Induction of high tolerance to artemisinin by sub-lethal administration: A new in vitro model of P

18 Pages·2017·5.77 MB·English

RESEARCHARTICLE
Induction of high tolerance to artemisinin by
sub-lethal administration: A new in vitro
model of P. falciparum
SerenaDeLucia1¤*,IoannisTsamesidis2,MariaCarminaPau3,KristinaR.Kesely4,
AntonellaPantaleo3,FrancescoTurrini1
1 DepartmentofOncology,UniversityofTurin,Turin,Italy,2 DepartmentofMedicine,SectionofInternal
Medicine,UniversityofVerona,Verona,Italy,3 DepartmentofBiomedicalSciences,UniversityofSassari,
Sassari,Italy,4 DepartmentofBiochemistry,PurdueUniversity,WestLafayette,UnitedStatesofAmerica
a1111111111 ¤ Currentaddress:DepartmentofClinicalandBiologicalSciences,UniversityofTurin,Turin,Italy
a1111111111 *[email protected]
a1111111111
a1111111111
a1111111111 Abstract
Artemisininresistanceisamajorthreattomalariacontrolefforts.Resistanceischaracter-
izedbyanincreaseinthePlasmodiumfalciparumparasiteclearancehalf-lifefollowing
treatmentwithartemisinin-basedcombinationtherapies(ACTs)andanincreaseintheper-
OPENACCESS
centageofsurvivingparasites.Theremarkablyshortbloodhalf-lifeofartemisininderivatives
Citation:DeLuciaS,TsamesidisI,PauMC,Kesely
maycontributetodrug-resistance,possiblythroughfactorsincludingsub-lethalplasmacon-
KR,PantaleoA,TurriniF(2018)Inductionofhigh
centrationsandinadequateexposure.Hereweselectedforanewstrainofartemisininresis-
tolerancetoartemisininbysub-lethal
administration:AnewinvitromodelofP. tantparasites,termedtheartemisininresistantstrain1(ARS1),bytreatingP.falciparum
falciparum.PLoSONE13(1):e0191084.https:// PaloAlto(PA)cultureswithsub-lethalconcentrationsofdihydroartemisinin(DHA).The
doi.org/10.1371/journal.pone.0191084
resistancephenotypewasmaintainedforover1yearthroughmonthlymaintenancetreat-
Editor:GeorgesSnounou,Universite´Pierreet mentswithlowdosesof2.5nMDHA.TherewasamoderateincreaseintheDHAIC in
50
MarieCurie,FRANCE
ARS1whencomparedwithparentalstrainPAafter72hofdrugexposure(from0.68nMto
Received:April12,2017 2nMDHA).Inaddition,ARS1survivedtreatmentphysiologicallyrelevantDHAconcentra-
Accepted:December18,2017 tions(700nM)observedinpatients.Furthermore,weconfirmedalackofcross-resistance
againstapanelofantimalarialscommonlyusedaspartnerdrugsinACTs.Finally,ARS1did
Published:January17,2018
notcontainPfk13propellerdomainmutationsassociatedwithARTresistanceintheGreater
Copyright:©2018DeLuciaetal.Thisisanopen
MekongRegion.Withastablegrowthrate,ARS1representsavaluabletoolforthedevelop-
accessarticledistributedunderthetermsofthe
CreativeCommonsAttributionLicense,which mentofnewantimalarialcompoundsandstudiestofurtherelucidatethemechanismsof
permitsunrestricteduse,distribution,and ARTresistance.
reproductioninanymedium,providedtheoriginal
authorandsourcearecredited.
DataAvailabilityStatement:Allrelevantdataare
withinthepaperanditsSupportingInformation
files.
