Genome scanning of Amazonian Plasmodium falciparum shows subtelomeric instability and ...

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Research
Genome scanning of Amazonian Plasmodium
falciparum shows subtelomeric instability
and clindamycin-resistant parasites
Neekesh V. Dharia,1 David Plouffe,2 Selina E.R. Bopp,1 Gonzalo E. Gonza´lez-Pa´ez,1
Carmen Lucas,3 Carola Salas,3 Valeria Soberon,3 Badry Bursulaya,2 Tadeusz J. Kochel,3
David J. Bacon,3 and Elizabeth A. Winzeler1,2,4
1DepartmentofCellBiology,ICND202,TheScrippsResearchInstitute,LaJolla,California92037,USA;2GenomicsInstitute
oftheNovartisResearchFoundation,SanDiego,California92121,USA;3ParasitologyProgram,NavalMedicalResearch
CenterDetachment,LimaAPOAA34031-3800,Peru
Here,wefullycharacterizethegenomesof14PlasmodiumfalciparumpatientisolatestakenrecentlyfromtheIquitosregion
usinggenomescanning,amicroarray-basedtechniquethatdelineatesthemajorityofsingle-basechanges,indels,andcopy
numbervariantsdistinguishingthecodingregionsoftwoclones.WeshowthattheparasitepopulationinthePeruvian
Amazonbearsalimitednumberofgenotypesandlowrecombinationfrequencies.Despitetheessentiallyclonalnatureof
someisolates,weseehighfrequenciesofmutationsinsubtelomerichighlyvariablegenesandinternalvargenes,indicating
mutationsarisingduringself-matingormitoticreplication.Thedataalsorevealthatoneortwomeiosesseparatedifferent
isolates,showingthatP.falciparumclonesisolatedfromdifferentindividualsindefinedgeographicalregionscouldbeuseful
inlinkageanalysesorquantitativetraitlocusstudies.Throughpairwisecomparisonsofdifferentisolateswediscovered
pointmutationsintheapicoplastgenomethatareclosetoknownmutationsthatconferclindamycinresistanceinother
species,butwhichwerehithertounknowninmalariaparasites.Subsequentdrugsensitivitytestingrevealedover100-fold
increaseofclindamycinEC instrainsharboringoneofthesemutations.Thisevidenceofclindamycin-resistantparasites
50
intheAmazonsuggeststhatashiftshouldbemadeinhealthpolicyawayfromquinine+clindamycintherapyformalaria
inpregnantwomenandinfants,andthatthedevelopmentofnewlincosamideantibioticsformalariashouldberecon-
sidered.
[Supplemental material is available online at http://www.genome.org. The microarray data from this study have been
submittedtotheNCBIGeneExpressionOmnibus(http://www.ncbi.nlm.nih.gov/geo)underaccessionno.GSE22861and
arealsoathttp://www.scripps.edu/cb/winzeler/resources/pf_peru.]
The World Health Organization (WHO) campaign to eradicate Thefactthatthegenesinvolvedinartemisininresistancearenot
malariainthe1950sand1960swasinitiallylargelysuccessfulin knownisduetoavarietyofproblems,includingthefactthatin
decreasingtheburdenofmalaria.Drugfailureeventuallyledtoa vivo resistance has not been replicated in vitro (Dondorp et al.
resurgenceinthenumberofmalariacasesinthe1990sandcaused 2009).Additionally,theassociationofphenotypeswithgenotypes
vastnumbersofdeathsthatcouldhavebeenavoidedthrougha in P. falciparum is hampered by the difficulties in performing
betterappreciationoftheprevalenceofdrug-resistantmalariaand complementationstudiesduetoextremelylowtransfectioneffi-
moreinformedchoicesoffirst-linedrugs(Greenwoodetal.2008). cienciesandthefactthatlaboratorycrossesofdrug-sensitiveand
Whilemalariadeathsarenowlikelytodecline,primarilybecause drug-resistant P. falciparum cannot be easily performed. Thus, it
of the introduction of artemisinin-based combination therapy tookmorethan40yrbetweentheidentificationofchloroquine
(ACT)aswellasincreasedinsecticidesprayingandtheuseofbed resistanceinthefield(Harinasutaetal.1965)andconfirmation
nets(Greenwoodetal.2008),thismayonlybetemporary.Indeed, thatresistanceisduetomutationsinthechloroquineresistance
therehasbeenageneralincreaseintheparasiteclearancetimesin transporter(pfcrt,MAL7P1.27)(Wellemsetal.1990;Fidocketal.
ACT-treated Plasmodium falciparum malaria cases from near the 2000;Sidhuetal.2002).Whiletherecombinantprogenyfromone
Thai–Cambodianborder,suggestingthatcasenumbersmaysoon of the three extant crosses (Walliker et al. 1987; Wellems et al.
beginincreasing(Dondorpetal.2009). 1990;Haytonetal.2008)havemostfamouslybeenusedtomap
Remarkably,althoughartemisininisusedontensofmillions chloroquineresistance(Wellemsetal.1990),theyhavebeenused
of individualsannually, we have little idea abouthow it acts or to map loci contributing to a wide variety of phenotypes that
whichgenescontributetoresistance,confoundingthecommun- distinguishparentalclones.Forexample,theyhavealreadybeen
ity’s ability to monitor the spread of resistance using molecular scoredforavarietyofdifferentphenotypesthatarerelatedtodrug
markersandtodeploynewtherapies(EastmanandFidock2009). sensitivity, including antifolate sensitivity (Wang et al. 2004b),
quininesensitivity(Ferdigetal.2004),expressionlevels(Gonzales
etal.2008),andplasmodialsurfaceionchannels(Alkhaliletal.
4Correspondingauthor. 2009),buttheycouldbescoredforanyphenotypethatquanti-
[email protected];fax[phone].
tatively distinguishes the parental strains Dd2 and HB3, such
Article published online before print. Article and publication date are at
http://www.genome.org/cgi/doi/10.1101/gr.105163.110. aspropensitytomutateinthelaboratory(Rathodetal.1997).
