRI,cystsfromPZQ+ABZ2dand PZQ+ABZ5dgroupshadlowercystvolumethancystsfromthecontrolgroup(48.64mm3vs. 97.92mm3,<0.001and24.36mm3vs.97.92mm3,p=0.03).Cystvolumealsodecreasedinthe 5-dtreatedcystscomparedtothe2-dtreatedcysts(48.64mm3vs.24.36mm3,p<0.001)(S1 Table). SimilartoGEI,pre-andpost-treatmentcystvolumesincontrolpigsweresimilar(106.16 vs.97.92,p=0.045),whilepost-treatmentcystvolumesweresignificantlysmallerintreated animals(PZQ+ABZ2d:74.56vs.48.64,p<0.001andPZQ+ABZ5d:114.18vs.24.36,p<0.001) (S1Table).Adjustmentforpre-treatmentmeasuresinANCOVAconfirmedthatcystsfrom bothtreatedgroupshadsmallervolumesthancystsfromthecontrolgroup(PZQ+ABZ2d: -62.117,p-value=0.014andPZQ+ABZ5d:-95.032,p-value<0.001).Similartoenhancement, GEEanalysisconfirmedthattheeffectoftreatmentoncystvolumewasmoremarkedatday5 (RCforinteractiontermbetweenpre-postmeasuresandgroups:-48.201,<0.001)(S2Table). Table2. Changesincontrastenhancementaroundcystsinpigbrainsasaneffectofpraziquantelplusalbendazoletreatment. Valuesrepresent mediansandrangesofratioGEIvaluesofeachbraincystsineachstratum. Imagingresults Treatmentconditions *p-value *p-valueCvsPA5d *p-valuePA2dvsPA5d CvsPA2d Control PZQ+ABZ(PA) Median PA2d PA5d (range) Median(range) Median(range) GEIratio(postGEI/preGEI) 1.01(0.81–1.23) 1.32(0.97–2.0) 1.47(0.89–2.32) 0.021 0.021 0.387 *MannWhitneytest(adjustedperpig) https://doi.org/10.1371/journal.pntd.0005624.t002 PLOSNeglectedTropicalDiseases|https://doi.org/10.1371/journal.pntd.0005624 June2,2017 6/14 Radiologicalevolutionofneurocysticercosisaftertreatmentwithpraziquantelandalbendazole Fig1.Imagingfindingsbytreatmentconditions.Dispersiongraphsofpost-treatment/baselineGEIRatio(A,left)andCystVolumeRatio(B, right)bytreatmentgroups,respectively.OrangelinesshowCI forthegroup.Asterisksindicatestatisticallysignificantdifferencesinratios 95 betweengroupsforMann-Whitneytest:*:p<0.05,**:p<0.01,***:p<0.005,****:p<0.001. https://doi.org/10.1371/journal.pntd.0005624.g001 Asimilareffectwasalsoseenwhenindividualcystvolumeratios(post-/pre-treatment) werecomparedbetweengroups.Cystvolumeratiowaslower(morereduction)incystsfrom bothtreatedgroupsthaninthosefromthecontrolgroup(0.60forABZ+PZQ2dand0.22for ABZ+PZQ5dvs.0.95forcontrols,<0.05)(Fig1),demonstratingcystvolumereductionafter treatment.However,cystsfrompigsinPZQ+ABZ5dgrouphadsimilarvolumereduction thandidcystsinthePZQ+ABZ2dgroup(0.22vs.0.60,p=0.248)(Table3). Table3. Cystvolumevariationaseffectofpraziquantelplusalbendazoltreatment. Valuesrepresentmediansandrangesofpost-/pre-treatmentratio ofvolumevaluesofbraincystsineachstratum. Imagingresults Treatmentconditions *p-valueCvsPA *p-valueCvsPA *p-valuePA2dvs 2d 5d PA5d Control PZQ+ABZ(PA) Median 2d 5d (Range) Median Median (Range) (Range) Cystvolumeratio(postVolume/ 0.95 0.60 0.22 0.04 0.021 0.248 preVolume) (0.72–1.23) (0.19–1.19) (0.10–1.13) *MannWhitneytest(adjustedperpig) https://doi.org/10.1371/journal.pntd.0005624.t003 PLOSNeglectedTropicalDiseases|https://doi.org/10.1371/journal.pntd.0005624 June2,2017 7/14 Radiologicalevolutionofneurocysticercosisaftertreatmentwithpraziquantelandalbendazole Fig2.Radiologicalevolutionafterantiparasitictreatmentwithpraziquantelandalbendazole. https://doi.org/10.1371/journal.pntd.0005624.g002 Furtheranalysisdemonstratedasignificantnegativerelationshipbetweencystvolumeratio withGEIratioafter5daysoftreatment(r=-0.412,p<0.001)(S3Table,Fig2)suggestingthat cystswithmoreenhancement(GEI)experiencegreaterreductioninvolume. Imagingfindingsandhistology Toconfirmtheradiologicalchanges,weperformedanex-vivoexaminationtomeasure