Introduction
Funding:Thisstudywassupportedbyagiftfrom
ARAM-Onlus.Thefundershadnoroleinstudy Plasmodiumfalciparumisthemostlethalofthemalariaknowntoinfecthumans[1],and
design,datacollectionandanalysis,decisionto wasresponsiblefornearly212millionnewcasesand429.000deathsin2016[2].Artemisinin
publish,orpreparationofthemanuscript. derivatives(ART)representthemostwidelyusedanti-malarialdrugs.Derivedfromtheplant
Competinginterests:Theauthorshavedeclared Artemisiaannua,ARTcontainanendoperoxideringessentialforitspotentandrapidantima-
thatnocompetinginterestsexist. larialactivity[3,4].Althoughring-stageparasitesaremostsensitivetodrugtreatment,ART
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AnewinvitromodelofP.falciparum
areactiveagainstallasexualstagesofparasitedevelopment[5].Unfortunately,theuseofART
asmono-therapieshascontributedtotheincreaseandspreadofartemisininresistantpara-
sites,andremainsamajorthreatformalariacontroleffortsinSouthEastAsia[6].Although
artemisininderivativesarenowonlyusedincombinationwithslow-actingpartnerdrugsas
ACTstodeterdrug—resistance,however,resistancetopartnerdrugshasbeendetected[7,8],
furthernecessitatingthedevelopmentofnewandeffectiveantimalarials[9,10].
Artemisininresistanceisdefinedbyadelayinparasiteclearancefollowing3days(DPC3)
oftreatmentwithACTs,but,manytimes,parasitesbecomeresistanttothepartnerdrugin
additiontoART[11].Partnerdrugresistanceischaracterizedbyrecrudescenceoftheinfec-
tionafter28oreven42daysfollowingACTtreatment[12].Forthisreason,malariaparasite
areevaluated3daysand28daysfollowingtreatmentforalackofadequateclinicalandparasi-
tologicalresponseatday28(ACPR28)[13,14,15,16].ARTrelyontheirrapidconversionto
reactivefreeradicalsforpotentantimalarialactivity.However,theirveryshortclearancehalf-
life(~1h)combinedwithunderdosageofinfectedpatientsandincompletedrugtreatment
maycontributetotheoccurrenceandspreadofARTresistance[17,18,19].Exacerbatingthe
situation,patientsfrequentlyundergoincompletetreatment[20,21].ARTresistantparasites
mayhaveacquiredtheabilitytosurviveunderthesub-lethaldrugconcentrationsorinsuffi-
cientexposuretoartemisinin,similartothatseeninbacterialresistancetoantimicrobial
agents[22,23,24].
StableARTresistantstrainsofP.falciparum,havebeenobtainedfrominfectedpatients
whileothershavebeenmanipulatedtoinduceartemisininresistancebysubjectingparasitesto
increasingconcentrationsofDHAinvitro[22,25,26,27].
TheheterogeneityandnumberoffactorsthatcancontributetoARTresistancehavebeen
evidencedbytheamountofstudiesperformedinasimilarmannerinvitro[28,29].Interest-
ingly,theover-expressionofP.falciparummalariagenesrelatedtocellrepairmechanisms
havebeenidentifiedandnotonlysupportresearchdemonstratingARTgeneratefreeradicals
forantimalarialactivity,butalsotherapidresponseofparasitestoincreasingdrugpressureby
inducingaseriesofprotectiveandrepairmechanisms[30,31].
Laboratory-adaptedstrainsofP.falciparumcontainphenotypesuncharacteristictothose
foundinclinicalisolates,orparasiteresistancefoundinpatients[32].Whileallthesestrains
maybeARTresistant,notallartemisininresistancephenotypesarethesame.Inaddition,the
typemanipulationusedtoinduceartemisininresistanceinvitrocaninduceasimilarunchar-
acteristicphenotype.Forexample,alargenumberofdormantparasitesareinducedbyshort
treatmentswithhighDHAconcentrations,butevidenceofsimilarlydormantparasitesduring
infectionislacking[33].
Finally,ithasbeenreportedthatmutationsintheP.falciparumkelch-13propeller(Pfk13)
geneareassociatedwithARTresistantmalariaintheGreaterMekongSub-regionandin
China-Myanmar[34,35,36].Ithasbeensuggestedthisresistanceisduetothedysregulation
ofPhosphatidylinositol3-phospatemetabolism,whichiscriticalforparasitesurvival[37,38,
39].AlthoughACTshavebeenwidelyusedforoveradecade,noPfk13propellermutations
associatedwithARTresistancehavebeenreportedforinAfricanP.falciparumpopulations
[40,41].Theseresultsstronglysupporttheinvolvementofmultiplefactorswithdelayedpara-
siteclearanceafterACTtreatmentandtheimportanceofpreventingparasitetolerancetothe
partnerdrug.