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Genome scanning of Amazonian P. falciparum
However, there area limitednumberof phenotypes that distin- withinformedconsentfromtheregionsurroundingIquitos,Peru
guishthesetwolaboratorystrainsthatwerederivedfrompatients during the peak malaria season (February through July) of 2006
40 yr ago. While more crosses would provide valuable data for (Supplemental File1).The isolatesweretakenintoshort-termin
manyresearchersinterestedinparasitegenetics,thereareethical vitrocultureforperiodsofweeksandsubjectedtogenotypingof
considerationsassociatedwithusingprimatesinresearch.Anal- known resistance genes by PCR. As indicated by increased sulfa-
ternativetocreatingnewrecombinantprogenyistofindexisting doxine-pyrimethaminesensitivityinPeru(Durandetal.2007),we
recombinantisolatesthatarosefromrecentmeiosesoccurringin detectedwild-typepfdhps(dihydropteroatesynthetase,PF08_0095)
humans. Such parasites might be identified by performing full- andsingleandtriplemutantalleles(NCNIandICNL,respectively)
genomeanalysesonparasitesfromalimitedgeographicalareaand ofpfdhfr-ts(bifunctionaldihydrofolatereductase-thymidylatesyn-
could provide the malaria community with an unprecedented thase,PFD0830w)byconventionalgenotyping,aswellastheK76T
numberofparasitesdifferinginavarietyofphenotypesforusein mutationthatconfersresistancetochloroquineresistancemuta-
linkageorquantitativetraitlocus(QTL)studies. tion(Sidhuetal.2002).Altogether,therewerefourgenotypesfor
Oneattractivegroupofparasitesforfullgenomeinvestigation pfmdr1(multidrugresistanceprotein,PFE1150w),threeforpfdhps,
isfromthePeruvianAmazon.Duetothelowtransmissionratesit threeforpfdhfr-ts,andtwoforpfcrt,suggestingfourparentalhap-
isexpectedthatparasitesisolatedfromanindividualwillbefrom lotypesinthePeruvianpopulation.
asinglecloneinfection.Inaddition,malariawaseradicatedinthe
1960sinthisregionbutre-emergedwithepidemicsintheearly
Genecopynumberanalysisrevealsonlywidespread
1990s (Aramburu Guarda et al. 1999; Branch et al. 2005), sug-
subtelomericvariants
gesting that the genomes might contain signatures of selective
sweeps(Woottonetal.2002;Roperetal.2004).Atfirst,malariain Because drug resistance can also be conferred by copy number
thisregionwastreatedwithchloroquine(first-line),sulfadoxine- variations(CNVs),theisolateswerenextexaminedbymicroarray.
pyrimethamine (second-line), and quinine with clindamycin or GenomicDNAfrom14isolateswasanalyzedonhigh-densityfull-
tetracycline (third-line) (Aramburu Guarda et al. 1999), but the genometilingmicroarrays(Table1;Dhariaetal.2009).First,10–15mg
emergenceofresistanceresultedin widespreadchloroquine and ofwholegenomicDNAfromparasitesgrowninleukocyte-depleted
antifolate treatment failure (Durand et al. 2007). Today, malaria redcellsfromeachPeruvianisolatewasfragmentedwithDNaseI,
remainshypoendemicwithlowlevelsofseasonaltransmissionof end-labeled, and hybridized to a tiling microarray that covers
P. falciparum and P. vivax parasites in the region surrounding ;90%ofcodingregions,whichcomprise53%ofthegenomeand
Iquitos,Peru(Roshanravanetal.2003).Previousstudiesofpara- 60%ofnoncodingregions.Thearraycontains4.8million,mostly
sitesintheregiondescribeonlyoneortwoindependenthaplo- 25-mer, single-stranded probes that are perfect matches to the
types for important drug-resistance genes, suggesting a limited reference3D7genomeandarespaced,onaverage,2–3ntaparton
numberof foundersfor this population (Baconet al. 2009) and alternatingstrands.Onlyprobesthatmaptoasinglelocationin
suggestthatwemightfindrecombinantprogenyinthisregion. thegenomewereusedfortheseanalyses.Althoughtherearefewer
Inthisstudy,weperformedgenomescanningon14P.falci- probestorepetitiveregions,eachnucleotideintheP.falciparum
parumpatientisolatesfromalimitedgeographicalregion.Weshow codinggenomegenerallycanbeprobednineto13timesonthe
thattheparasitepopulationinthePeruvianAmazonisveryclosely microarray.
related,withcombinationsofonlytwotofourdifferentgenotypes The advantage of using microarrays is that they can easily
fordrugresistancegenes,suggestingatmostfourparentalhaplo- detect copy number variations and deletions. Recent reports in-
types. Furthermore, some parasites taken from different patients dicateCNVscanbeawayformalariaparasitestoprovidedosage
whopresentedwithsymptomswereessentiallyclonesofonean- compensationforaproteinwhoseactivityiseitherinhibitedbya
other,whileotherswererecentmeioticsiblingsthatcouldbeuseful drug or whose expression otherwise increases resistance (Kidgell
inlinkagestudiesoreQTLanalyses.Unexpectedly,genomescan- etal.2006;Sidhuetal.2006;Nairetal.2008).Wethereforefirst
ningalsorevealeduncharacterizedmutationsintheapicoplast23S testedtoseewhetherthePeruvianstrainswouldalsoharborCNVs
rRNAthatdistinguishedsomePeruvianstrainsfromthereference by examining the genome-wide hybridization patterns. As de-
strain,3D7.Becauseoneofthesemutationshadbeenpreviously scribedpreviously(Kidgelletal.2006;Dhariaetal.2009),wecal-
associatedwithlincosamideantibioticresistanceinthechloroplast culatedthelog ratiosfortheaverageintensityoftheprobescor-
2
andmanybacterial species(VesterandDouthwaite 2001),sensi- responding to each gene in the genome versus a 3D7 reference
tivitytestingwasperformedrevealingthattheparasitesharboring hybridization and systematically lookedin allisolates for sets of
themutationhadindeedbecomeresistanttoclindamycin,adrug morethanthreeconsecutivegeneswithincreasedlog ratiosabove
2
usedincombinationwithquininetotreatpregnantwomenand 0.5. We did not see large-scale amplifications surrounding the
infantsformalariainPeru.Thesearethefirstdocumentedcasesof pfmdr1locus,whichhavepreviouslybeenobservedinlaboratory
resistancetothisclassofdrugsinmalariaandsuggestthattheuseof strains(Kidgelletal.2006).Thiswasexpectedastherewerepoint
lincosamidedrugsintreatingmalariashouldbereconsidered. mutationsinpfmdr1,andwhileamplificationofwild-typepfmdr1is
associatedwithmefloquineresistance(Priceetal.2004),therehave
beennoreportsofamplificationofmutantpfmdr1.Surprisingly,we
Results
didnotseeamplificationaroundthepfgch1(GTPcyclohydrolaseI,
PFL1155w)locusinresponsetoantifolateusedespitethehistorical
Initialgenotypingshowsalimitednumberofparental
useofthistreatmentinPeru(Kidgelletal.2006;Nairetal.2008).
haplotypes
Thismayberelatedtothere-emergenceofantifolate-sensitiveal-
Aninitialmotivationforconductingthisstudywastoexaminefield lelesofpfdhfr-tsandpfdhps,oritmayberelatedtothelackofthe
isolatestodeterminewhetherthesametypesofgenomicchanges quadruplepointmutationpfdhfr-tsinPeru(Baconetal.2009).Al-
typicallyfoundinlaboratorystrainsofP.falciparumwouldbedis- though single-gene events may have been missed, we only saw
covered.Tothisend,patientisolatesofP.falciparumwereobtained evidenceofwidespreadmultigeneCNVsinthePeruvianisolates
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Dharia et al.