inflammationandthecystdamageusingtheISC(inflammatoryscore-composite)andthe CDSC(cystdamagescore-composite),thenwecorrelatedthosehistologicalparameterswith GEIandcystvolumeratios(radiologicalparameters). Cystsfromrightbrainhemispheres(n=165)wereselectedforhistopathologicalstudies.Of these,only105cystshadacompletecyststructureandcapsuleandwerethereforeevaluable. BothtreatedgroupshadhigherISCandCDSCthanthecontrolgroup(p<0.001,MannWhit- neytest).Bothscoreswerehigherat5dcomparedto2d,buttherewerenosignificantdiffer- encesinthesevariablesbetweenbothtreatedgroups(ISC:352vs304,p=0.364;CDSC:388vs 336,p=0.405forCDSC)(S4Table). HigherISCsweresignificantandpositivelyassociatedwithGEIratio(r=0.002,p=0.028), meaningthatcystswithhigherincreasesinenhancementhavemorepost-treatmentpericystic inflammation.However,therewasnosignificantcorrelationbetweenGEIandCDSC(r= -0.001,p=0.286)(S3Table). Interestingly,therewasasignificantnegativerelationshipbetweencystvolumeratio(post-/ pre-treatmentmeasure)andpost-treatmentinflammation(ISC)atday5(RC=-0.002,p=0.004), PLOSNeglectedTropicalDiseases|https://doi.org/10.1371/journal.pntd.0005624 June2,2017 8/14 Radiologicalevolutionofneurocysticercosisaftertreatmentwithpraziquantelandalbendazole Fig3.Histologicalchangesandcystvolumevariationatday5.AandBshowstwocystsfrompigsof PZQ+ABZ5dgroup.Arepresentativecystwithhighvolumeratio(morecystvolumereductiononscan images)isseeninA,whereasacystwithlowvolumeratioisshowninB.BlackdottedlinesindicateIS4(higher stageofinflammation),reddottedlinesdelineateCD3(higherstageofcystdamage),s:scolexandcw:cyst wall.CystsinAandChadthesameproportionofCD3(100%),butAhadmoreofIS4(100%)thanC.Thus, bothcystshavethesameCDSC(300)butdifferentISC(A:400andC:360).BandDarethemagnifications withintheredcirclesinAandC.BandDshowCD3,wherethecystwallisswollenwithlossofitsarchitecture andpenetratedbyeosinophils(bars=50um). https://doi.org/10.1371/journal.pntd.0005624.g003 suggestingthatcystswithincreasedinflammationshowedincreasedreductioninvolume.Slides ofcystswithhighvolumeratio(higherreductionofcystvolume)showedlossofvesiculararea andexcesscystwallfoldinguponthemselvesorwrinklingaccompaniedbygranulomatousreac- tion(Fig3Aand3C).Inbothcases,eosinophilshaveinvadedtheparasite’swallasaneffectof treatment(Fig3Band3D).Thiseosinophilicinvasionhasbeenobservedbeforeatpointsofhigh inflammation[26]anditisademonstrationofanacuteresponse. However,therewasnosignificantrelationshipbetweenvolumeratioandcystdamage (CDSC)inanygroup(S3Table). Discussion CombinedtreatmentofparenchymalNCCwithpraziquantelandalbendazoledestroysbrain cystsinhumansandpigs[1,10],whichisassociatedwithabetterclinicalevolutionincasesof humanNCC[10].However,afteranthelmintictreatmenthumansarenotusuallyreimaged untilsixor12monthsaftertreatmentsoearlyeffectsarenotmeasured. Despitetheefficacyofcombinedtreatment,inhumanstherapycausesanexacerbationof symptoms,usuallyseizures,duetoacuteinflammatoryresponsetodegeneratingordying cysts[27].Toassessearlyradiologicalchanges,weexaminedMRIparametersofenhancement PLOSNeglectedTropicalDiseases|https://doi.org/10.1371/journal.pntd.0005624 June2,2017 9/14 Radiologicalevolutionofneurocysticercosisaftertreatmentwithpraziquantelandalbendazole andcystsizeandconfirmedthosefindingswithanex-vivohistopathology(tissue-basedsemi- quantitativeestimatesofinflammation,andcystdamage)innaturallyT.solium-infectedpigs treatedwithalbendazoleandpraziquantelat2and5dayspostinitiationoftreatment,com- paredtountreatedcontrolanimals. EnhancementhasbeenassociatedtothedisruptionoftheBBBinporcineNCC[22,28,29] asithappensinotherdiseasessuchasmultiplesclerosis[30–32],gliomas,metastasesand