Withthesefindingsinmind,wesetouttoselectanartemisininresistantstrainbyexposing
parasitestosub-lethalDHAconcentrationsandthenprogressivelyshorteningthetimeinter-
valsbetweendrugtreatments.Culturesweremonitored,takinggreatcaretopreventtheaccu-
mulationofdormantparasites.WenowpresentanovelP.falciparumartemisininresistant
strain(ARS1)thatpossessahightoleranceagainstsub-lethalDHAconcentrationsandthis
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AnewinvitromodelofP.falciparum
stablestrainwillhelpelucidateARTresistancemechanismsanddirecteffortstotestnewanti-
malarialcompounds.
Materialsandmethods
Plasmodiumfalciparumcultures
ThreeP.falciparumparasitesstrainswereusedinthisstudyandobtainedfromfieldisolate.
ThePaloAlto(PA)strainwasisolatedfromaUgandanpatientandisconsideredasareference
strainduetoitshighgeneticstability.PAhashistoricallybeenusedasastraintostudydrug
susceptibilityinP.falciparuminvitro[42].TheFCR3strainderivedfromGambianparasites
andisgeneticallyrelatedwithPAandithasbeenusedtogenerateachloroquine(CQ)and
pyrimethamine(PM)resistantstrain[22,42].TheHB3AparasitesstrainderivedfromHondu-
rasandhasbeenusedtoinducechloroquineresistance[43,22].
Plasmodiumfalciparum-infectedredbloodcell(I-RBC)culture
maintenance
P.falciparumstrainsPA,FCR3,HB3AandtheARS1strain,selectedinthisstudy(mycoplasma
free),weremaintainedinculturewithfreshredbloodcellsRBCs(Rh+)fromhealthyvolun-
teersofbothsexes.Humanbloodsampleswereusedonlytosustaintheparasitesinvitrocul-
tures.BloodsamplesusedtoperformthepresentstudywereobtainedfromBancadelsangue
C/OOspedaleMolinette,Torinowithwritteninformedconsent.Thisstudywasconducted
inaccordancewithGoodClinicalPracticeguidelinesandtheDeclarationofHelsinki.Noethi-
calapprovalhasbeenrequested.RBCswereseparatedfromplasmaandleukocytesbythree
washesinRPMI1640mediumcontaining25mMHEPES(R5886SigmaAldrich),andthen
resuspendedinRPMI1640+HEPESmediumsupplementedwith200mML-glutamine
(59202CSigmaAldrich),2mMglucoseandgentamicin(80mg/mL).Forcompletegrowth
medium,thiswasfurthersupplementedwith1%SAG-Msolution,(150mMNaCl,1.25mM
adenine,30mMglucose,145mMmannitol)andheat-inactivatedhumanserum.I-RBCcul-
turesweremaintainedat2–5%parasitemia(1%haematocrit)at37˚Cinanair/CO -athmo-
2
sphereof95/5%(vol/vol).Allassayswereperformedatthisparasitemiaandhematocritunless
otherwisedescribed.
CulturesweresynchronizedweeklybyPercollseparation[44]or5%sorbitolsolutiontreat-
ment[45].Thesynchronizationprocessdidnotinfluencethegenerationorphenotypeofthe
artemisininresistantstrain.ParasiteviabilityandparasitemiaweremonitoredbyDiff-Quick
stainedthinbloodsmearsandlightmicroscopy(CarlZeissStandardMicroscopeLamphouse
467230).Parasitemiawasdefinedasthenumberofparasites/numberofRBCscounted,fora
totalof5000RBCs.Twothinsmearsperconditionwerecounted3separatetimesbyeachof
threeoperators.
Selectionofartemisininresistantstrain1(ARS1)followingDHA
treatment
DHA(D7439Sigma-Aldrich)wassuspendedinsteriledimethylsulfoxide(DMSO)andsubse-
quentlydilutedinRPMI1640+HEPESpriortoI-RBCtreatment.