Table1. Patientisolatesandgenotypes
ID Date Healthcarecenter pfdhfr-ts pfdhps pfmdr1 pfcrt api23S
IQE-2924 Feb-06 BellavistaNanay NCNI AKA GFSDD CVMNTH Wildtype
IQE-2980 Feb-06 SanJuan NCNI AKA DFCDD SVMNTH Wildtype
IQE-3296 Mar-06 SanJuan NCNI AKA DFCDD SVMNTH Wildtype
IQE-3416 Mar-06 HospApoyoIquitos NCNI AKA GFSDD CVMNTH Wildtype
IQE-3435 Mar-06 MoronaCocha NCNI AKA DFCDD CVMNTH Wildtype
IQE-3466 Mar-06 MoronaCocha NCNI AKA GFSDD CVMNTH Wildtype
IQE-3502 Mar-06 MoronaCocha ICNL AKA GFSDD CVMNTH A2058C
IQE-3648 Apr-06 ZungaroCocha ICNL AKA GFSDD SVMNTH Wildtype
IQE-3785 May-06 SanJaun ICNL GKG DFCDY SVMNTH A2058C
IQE-3848 May-06 SanJuan ICNL GKG DFCDY SVMNTH A2058C
IQE-3866 May-06 SanAntonio NCNI AKA DFCDD SVMNTH Wildtype
IQE-4006 Jun-06 BellavistaNanay ICNL GEG DFCDD SVMNTH A2612T
IQE-4015 Jun-06 SanAntonio NCNI* GEG DFCDY SVMNTH A2612T
IQE-4081 Jun-06 TupacAmaru ICNL GKG GFSDD SVMNTH A2058C
GenomicDNAfrom14patient-derivedP.falciparumisolateswasappliedtothemicroarrayfromthroughoutthe2006malariousseasonfromvarious
healthcarecenterssurroundingIquitos,Peru.Standardgenotypingwasusedtodeterminethedrugresistanceallelesforpfdhfr-ts(N51I,C59R,S108T/N,
I164L,*=C50R),pfdhps(A437G,K540E,A581G),pfmdr1(N86Y,D142G,Y184F,S1034C,N1042D,D1246Y),andpfcrt(C72S,V73,M74I,N75E,K76T,
H97Q).Theapicoplast23S(api23S)rRNAgenotypewasdeterminedbymicroarrayanalysiscombinedwithPCRanddirectsequencing.Mutationsare
underscored.
in subtelomeric regions like the deletions described below (Sup- (PF11_0344), merozoite surface protein 1 (PFI1475w), thrombo-
plementalFile2),indicatingthattheyappeartobelesscommon spondin-related anonymous protein (PF13_0201), and CelTOS
infieldisolatesthaninlaboratorystrains. (PFL0800c), allranked amongstthe top10% mostvariableofall
genes in the genome including subtelomeric genes, highlighting
the difficultyinfindingantigensthatwillprovideuniversalpro-
Deletionsindiagnosticgenesmayconfound
tection(Winzeler2008).
P.falciparumdiagnoses
The MOID algorithm was also used as previously described to
Microarrayanalysisdemonstrateslimiteddiversity
identify regions that were likely to be deleted in the Peruvian
strains(Dhariaetal.2009).Wefounddeletedregionscovering20– Our next goal was to determine whether or not there might be
25kbinsubtelomericregions,includingtheapparentdeletionof haplotypesinlinkagedisequilibriuminthepopulationthatmight
important diagnostic loci like histidine-rich proteins II and III revealnoveldrugresistanceloci.Wethusfirstperformedpairwise
(HRP2,MAL7P1.231andHRP3,MAL13P1.480,respectively)(Sup- analysisonthepatientisolatesbyfirstcomputationallyidentifying
plementalFile2;SupplementalFig.S1;Wongsrichanalaietal.2007; allpolymorphismsthatdistinguishedeachisolateofthepair.We
Martin et al. 2009). Because many malaria diagnostic tests that thenplotted100-kbblocksthatappearedtosharethesamegeno-
distinguishbetweenP.falciparumandP.vivaxarebasedonimmune typeasafunctionofgenomeposition.(Fig.1A–I).Unexpectedly,
recognition between commercial antibodies and these proteins pairwisecomparisonsofisolatehybridizationintensitiesrevealed
(Wongsrichanalaietal.2007),itislikelythatfalse-negativeread- thatmanyisolatessharedthesamegenotypeforlargeportionsof
ingscouldbeobtained.ThesedatasuggestthattheuseofHRP2-or chromosomesorwerevirtually‘‘clones’’ofoneanother.Thelow
HRP3-baseddiagnostictestsinthisregionshouldbereconsidered. endemicity, low rates of multiple infections, and the spread of
‘‘clonal’’ parasite genotypes suggest high rates of self-mating as
previouslydescribedinVenezula(Urdanetaetal.2001).However,
Genepolymorphismfrequenciesrevealedexpectedlevels
with‘‘clonal’’genotypesisolatedoveraperiodofmonthstherewas
ofvariationinthepopulation
agreatamountofdiversityinsubtelomericregions,includingvar,
Themicroarraydesignusedherecanalsobeusedtodetectsingle rifin,andstevorgenes,andinternalvargeneclusterssuggestinghigh
nucleotidepolymorphisms(SNPs).Wepreviouslyusedthisdesign levels of subtelomeric mutations (Fig. 1D,E) introduced by self-
todetectover90%ofSNPsprobedbythemicroarraythatdistin- mating ormitoticrecombination thatmay be influencedbyim-
guisheddifferentstrainsofP.falciparumwitha10%false-positive mune selection given that antigenic genes often reside in these
rate(Dhariaetal.2009).Inordertogloballydetectpolymorphism regions.Cloningandsequencingofamemberofthevargenefamily
frequenciesfordifferentgenefamilies,weranourpolymorphism (PFD1015c;SupplementalFig.S2)revealeddifferentsequencepat-
detection algorithm for each isolate with the 3D7 reference hy- ternsbetween‘‘clonal’’isolates,thusconfirmingtheexpecteddif-
bridization (Supplemental File 3). We saw an expected high fre- ferences. While the lack of diversity could be due to array in-
quencyofpolymorphismsingenesannotated‘‘antigenicvariation’’ sensitivity,especiallyasothercomparativegenomehybridization
(GO:0020033),with60.2%averagepolymorphicprobefrequency approacheshaveshownthatonlyafractionofSNPs(12%–24%)are
acrossall14isolatesandalowfrequencyofpolymorphismsingenes typicallydetectedusingarrayscomplementarytotheP.falciparum
annotated‘‘metabolicprocess’’(GO:0008152),with2.6%average genome(Tanetal.2009),thisseemsunlikely.First,Tanetal.(2009)
polymorphicprobefrequencyacrossall14isolates.Thisdistribu- also showed that sensitivity is inversely proportional to probe
tionissimilartofrequenciesdetectedintheanalysisofavarietyof length,andthusitisdifficulttocomparetheperformanceof25-mer
different laboratory strains (Kidgell et al. 2006). In the Peruvian arrays,suchasthoseusedhere,to45–65-merarrays.Inaddition,
population,importantpossiblevaccinecandidates,includingthe probedensityisalsoa factor,asthereare 103more probeson
circumsporozoiteprotein(PFC0210c),apicalmembraneantigen1 thearrayusedherethanthoseusedintheTanetal.(2009)study.
