Radiological evolution of porcine neurocysticercosis after combined antiparasitic treatment with

14 Pages·2017·7.2 MB·English

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
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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
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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
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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
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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
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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)
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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)
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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),
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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
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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
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