Forthefirstweek(D1–D7):i)synchronousculturesofPA,FCR3andHB3Aweresupple-
menteddailywiththreeconcentrationsofDHA(1.25,2.5and5nM)alongwithanuntreated
control.Forcultureswithapercentageofviableparasiteslowerthan1%,thedailytreatments
werestoppedandonlyre-appliedwithaparasitemiaofgreaterthan2.5%.ii)Beforeeverynew
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treatment,I-RBCcultureswerewashedonceinRPMI1640+HEPESandresuspendedin
freshgrowthmediumcontainingthesameconcentrationofDHA.
Forthenext7weeks(D8–D54):i)PA,FCR3andHB3Aweresynchronizedwith80%Per-
collevery7–8days,ii)treatedwiththeirrespectiveconcentrationofDHAonceaweek(the
dayfollowingPercollsynchronization),iii)thegrowthmediumwaschangedevery24h.Para-
sitemiawasdeterminedeverydayfromthestartofthestudy.Whencultureswerecompletely
devoidofviableparasitesforatleast7days(analysingtwothinsmearseveryday),thecultures
wereconsideredparasitefree.Fig1demonstratesparasitemorphologyfromD0toD54ofthe
parentalPAstrainandthesurvivingARS1strainwiththeaverageparasitemiareportedaspre-
viouslydescribedwithtwooperatorsandasapercentoftheuntreatedcontrol.Tomaintain
theARS1strainselectedfromPA:i)ARS1culturesweresynchronizedevery7dayswith80%
PercolltoisolatetrophozoitestageparasitesandtreatedwithSorbitol5%24hlatertoisolate
ringstageparasites;ii)culturesweretreatedwith2.5nMofDHAmonthlyatringstage24h
followingsorbitolsynchronization;iii)parasitecultureswereincubatedwithDHAfor48h
andthenreplacedwithfreshgrowthmediumwhichwaschangedevery48h;iv)thismonthly
DHAtreatmentwascontinuedforover1-yearperiod.
Analysisoflong-termtreatmentofstrainsPAandARS1withrepeated
DHAtreatments
PAandARS1weretreatedwith1.25and2.5nMDHA,respectivelyevery48hforatotalof
144hinordertoevaluatethePAandARS1behaviourassessedwithdifferentfrequencyDHA
pressure(Fig2)orwith2nMDHAevery12or24hfora72h.Parasitemiawasdeterminedas
previouslydescribed.
AnalysisofDHA’seffectonparasitematurationanddeath
PAandARS1ringstageculturesweretreatedwith0.6,1.25,2.5,5and10nMofDHAand
evaluatedat24and48hfollowingdrugaddition.TheeffectofDHAtreatmentonP.falcipa-
rummaturationanddeathwasevaluatedbydeterminingtheparasitemiaofeachmaturation
stages:ring,trophozoiteandschizont,andforintracellulardeadparasites(pyknotic)inboth
DHA-treatedanduntreatedcontrolcultures.Thepercentageofeachstageofliveparasiteand
thenumberofdeadparasitespresentinDHAtreatedcultureswereexpressedasthe%parasi-
temiarelativetotheuntreatedcultures.
Rings-stageSurvivalAssay(RSA0-3h)
TheinvitroRSA0-3hassaywasperformedusingthemethoddescribedbyWorldwideAntima-
larialResistanceNetwork(WWARN)(2013)andbyWitkowskietal.todemonstratethepres-
enceofreducedsensitivitytoDHAinthenewlyselectedARS1strain[46,47,48].
Briefly,the0–3hpostinvasion,ringstageparasiteswereexposedto700nMDHAfor6h.
Thecultureswerewashedtoremovethedrugandthenculturedwiththecompletemedium
foradditional66h.Theproportionofviableparasiteswascountedinthesecondgeneration
parasiteswithnormalmorphology.
AsdescribedbytheWHOguidelines,wecalculatedthesurvivalrate(%)andthegrowth
rate:i)thepercentageofsurvivalratewasdefinedastheDHA-exposedparasitemiaat72h/
non-exposedparasitemiaat72hx100;ii)thegrowthratewasdefinedasthenon-exposed
parasitemiaat72h/theinitialparasitemiaat0h.Everythinsmearwerecountedbymicroscopy
viaGiemsastainedevaluatingatotalof10000RBCs.Twothinsmearsperconditionwere
counted3separatetimesbyeachofthreeoperators.Valuesobtainedfor%ofsurvivalrateof
DHA-treatedareinterpretableiftheycorrespondtoagrowthrate(cid:21)1.5%.