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Genome scanning of Amazonian P. falciparum
Indeed,previousworkhasshownthatupto90%oftheexisting analysis of the parental and offspring genotypes demonstrated
variationinuniquecodingregionscanbedetectedusingourar- thatthematernalgenotypewasIQE3848/3785andthepaternal
ray(Dhariaetal.2009),andthisarrayhasbeenusedtosuccess- genotype was IQE2924/3416/3466 (Fig. 1I). The pairwise com-
fully discover newly emerged SNPS in the P. falciparum genome parisondata,aswellasthefactthatthereareatmostfourdifferent
(Rottmanetal.2010). drug resistance alleles for each gene examined, indicated that
Searchingforisolatepairsthatcoulddefinetheentiregeno- P.falciparumparasitesintheareasurroundingIquitosarelikelythe
typeofathirdisolate,wediscoveredthatisolateIQE3502wasthe descendentsofonlyfourmultidrug-resistantparentalclones.
productofacrossbetweenparasitessharingtheIQE2924/3416/
3466andIQE3848/3785genotypes(Fig.1F–H).Intheprogeny,we
ThePeruvianisolateshavetwomitochondrialandfour
detected between 13 definitive and 19 possible recombination
apicoplastgenotypes
breakpoints in this cross that corresponds to a recombination
distanceof12–18kbpercentimorgan,correlatingwellwiththe Theextensivemitochondrialgenotypingcarriedouttodetermine
reportedvalueof;15kbpercentimorgan(Suetal.2007).Dueto theage ofP. falciparum demonstrates a singlemitochondrialge-
the maternal inheritance of the nonnuclear mitochondrial and notype(haplotype5)inPeruvianparasitepopulationsin1999(Joy
apicoplast genomes (Creasey et al. 1994; Okamoto et al. 2009), etal.2003).Weconfirmedthatourmicroarraycouldbeusedfor
limited mitochondrial genotyping by validating Dd2 and HB3
hybridizations(Dhariaetal.2009)withdatafromJoyetal.(2003).
Oursamplesfrom2006indicatethepresenceoftwomitochondrial
haplotypesinPerubymicroarraywithmutationsnearbasepair
positions1690(Fig.2Ad),4807,and4947(Fig.2Ae;Supplemental
File1),butallelesmayhavebeenmissedduetoalackofunique
probesinrepetitiveregionsofthemitochondrialgenome.
Littleworkhasbeendonetolookatmutationsintheapicoplast
genome in P. falciparum, an essential chloroplast-like organelle
thoughttobeofalgalorigin(DahlandRosenthal2008).Duetothe
high AT content (85.75%) of the plastid genome, we only have
uniqueprobesto55.9%ofthe34-kbapicoplastgenome.Likethe
chloroplast genome, the circular apicoplast genome has inverted
repeatregionsoflength;5kb,containingribosomalRNAandtRNA
genes.Becauseoftheprobablecopycorrectionoftheinvertedre-
peatslikeinthechloroplast(Myersetal.1982),weconsideredprobes
thatmaptotheinvertedrepeatsasunique.Uponhybridizationand
analysisofthePeruvianisolates,wedeterminedthepresenceoffour
different apicoplast haplotypes characterized by combinations of
twomutationsinthe23SrRNAgene(PFC10_API0010)andone
polymorphism in an apicoplast-encoded molecular chaperone/
AAA+ gene (PFC10_API0060) (Supplemental File 1). PCR ampli-
fication and sequencing revealed these polymorphisms to be
Figure1. Pairwisecomparisonofisolatesrevealprogenyofanatural
cross.(A)Log ratiosofprobeintensitiesplottedalongchromosome14for
2
thehybridizationofgenomicDNAfromisolatesIQE2924andIQE3502
displaypolymorphisms,indicatedbyupwardanddownwardspikesfor
thelefthalfofthechromosome.Thenormaldistributionoflog ratios
2
around0fortherighthalfofthechromosomeindicatethatthesetwo
isolatessharethesameDNAsequenceforthishalfofthechromosome.(B)
LogofP-valueforpolymorphismdetectionplottedonchromosome14
showspolymorphismspredictedonlyforhalfofthechromosome.The
bluelinesindicatepolymorphismsinIQE2924usingIQE3502astheref-
erence, and the green indicates polymorphisms in IQE3502 using
IQE2924asthereference.ThehorizontaldashedlineindicatesaP-value
cutoffof1310(cid:2)5.(C)Blocksof100kbofsequencethatdisplayedno
polymorphismsbypairwisecomparisonofisolatesweredrawn(colored
blueorredinpanelsC–H),andchromosome14isdisplayed.(D,E)Es-
sentially‘‘clonal’’parasites,IQE2924andIQE3416(D)andIQE3848and
IQE3785 (E) show very few polymorphisms forthe core chromosomal
region.(F,G)Otherpairwisecomparisonsshowedparasitesthatsharedthe
samegenotypeblocksforapproximatelyhalfofthecorechromosomal
regions.IQE2924andIQE3502shared;60%,whileIQE3848andIQE3502
shared;40%.(H)OverlayingthegenotypeblockscomparingIQE3502to
IQE2924andIQE3848revealedthatthelattertwogenotypescouldexplain
thegenotypeofIQE3502,indicatingthatitwastheprogenyofanatural
crossbetweenthesetwogenotypes.Magentaregionsdidnotcontainany
polymorphismsbetweentheparentalandprogenygenotypes,andthusare
ofuncertainassignment.(I)Analysisofinheritanceoforganellargenomes
revealedthatIQE3502inheritedtheIQE3848genotype,suggestingthat
thiswasthematernalgenotype,whileIQE2924wasthepaternal.