abscesses[33].EarlierstudiesinNCCemployingcontrast-enhancedcomputedtomography (CT)inpigs[16–18]describedtheappearanceofpericysticenhancementtwoweeksfollowing praziquanteltreatment.Inhumans,anthelmintictreatmentalsoexacerbatesgadolinium(Gd) enhancementduringthefirstdaysoftherapy[7],causingachangefromaninitialringpatternof enhancementtoadiscpattern,asseenusingGdT1-MRI[34].Theseresultsarecoherentwith thepost-treatmentincreaseofenhancementreportedinthisstudy.Weobservedthattheeffect oftreatmentonenhancementincreaseswithtimealreadyonday2andisfurtherincreasedon day5.Also,therewasapositivecorrelationbetweenincreaseofenhancementandinflammation. AsenhancementisassociatedwithBBBdisruption,thefollowingorparallelprocessthatoccurs istheextravasationofimmunecellsintotheinjuredareaandtheincreaseoftheinflammatory response.Thisagreeswithpreviousstudieswherepro-inflammatorycytokines[14]andeosino- philswheremoreabundantinpericystictissueswheretheBBBhadbeendisrupted[26]. Unexpectedly,wefoundthatcystvolumewasreducedveryearlyaftertheonsetofantipara- sitictreatment.Reductionsincystvolumewereevidentinbothtreatedgroupsondaytwoand weremorepronouncedfivedaysaftertreatment,whenthemedianofcystvolumelosswas almost78%([1–0.22](cid:3)100;prevs.post-treatment).Changesinthesizeofbraincystsinpigs hadpreviouslybeenreportedaftertwoweeksofpraziquanteltreatment[16];howeverour findingssuggestthatsizablechangesinthecystsizeoccuralreadybytheseconddayoftreat- ment.Theseresultsmighthavebeenmoremarkedbecauseweusedcombinedtherapy,and areconsistentwithearlycystsizedecreaseobservedonday3[21]andafteroneweek[20]of antiparasitictreatmentinhumans.Thereductionofthesizeoftheparasitelikelyresultsfrom treatment-inducedcystdamageandassociatedincreasedpermeabilityofthecystmembrane, withaconsequentincreaseindensityofthecystcontentsduetotheinfluxofhostalbumin, proteincoagulation,andlossofwater[35]. Thereductionofthesizeofthecystwasalsoaccompaniedwithincreasedenhancement andinflammation.Apreviousstudyfromourgroupreportedthatenhancementwasassoci- atedwithgranulomaformation[22];inthisstudywefoundsimilarresultsbutadditionally accompaniedwithcystreduction.However,therewasnoassociationwithcystdamagescore (extensionofthedamage).Apossibleexplanationcouldbethatthecombinedtreatmentdam- agesthescolexfirst,beforedamageishistologicallynoticeableandextendedatthecystwall level.Onlyafterwardswouldthecystshrinkandreleasefluidthroughthemostheavilydam- agedregionsofitswall.Similarly,apreviousstudyconcludedthatthescolexistheprimarytar- getanditsdissolutioncarriesthecompleteresolutionofthecyst[36].Asforcystswithlittleor noenhancementwithanegligiblechangeinsize,theywouldrepresentthosecystsinpatients thatdonotrespondtodrugtherapy,althoughoursuggestionisvalidonlyuptofivedays. Despitethesesignificantfindings,ourstudyhassomelimitations.Weusedasmallnumber ofanimalsandtheparasiteloadperpigbrainwasveryvariable,makingitdifficulttocompare groups.However,weusedthreestatisticalanalysestohandlebaselinedifferencestotrulymea- suretheeffectoftreatment.ThevariablethicknessofMRIscansintroducedsomenoiseinthe measurementsofenhancementandvolume;nevertheless,cystvolumeandenhancementwere significantlydifferentinthetreatedgroups.Minordrawbacksincludeuseofonlyonehemi- sphereforhistopathologicalassessments;however,differencesincystloadbetweenhemi- sphereswerenotdiscernable[37].Also,weusedonlytworepresentativeslidestoassessthe PLOSNeglectedTropicalDiseases|https://doi.org/10.1371/journal.pntd.0005624 June2,2017 10/14