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Fig1.ResponseofP.falciparumculturesPA,FCR3,HB3AandARS1toDHA.PA(A),FCR3(B),HB3A(C)andARS1(D)cultureswerefirsttreateddailywith
1.25,2.5and5nMfor1weekandthentreatedweeklyfor7weeks,foratotalof54days.Matureandimmatureparasiteswerecounted.Datawerenormalizedthe
parasitemiaofuntreatedculturesdeterminedbylightmicroscopyofGiemsa-stainedsmears.Mean+/-SDof5independentexperimentsisshown.
https://doi.org/10.1371/journal.pone.0191084.g001
IC ofP.falciparumstrainstreatedwithantimalarialdrugs
50
SynchronousringstageculturesofstrainsPA,ARS1,FCR3andHB3Aweretreatedwith
DHA,mefloquine(MQ),piperaquine(PPQ),amodiaquine(AQ)andlumefantrine(LMF)and
parasitemiacheckedevery24hfollowingdrugtreatment.Thescalarconcentrationsofeach
drugincluded0.6,1.25,2.5,5,10,20and40nMforDHAand5,10,25,50,75,100and200nM
forAQ,MQ,PPQandLMF.TodeterminetheIC ofeachdrug,culturesweretreatedwitha
50
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Fig2.SurvivalofARS1toincreasedfrequencyofDHAtreatmentsevery48h.PAandARS1wereanalysedwithout
treatment(A)andafterrepeatedtreatmentsevery48hwith1.25nM(B)or2.5nM(C)DHA,bycountingmatureand
immatureparasites.DatawerenormalizedtothecontrolparasitemiaandanalysedbylightmicroscopyofGiemsa-
stainedthinsmears.Themediumwaschangedevery48h.Mean+/-SDof5independentexperimentsisshown.
Significantdifferencesbetweenstrainsareindicatedby((cid:3))atp<0,05and((cid:3)(cid:3))atp<0,01.
https://doi.org/10.1371/journal.pone.0191084.g002
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single-pulseofdrugandincubatedfor48h[49,28,50].IC valuesweredeterminedusing
50
ICestimatorsoftwarebyschizontcounting(http://www.antimalarial-icestimator.net/
runregression1.2.htm)[51].Thehalf-lifewasconsideredrelevantwhentheR2valueofthe
sloperegressionlinewashigherthan0.8.
TreatmentswithDHAandMQcombined
PAandARS1culturesweretreatedwithDHAaloneorincombinationwithMQtodetermine
ifthereissynergismbetweenthetwodrugsandwhetherARS1issensitivetothistypeofACT.
CulturesweresynchronizedusingPercoll/5%sorbitoltreatmentandassayinitiatedata5%
parasitemiaand1%haematocrit.IC valuesdeterminedat24,48and72hfollowingdrug
50
treatment:DHA(0.6,1.25,2.5,5,10,20and40nM)+/-supplementedwith5or10nMMQ
wheredescribed.
P.falciparumDNAextraction
AsaltingoutmethodwasusedtoextractparasiteDNAfrom100μLofI-RBCsaccordingto
standardproceduresbyMilleretal.[52].Briefly,RBCswerelysedwithRedCellsLysisBuffer
(RCLB)solution(Tris-HCl10mM,MgCl 5mMandNaCl10mM,pH7.6)andthecellpellet
2
isolatedandincubatedat55˚Cfor20’withWhiteCellsLysisBuffer(WCLB)solution(Tris-
HCl10mM,EDTA10mMandNaCl50mM,pH7.6)inthepresenceofSDS(10%)andpro-
teinaseK(20mg/ml).Theadditionofasaturatedsaltsolution(NaCl6M)purifiedtheDNA,
whichwasthenprecipitatedfollowingtheadditionofisopropanol.Aftera70%ethanolwash,
thefinalDNApelletwasre-suspendedinsterileH Oandanalyzedforpuritybydetermining
2
theratioofabsorbanceat260and280nm.