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Dharia et al.
Antimalarialsthatarethoughttoactagainsttheapicoplastdisplay
a ‘‘delayed-death’’ phenotype, where parasite development is
inhibited during the second cycle of erythrocyte invasion (Dahl
andRosenthal2008).Completeparasiteresistancetoclindamycin
has not been reported, but in vitro studies have suggested that
mutationsintheapicoplastgenomeconferresistancetothemac-
rolideantibioticazithromycin(Sidhuetal.2007).
TomeasuretheconservationofthisRNAgeneinPlasmodium
species,weusedpubliclyavailablereadsfromgenomesequencing
projectsforP.falciparumisolatesandotherPlasmodiumspeciesto
assemble the apicoplast-encoded sequence. In fact, all of the
worldwideP.falciparumisolatesthatwecouldassemblehad100%
identityfortheentire3923SrRNA(Africa:D6,FCR3,106-1,R033,
SEN_34_04; South East Asia: D10, FCB, FCC-2, K1, VS-1; India:
IGH-CR14,RAJ116;CentralAmerica:HB3,SANTA_LUCIA;South
America:7G8;Uncertainorigin:3D7).Notonlyisthesequence
highlyconservedwithinP.falciparum,theregionssurrounding
themutations,includingeveryresiduedisplayedinFigure2B,are
100%conservedacrossallspeciesforwhichwecouldassemblethe
RNA gene (P. vivax, P. ovale, P. reichenowi, P. knowlesi, P. berghei)
(SupplementalFiles4,5).
CrystalstructuresoftheHaloarculamarismortuiandDeinococcus
radiodurans large subunit ribosomal RNA (LSU) with clindamycin
bounddemonstratethatA2058-Ecisanimportanthydrogen-bond
partner(Schlunzenetal.2001;Tuetal.2005).Specifically,Tuand
colleaguessuggestthatthedesolvationoftheN2ofaguanineat
position2058-Ecaccountsformorethana10,000-folddecreasein
affinitytomacrolidesandlincosamides,andthus,theA2058G-Ec
Figure 2. Apicoplast polymorphisms correlated with clindamycin re-
sistance.(A)Polymorphismdetectionwasperformedfortheorganellar mutationconfersresistanceineubacteria(Tuetal.2005).Similarly,
genomes(api,apicoplast;mt,mitochondrion)versusthereference3D7. wehypothesizedthatanA2058C-Ecmutationwouldresultinan
IQE3502 and IQE4006 represent the mutations we saw in the Peru- analogousdesolvationeffectforoneofthethreehydrogen-bonding
vian parasites. PCR amplification and sequencing revealed these poly-
groups in cystosine and, thus, would result in clindamycin re-
morphisms to be point mutations: A4210C (a) and A4743T (b) in
sistanceinP.falciparum.Weconducteddockingstudiesofclinda-
PFC10_API0010,agenelocatedintheinvertedrepeatregionoftheapi-
coplastgenome(IRA,IRB).MutationT16659C(c)wasasynonymousSNP mycin and a related lincosamide proposed antimalarial, mir-
inapicoplastgenePFC10_API0060.Themitochondrionpolymorphisms incamycin(Olliaro and Wells 2009),toaP.falciparum homology
werepredictedtobenearbasepairpositions1690(d),4807(datanot model of the 23S peptidyl transferase domain based on the D.
shown),and 4947 (e). (B) The peptidyltransferase domain of the 23S
radioduransstructure(SupplementalFig.S3;Schlunzenetal.2001).
rRNA secondary structure was modeled based on E. coli. Mutations of
residues(blueletters)wereimplicatedinbacteriaandalgalchloroplasts Weobservedthatbothdrugsbindinthesameorientation,inter-
resistance to lincosamides. The red circles indicate residues that were actingsimilarlywithA2058-Ec(Fig.3A,B),andthustheA2058C-Ec
mutatedinourpatientisolates.WhileA2612Uwasnotcorrelatedwith mutationshouldgreatlydecreasetheaffinityofbothcompounds.
resistance,A2058Cwascorrelatedwithgreaterthan100-foldincreasein
IncontrasttoA2058-Ec,theothermutationweobserved,A2612-Ec
clindamycininvitroEC .
50
is distant from the clindamycin-binding pocket, suggesting that
Peruvian parasitesharboringthe A2612U-Ec mutation wouldnot
beresistanttoeitherclindamycinormirincamycin.
point mutations: A4210C (Fig. 2Aa) and A4743T (Fig. 2Ab) in
PFC10_API0010 and T16659C (synonymous SNP) in PFC10_
API0060(Fig.2Ac). Invitrodrugsusceptibilitytestsrevealclindamycinresistance
Althoughnotallstrainscouldberevivedfordrug-sensitivitytest-
ing,wewereabletodeterminetheEC forclindamycinusingSYBR
Clindamycinresistanceispredictedbyapointmutation 50
GreenIassayswith144-hincubationsforthereferencestrain3D7
intheapicoplast-encoded23SrRNA
and for one isolate that had wild-type 23S rRNA and four that
ThepolymorphismsA4210CandA4743TcorrespondtoA1875C-Pf carriedtheA2058C-EcorA2612U-Ec(Table2).Treatmentofpara-
andA2409U-Pfinthe23SrRNA(positionsA2058-EcandC2612-Ec, sites with clindamycin is thought to result in a ‘‘delayed-death’’
respectively,EcdenotingEscherichiacoli andPfdenotingP. falci- phenotypeduetothelossoftheapicoplastinthenextgeneration
parumnumbering)(Fig.2B,basedonE.coli secondarystructure) afteradministrationofthedrug(Ramyaetal.2007).Theeffectof
(Cannone et al. 2002). The A2058C-Ec mutation is in the same thedrugisthereforeonlymeasurableafterthesecondgenerationof
locationasadescribedmutationinthepeptidyltransferasecavity parasites,andalongerincubationperiodisneeded.Aspredicted,
ofthe23Sribosomalsubunitofbacteriaandalgalchloroplaststhat wediscoveredthattheA2058C-Ecmutationwascorrelatedwith
confersresistancetomacrolidesandlincosamides(lincomycinand clindamycin resistance, while the A2612T-Ec mutation was not.