Nested-PCRanalysisofstrainsPAandARS1cultures
SequencingthePfk13propellerdomain. TheP.falciparumk13propellerdomaingene
wasevaluatedforanymutationsbyusingnestedPCRtoamplifythegenepreviouslydescribed
byArieyetal.[32].AmplifiedPCRproductswerepurifiedusingExoI&FactAP(CarloErba)
andsequencedbyMacrogen,Inc.(Netherlands).Electroferogramswerevisualizedandana-
lyzedwithApoEsoftware,andalignmentswereperformedwithMuscle3.8softwareusingthe
k13sequenceofthe3D7clone(PF3D7_1343700)asthereferencestandard.
Genotypingofmsp1,msp2andglurpwithnested-PCRinPAandARS1strain. The
geneticcharacteristicsofP.falciparumpolymorphicgenes,suchastheMerozoiteSurfacepro-
teins1and2(msp1andmsp2)andtheGlutamate-richprotein(glurp)arecommonlyassessed
inmalariaendemicareastodiscriminaterecrudescencefromre-infectingparasitesalleles.
NestedPCRwasusedtoamplifythepolymorphicregionsofmsp1,msp2andglurpusingfam-
ily-specificprimers,previouslydescribedbySnounou[53].
Briefly,intheprimaryreaction,oligonucleotideprimerscorrespondedtoconserved
sequenceswithinmsp1(block2),msp2(block3)andglurpintheprimaryreaction.Inthe
nestedreaction,separateprimerpairswereusedthattargetedtherespectiveallelictypesof
msp1(K1,MAD20,andRO33)msp2(FC27andIC3D7)andglurpwereused[54].ThePCR
productswereseparatedon1.5%agaroseandvisualizedunderUVillumination.
Statisticalanalysis
Errorbarsindicatetheaverage±SD.Thestatisticalsignificanceisestimatedwithtwo-tailed
Student’st-testperformedwithMicrosoftExcelsoftware.
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Table1. IC50valuesofDHA,MQ,PPQ,AQ,LMFtreatedculturesofP.falciparumstrainsPA,ARS1,FCR3andHB3A.
StrainofP.falciparum Druginvitrotested IC50±SD(24h) IC50±SD(48h) IC50±SD(72h)
PA DHA 2.21±0.4nM 0.94±0.1nM 0.68±0.2nM
MQ 19.75±0.3nM 6.30±0.1nM 5.49±0.92nM
PPQ 20.93±1.4nM 19.19±1.9nM 16.20±1.1nM
AQ 21.03±1.4nM 15.80±1.8nM 14.05±2.1nM
LMF 22.64±0.2nM 18.50±2.4nM 17.12±0.8nM
ARS1 DHA 3.17±0.5nM 2.5±0.2nM 2.0±0.3nM
MQ 18.66±1.8nM 5.54±0.7nM 4.78±0.6nM
PPQ 19.90±1.7nM 11.66±0.8nM 8.65±0.3nM
AQ 19.46±0.6nM 14.45±1.4nM 14.2±0.8nM
LMF 21.29±1.3nM 17.55±2.4nM 16.99±1.52nM
FCR3 DHA 1.73±0.6nM 1.60±0.3nM N/D
HB3A DHA 2.14±0.3nM 2.27±0.05nM N/D
DHA,dihydroartemisinin;MQ,mefloquine;PPQ,piperaquine;AQ,amodiaquine;LMF,lumefantrine.
Allexperimentswereperformedatleastfivetimesindependently.Valuesareexpressedinpercentageofviableparasites±SD.
https://doi.org/10.1371/journal.pone.0191084.t001
Results
Selectionforanewartemisininresistantstrain
Wefirstperformedexperimentstoselectforartemisininresistantparasitesbyexperimentally
determiningthelowestconcentrationofDHAthatwouldenablecontinuouscultureofP.fal-
ciparumstrainsPA,FCR3andH3BAwhensubjectedtointermittentsub-lethaldrugexposure.
TheIC values(Table1)amongthethreestrainsrevealedslightvariationsinsensitivityto
50
DHAtreatment.TheIC valuesofDHAinHB3Aafter24and48h,2.14and2.27nM,respec-
50
tively,werehigherthanPAandFCR3andsupportedbyDingetal.andCuietal.[28,29].Var-
iationsinDHAsensitivityamongthemalariastrainscanbeattributedtothenatureofeach
individualstrainsuchastheirorigin,laboratoryadaptation,andlengthofpropagation[22].In
additionweobtainedIC valuesat24hinadditiontothe48hand72hduetothecharacteris-
50
ticshorthalf-lifeofDHAinpatientsandinculturesandtoevaluateitsfinalantimalarialeffi-
cacy[55].TheseIC valuesprovidedabaselinethatwasusedtoevaluateparasitesensitivityto
50
DHAduringtheselectionprocess.
Fortheselectionprocess,parasitecultureswereexposedto1.25,2.5and5nMDHAevery
24hfor1week,thenwashedandresuspendedindruglessmediumforatmost7weeks,which
includedasingleexposurefor24heveryweek(seeMaterialsandmethods).Overtime,PA
culturesgainedtheabilitytogrowataconsistentrateduringtheweeklytreatmentswith1.25
nMDHA(Fig1A).Weneverobservedthesamedrug-adaptationwithFCR3orHB3A(Fig1B
and1C).Aslightgrowthrecoverywasobservedduringweek2inHB3Abutparasitemia
declinedsharplyandflatlinedbyday12(Fig1C).ThehigherDHAconcentrations(2.5and5
nM)weretoxictotheparasitesandfailedtoselectforanydrugresistantparasites.Wemoni-
toredtheparasitemiaofPA,FCR3,andHB3Asolongas:i)therewereviableparasitesgrowing
incultureandii)thegrowthratewasconsistentorincreasedoveraperiodtime.Theevalua-
tionofFCR3andHB3Awereterminatedprematurelyduetothecompletelackofviable
parasites22and38daysfollowingDHAtreatment,respectively.PAwasmonitoredforthe
durationofthe8-weekstudy.AfterdailyapplicationofDHAduringthefirstweek,PAcultures
alonecontainedviableparasitesandtheculturesappearedtobedesensitizedtoDHAand,
thus,wererenamedtoartemisininresistantstrain1(ARS1).ARS1wasevaluatedforatotalof
54daysafterwhichthegrowthratestabilized(thestabilizationoftheculturestartedon32nd
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AnewinvitromodelofP.falciparum
day)andparasitesconsistentlydisplayedlowsusceptibilitytothesameDHAconcentration
usedduringtheselectionperiod.At5nM,ARS1wasstillcapableofmaintainingasurvival
rateof20%,corroboratingourinitialfindings.ItappearedthattheactualselectionforARS1
wasmadeafterthefirstweekofdailyDHAtreatmentsandthefollowing7weeksofweekly
1.25and2.5nMDHAtreatmentsservedtomaintainthisnewstrainwithdecreasedsensitivity
toDHA(Fig1D).Similarly,a40%increaseinIC wasobservedinARS1whencompared
50
withvaluesfromparentalstrainPA,48hfollowingexposuretoDHA(Table1).
Akeyresponseofresistantparasitepopulationstosub-lethaldrugconcentrationswasto
recoverfollowingtreatment,evidencedbyanincreaseinparasitemia.Recoverytimesarespe-
cifictoeachstrainusedtotheantimalarialmechanism,andso,wereducedthetimeinterval
betweenDHAtreatmentsfromweeklytoevery48h(Fig2).GrowthcurvesofARS1andPA
arecomparableintheabsenceofDHA(Fig2A).Followingtheadditionof1.25nM(Fig2B)
and2.5nM(Fig2C)DHAtreatedevery48h,onlyARS1parasitemiaremainedincreasedby
400%and80%,respectively,fromtheinitialvalue.Ontheotherhand,PAculturesdidnotsur-
vivesimilarDHAtreatmentsandparasitemiaprogressivelydecreased.Thesefindingssuggest
thatdamageinflictedonARS1isreversiblesincetheparasiteswereabletoproliferateunder
theseDHAconcentrations.