clindamycin) (Harris et al. 1989; Vester and Douthwaite 2001). AlthoughthemutationsintheapicoplastrRNAmayhavearisen
Clindamycin,likeseveralantibiotics,caneffectivelytreatmalaria, in response to other antibiotics that are used to treat bacterial
presumablythroughtheinhibitionofprocessesintheapicoplast. infections in the region, Peruvian P. falciparum isolates with
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Genome scanning of Amazonian P. falciparum
evidencethatthe2058-Ecmutationconferssomelevelofclinda-
mycinresistanceintheseisolates.First,thereistheevidencefrom
E. coli, Brachyspira hyodysenteriae, Helicobacter pylori, Mycoplasma
pneumoniae,Propionibacteria,Streptococcuspneumoniae,Streptomyces
ambofaciens,andtheChlamydomonasreinhardtiichloroplastthata
mutation at position A2058-Ec to G, C, or U confers licosamide
resistance(VesterandDouthwaite2001).Second,thereistheevi-
dence from the clindamycin-bound crystal structures of the 23S
rRNA from H. marismortui and D. radiodurans discussed above
(Schlunzenetal.2001;Tuetal.2005).Third,treatmentwithclin-
damycinresultsina‘‘delayed-death’’ phenotype consistentwith
inhibition of apicoplast function, suggesting that the apicoplast
genomeisalikelyplacetolookforresistance-conferringmutations.
Fourth,clindamycin-resistantmalariahasnotbeenreportedbefore
andnoneoftheavailablesequencesforthePlasmodiumapicoplast
havethismutation(SupplementalFile5).Finally,therelatedApi-
complexanparasiteToxoplasmagondiiwasselectedforresistanceto
clindamycinandhadacquiredamutationat2061-Ecinthesame
conservedregionofthepeptidyltransferasedomainoftheapico-
plast-encoded rRNA (Camps et al. 2002). Although additional
testingandgenotypingofadditionalstrainswillbeneededtoab-
solutely rule out a chromosomal contribution, it seems highly
likelythatclindamycinresistanceiscausedbythismutation.
Figure 3. Structures of clindamycin and mirincamycin and the pre-
dictedinteractionswith23SrRNApeptidyltransferasecavity.(A)Previous
TheK76Tmutationgiveslowlevelsofchloroquineresistance
crystallography studies have shown A2058-Ec (red) is important for
makingcontactswithclindamycin(Schlunzenetal.2001),andthesimi- inPeruvianstrains
larityofchemicalstructureofclindamycinandmirincamycinsuggested
that cross-resistance may occur. (B) Lowest energy dockings of clinda- While testing for clindamycin resistance we also examined the
mycin(green)andmirincamycin(magenta)toahomologymodelofthe isolates’ sensitivites to other drugs using the SYBR green pro-
P.falciparumpeptidyltransferasedomaindemonstratedthatbothmole- liferationassaythatweroutinelyuseindrugdiscovery(Wuetal.
culesinteractwiththe23SrRNAinthesameway,andthus,A2058C-Ec
2009),whichgivesaccurateestimatesoftheEC s(Table2).Sub-
(red)waspredictedtoconferresistancetobothlincosamidedrugs. 50
stantialpyrimethamine resistancewas confirmed as predicted by
thesingle-mutantpfdhfr-tsalleleandtoagreaterextentbythetri-
A2058C-Ecwereclindamycinresistantwithgreaterthana100-fold ple-mutantpfdhfr-tsallele.ThoseisolateswhoseEC swereabove
50
increaseinEC . 5mMallcontainedthepfdhfr-tsquadruplemutant.ThreePeruvian
50
Unfortunately, since there is no method for allelic re- isolates showed slightly decreased susceptibility to quinine, con-
placementofapicoplastrRNAgenestoconfirmthattheA2058C- sistentwiththepointmutationN1042Dinpfmdr1conferringqui-
EcmutationconfersclindamycinresistanceinP.falciparum,itis nineresistance(Sidhuetal.2005).Aspredicted,allofthePeruvian
formallypossiblethatclindamycinresistancemaybe,inpart,due isolates had greater than 10-fold increase in chloroquine EC
50
toanothermutationinthegenome.Inordertoidentifyportionsof comparedwiththechloroquine-sensitivereference3D7consistent
thegenomethatmayconferresistance,weassumedresistancewas with genotyping, indicating that all Peruvian isolates had the
inheritedthroughsharedhaplotypeblocks.Thus,bycomparing hallmarkK76Tmutationinpfcrt(Sidhuetal.2002).Nevertheless,
theresistantisolatestoeachotherandtothesensitiveisolates,we themeasuredEC sof10–20nMdoesnotapproachthatofthewell-
50
wereabletoexclude83.4%oftheparasitenucleargenome(Sup- characterizedstrainDd2(typically179nMinourhands),which
plementalTableS1).Whiletheremaining16.6%ofthegenome also bears the K76T allele, suggesting that additional genetic
(Supplemental File 6) does encode rRNA methylases, efflux pro- determinants contained in laboratory strains are important for
teins,orinactivatingenzymessimilartothosedescribedinbacte- levels of chloroquine sensitivity. Likewise, all Peruvian isolates
rialclindamycinresistance(Roberts2008),thereisamultitudeof testedwerealsomoremefloquine-andartemisinin-sensitivethan
Table2. DrugEC s
50
EC [nM](95%CI)
50
Isolate Chloroquine Pyrimethamine Artemisinin Mefloquine Quinine Clindamycin
3D7 1.69(1.43–2.00) 2.25(1.49–3.42) 3.95(3.16–4.93) 8.16(7.47–8.90) 7.15(6.22–8.21) 1.25(0.932–1.68)
IQE3296 10.2(8.00–12.9) 1310(599–2860) 0.835(0.755–0.923) 2.04(1.57–2.65) 18.8(14.4–24.7) 2.81(1.62–4.87)
IQE3502 7.13(4.93–10.3) >5000 0.439(0.358–0.537) 1.63(1.33–2.00) 7.72(5.28–11.3) 951(357–2530)
IQE3785 15.7(13.1–18.7) >5000 0.739(0.657–0.831) 1.02(0.852–1.21) 15.2(9.44–24.4) 408(228–731)
IQE3848 17.2(13.9–21.4) >5000 0.743(0.541–1.02) 1.44(1.22–1.70) 22.6(14.5–35.2) 520(337–801)
IQE4006 19.215.0–24.8) >5000 1.05(0.871–1.27) 1.82(1.23–2.68) 21.1(12.5–35.8) 4.07(2.68–6.19)
EC sasdeterminedbyfittingsigmoidcurvesusingGraphPadPrism(95%confidenceintervalsshowninparenthesis)withdataobtainedusingSYBR
50
GreenIassays(96hforchloroquine,pyrimethamine,artemisinin,mefloquine,quinine,and144hforclindamycin).