WhilemonitoringparasiterecoveryduringthepreviousDHAtreatments,thestagesyn-
chronicityofARS1decreasedcomparedtoPA.TobetterunderstandtheeffectofDHAon
parasitematurationanddeath,ring-stagecultureswereexposedtoincreasingconcentrations
for48h(Fig3).Asindicatedbytherelativepercentageofrings,trophozoites,schizontsand
deadparasites,ahighnumberofparasitessurvivedinARS1atconcentrationsupto10nMby
24h,butdecreasedtolevelsobservedinPAby48h.AsignificantamountofARS1parasites
survivedatlowerDHAconcentrationscomparedwithPA.Untreatedcultureswereatthe
expectedringstage.ThepresenceoftrophozoitesandschizontsinARS1signifiedparasite
maturationwasnegativelyaffectedandslowerthanuntreatedcultures.Overall,thepercentage
ofimmatureparasiteswashigherinARS1thaninPAatboth24h(Fig3Aand3B)and48h
(Fig3Cand3D).It’spossiblethattheDHAtreatedARS1parasitesenteredastateofquies-
cenceattheringstageortheirdevelopmentisdelayedafter24hwhichcouldconferthearte-
misinintolerance.
Thissetofexperimentsdemonstratestheabilitytoselectforanartemisininresistantstrain
fromaninitiallysensitivetoDHAbyusingsub-lethalconcentrations.Theresultssuggestthat
resistantparasitesmaybeselectedforduringthefirstdivisioncyclesthatfollowsub-lethal
damageexertedbyDHA.
CharacterizationofthenewlygeneratedARS1strain
WenextsoughttoevaluateARS1resiliencetocontinuousdrugpressure,sowereplenished
cultureswith2nMDHAevery12or24h.Replacementevery12hwasnearlylethaltoARS1,
whichappearedtorequirealongerperiodoftimeforparasiterecoverybetweendrugtreat-
ments.ThedecreaseinparasitemiaofARS1paralleledthatofPA(Fig4A).Replacementof
DHAevery24hreflectsdrugtreatmentscheduleinpatients.ARS1survivedthistypeoftreat-
ment,whileitwaslethaltoPA,killingnearly100%ofparasitesafter3daysoftreatment(Fig
4B).Onthecontrary,ARS1hadagrowthratesimilartothatofuntreatedARS1cultures.
Tomaintainadrugresistancephenotypeinvitro,itiscriticaltoknowthemaximumtime
intervalthatcanbeallowedbetweendrugtreatments.Thishelpstoevaluateastrain’sstability
ofdrugresistance.Over1-yearperiod,wetreatedARS1withDHAconcentrationsupto20
nMtodeterminetheminimumconcentrationandmaximumperiodoftimeARS1parasites
maintaintheartemisininresistantphenotype.
PLOSONE|https://doi.org/10.1371/journal.pone.0191084 January17,2018 9/18
AnewinvitromodelofP.falciparum
Fig3.EffectofPAandARS1onparasitematurationfollowingDHAtreatment.Thepercentagesofrings,trophozoites,schizontsanddeadparasiteswere
determinedaftertreatmentofringstageparasitizedculturesofthePA(A,C)andARS1(B,D)strainsexposedto0.6,1.25,2.5,5and10nMofDHAfor48h.
Parasitemiawasmeasuredatboth24h(A)(B)and48h(C)(D)aftertreatmentvialightmicroscopyofGiemsa-stainedthinsmears.Datawerenormalizedtothe
untreatedcontrolparasitemia.ThedifferencesofimmatureparasitesstageofARS1comparedtoPAareindicatedby:(a)p<0.05at24h,(b)p<0.05at48h.
https://doi.org/10.1371/journal.pone.0191084.g003
After2–3monthswithoutDHApressure,ARS1lostitsresistantphenotype.Parasitescon-
tinuetocarrythepreviouslycharacterizedphenotypewithatleast1.25nMDHAadministered
monthly.Itwasundermonthlytreatmentswith2.5nMDHAthattheresistancephenotype
wasconservedforover1year.Repeatedfreezingandthawingofculturesusingthestandard
protocolsthatemployhighconcentrationsofglyceroldidnotaffectthisresistanceeither.
TheRings-stageSurvivalAssay(RSA0-3h)isacommonassaytoassessaparasitetolerance
toDHAorotherantimalarialdrug.Briefly,parasitecultureswereexposedtoasinglepulse
of700nMDHAfor6husingyoungring-stageparasites0-3hpost-invasionafterculture
PLOSONE|https://doi.org/10.1371/journal.pone.0191084 January17,2018 10/18