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Dharia et al.
3D7.Thismayberelatedtothefactthattheseisolatescontaindif- such as the histidine-rich protein II, encoded by a subtelomeric
ferentpfmdr1allelesfrom3D7(Sidhuetal.2005),oritmaybedueto regionthatappearstobedeletedinasubpopulationofPeruvian
thepresenceofallelesinotherpumpsortransportersthathavebeen P.falciparum.Thisdeletionhasbeenpreviouslydescribed(Pieroni
implicatedinmalariaparasitedrugresponses,includingtheP.fal- etal.1998;Bell etal.2005; Wongsrichanalaietal.2007; Martin
ciparum sodium hydrogen exchanger, pfnhe (Ferdig et al. 2004; etal.2009),andthepresenceofthisdeletionintheAmazonsug-
Bennettetal.2007),pfatpase6(Eckstein-Ludwigetal.2003;Jambou geststhatwidespreaduseofmalariarapiddiagnosticteststhatde-
etal.2005;Uhlemannetal.2005),pfmdr2(Rosenbergetal.2006), pendonHRP2willresultinmisseddiagnoses.
orthemultidrugresistance-associatedprotein(Rajetal.2009). Second,weshowthatamutationintheapicoplast23SrRNA
correlateswitha100-foldincreaseinEC ofclindamycin.Because
50
Discussion itisrelativelydifficulttotestforclindamycinsensitivityinvitro,
activemonitoringforthe23SrRNAmutationmaybeadvisablein
OurfindingsthatthePeruvianparasitepopulationishighlyrelated regionswherelincosamideantibioticsareusedtotreatmalaria.Our
and that there is an apparent low frequency of meiotic re- analysisofavailablesequencedatasuggeststhatmutationsinthe
combinationisnotentirelysurprising.Previouspopulationstudies 23S rRNA are uncommon, and clindamycin resistance is not
inareasoflowendemicityintheAmericasshowedlimitedgenetic widespread.However,theparasiteisolatesusedtodrawthesecon-
diversityandhighself-matingrates,althoughtheywerebasedon clusionswere,inmanycases,obtainedmorethan10yrago.Onthe
veryfewmarkers(Arieyetal.1999;Haddadetal.1999).Peruwas otherhand,apreviousstudyinwhichPCRamplificationofa595-
alsoexpectedtohaveaparasitepopulationwithlimiteddiversity, bp fragment of the apicoplast RNA gene from Indian, Pakistani,
andindeed,genotypingofmajorvaccinecandidatesshowedless Laotian,andSingaporeanbloodspotsin1997(Tanetal.1997)was
diversity than populations in Africa (Chenet et al. 2008). Addi- conducted,showedevidenceofseveralmutationsneartheA2058-
tionally, a study of the malaria drug-resistance genes using 167 Ecregion,suggestingthatclindamycinresistancemayexistinAsia.
P.falciparumsamplescollectedbetween1999and2007hasfound Indeed,therearereportsinSouthEastAsiaofpregnantwomenwith
onlyalimitednumber(twotofive)ofhaplotypesinthisregion recurrentP.falciparuminfectiondespitetreatmentwithquinineor
(Baconetal.2009).Whilethishighlystructuredpopulationmay artesunate(plusorminusclindamycin)(Halletal.1975).Although
notbeidealforlookingforgenomicregionsinlinkagedisequilib- nogenotypingisavailable,Burkhardtetal.(2007)recentlyshowed
rium(Volkmanetal.2007),strainscollectedfromtheregionscould that some individuals infected with Amazonian parasites from
beusedinlinkageanalyses,andinQTLstudies.Thehybridization Braziltakemuchlongertoclearinfectionsafterclindamycintreat-
datacollectedhereconstitutesasetofgeneticmarkersthatcouldbe mentthanindividualsinfectedwithAfricanparasites.Further
usefulinfurtheranalyses. studies,especiallyinSouthAmerica,willbeneededtoassesshow
While our findings that parasite strains are separated by a prevalentthemutationisandfromwhenceitcame,especiallyas
limitednumberofmeiosesmayonlyapplytoregionsoflowende- recentclinicaltrialsoffosmidomycin+clindamycinshowitspo-
micity,previouspopulationstudiesinAfrica,whererecombination tentialasanefficaciouscombinationtherapy(Borrmannetal.2004;
frequencies are expected be much higher, have mostly relied on OlliaroandWells2009).
smallnumbersofmarkerstounderstandhowdrug-resistantpara- Third,anewlincosamidedrug,mirincamycin,isinpreclini-
sitesspreadanddetermineratesofsexualrecombinationbetween cal development, being prepared by the Medicines for Malaria
differentgenotypes. Forexample,microsatellite genotypingat12 Venture for Investigational New Drug status (Olliaro and Wells
loci of worldwide isolates suggested that the genetic structure is 2009)andhasbeenshowntohavegoodinvitroactivityagainst
linked to epidemiology with low heterozygosity in areas where P.falciparumisolatesfromGabon(Heldetal.2010).Whilethisdrug
transmissionislow(Andersonetal.2000).Astudyofthebreakdown ispromising,becauseithasbeenshowntohavesynergisticeffects
oflinkagedisequilibriumovera5-kbregionofthegenomedem- withprimaquineforclearingliver-stageparasitesinanimalmodels
onstratedthatmeioticrecombinationishighinP.falciparum,and (Schmidt1985),thereisa concernforcross-resistancewith clin-
theapparent‘‘clonality’’ofparasitesisprobablyduetoinbreedingin damycin.Comparingthestructureofmirincamycintoclindamy-
areaswithlowendemicity(Conwayetal.1999).Morerecentap- cinrevealsthatthesetwomoleculessharethemoietyimplicated
proachesusingsequencing(Jeffaresetal.2007;Volkmanetal.2007) formakingcontactswiththe23SrRNAatthepeptidyltransferase
orSNPchips(Neafseyetal.2008;Muetal.2010)haveidentified cavity, as demonstrated by the crystal structure of clindamycin
recombinationratesandregionsunderselectionwithmuchgreater boundtobacterialribosomalLSU(Schlunzenetal.2001;Tuetal.
precision. Similarly, our whole-genome approach may allow for 2005). Thus, both clindamycin and mirincamycin may make
amorecomprehensiveviewofgeneticstructureandrecombination contacts with residue A2058-Ec (Fig. 3A,B) suggesting that there
inlocal,andmoredispersedparasitepopulationsinregionsofhigh may be cross-resistance to mirincamycin in the Amazon region
endemicityinAfricabyclassifyinghaplotypesforeachcorechro- with the A2058C-Ec mutation. In light of this evidence that
mosomalgeneinanisolate. mirincamycin resistance may already exist, the development of
Additionally, our study has several implications for the di- this and future lincosamide antimalarialdrugs should be recon-
agnosisandtreatmentofmalaria.First,thestudydocumentsthe sidered.
inherentgeneticvariabilityandlong-terminstabilitythatisfound
in many subtelomeric genes. While the isolates used in these Conclusions
studieswerealltakenintoshort-terminvitrocultureforaperiodof
weeksinordertoisolatesufficientquantitiesofgenomicmaterial,it The methods described here are useful for broadly looking at
isunlikelythatthisinstabilityaroseinvitro,sincemultipleclonal populationstructure,andourdatashowhownovelinsightscanbe
isolates shared the same patterns of variability in subtelomeric gainedthroughfullgenomeapproaches.Insomecasesitmaybe
regions,andithasalsosubsequentlybeenconfirmedinuncultured possibletodeterminewhetherthepopulationhasbeenstructured
isolates(Gamboaetal.2010).Thisvariabilitymaycontributetothe throughimmuneselectiononspecificantigens.However,because
failureofrapiddiagnosticteststhataredirectedtowardantigens microarrays offer a lower resolution view, analyses of specific
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Genome scanning of Amazonian P. falciparum
antigensarestillbestperformedthroughsequencingorconven- themicroarraysovernightat45°CinAffymetrixbuffers,washed,
tionalgenotyping.Furthermore,thesemethodscanalsobeusedto andscannedusingamodifiedprotocolwithwashtemperaturesof
discovernewallelesinvolvedinresistance.Ourfindingthatalleles 23°CtoaccountforthehighATcontentofP.falciparum(Supple-
associated with clindamycin resistance exist in South America mentalFile7:Dhariaetal.2009).
highlightstheimportanceofdevelopingdrugswithnovelmech-
anismsofactionandpresentsnewmolecularstrategiesformoni- Microarrayanalysis
toringclindamycinresistanceinSouthAmericaandalsointherest
Briefly, for polymorphism detection, using a sliding window of
oftheworld.
threeoverlappingprobes,wescannedforsetsofprobesthathad
significantly lowerhybridization when comparedwith the refer-
Methods enceindicativeofapolymorphismasdeterminedbyZ-testusing
a P-value cutoff of 1 3 10(cid:2)5 and a reference 3D7 hybridization.
Studysiteandcollectionofmalariapatient-derivedisolates Genecopynumbervariationscanningwasperformedbycalculat-
ingthelog ratioofthemeanintensityforprobestoagenedivided
Briefly,theDepartmentofLoretocomprisesalmostone-fourthof 2
bythemeanintensityoftheprobesfor a reference 3D7hybrid-
thelandmassofPeruandhasanecosystemcharacteristicofthe
izationaspreviouslydescribed(Dhariaetal.2009).
Amazonlowlands(AramburuGuardaetal.1999).Theruralpop-
Pairwise comparisons were performed with microarray hy-
ulationofLoreto,estimatedat;474,000inhabitants,isclustered
bridizationsof14patientisolatesusingoneisolateasareferencefor
intownsandvillageslocatedintheAmazontributarysystem.The
another, thus producing 182 comparisons. Polymorphisms were
cityofIquitos,thelargesturbancenterinLoretowithapopulation
calledusingP-valuecutoffsof1310(cid:2)5,andwedefinedisogenic
of;345,000,islocated120mabovesealevel,atthejunctureofthe
blocksasall100-kbwindowsthathadnopolymorphismsbetween
UcayaliandNapoRivers,formingtheAmazonRiverproper,andis
apairofisolates.Next,weidentifiedtheprogenyofanaturalcross
accessibleonlybyairorriver.Iquitoshasatropicalclimate,with
by scanning through all possible combinations of three isolates.
ameantemperatureof27.5°C,ameanannualrainfallof4m,and
Specifically,welookedfortwoisolatesthateach,separately,were
ameanannualprecipitationof2.7m.Theclimateinthisareais
;50%isogenictoathirdisolate,butincombinationwereisogenic
typicaloftheAmazonbasin,witharainyseasonfromNovember
for >90% of the third isolate. To detect polymorphisms in the
to May and a dry season from June through October. The peak
organellar apicoplast and mitochondrial genomes, the Peruvian
transmissionrateforP.falciparumoccursduringtherainyseason
isolateswerecomparedwith3D7referencehybridizationsusinga
(AramburuGuardaetal.1999).Thepopulationispredominantly
P-valuecutoffof1310(cid:2)5.
mixedSpanishandAmericanIndian(mestizo).Householdecon-
omiesaresustainedbyagriculture,fishing,ortourism.The2006
studysamplescamefromIquitos. EC determination
50
The2006malariaisolateswerecollectedaspartofanongoing
febrile surveillance protocol (approval no. NMRCD.2000.0006 96hEC50assayswereperformedforchloroquine,pyrimethamine,
fromtheNavalMedicalResearchCenterDetachment,Lima,Peru) mefloquine,artemisinin,andquinineusinga384-wellformat.A
frompatientswithsymptomstypicalofmalariawhovisitedclinics prolongedincubationperiodwasrequired,asthePeruvianisolates
locatedinthejunglecityofIquitos.Oncetheyhadbeendiagnosed grewatslowerratesthanstandardlaboratorystrains,anda10-to
withmalariabymicroscopy,bloodwasspottedontofilterpaperfor 20-fold difference in signal between control DMSO treated and
characterization of SNPs and microsatellites. Venous blood was high-dosedrug-treatedwellswasnecessary.ParasitesofeachPeru-
collectedinto3-mLEDTAtubesforlaboratorystudiesfromfebrile vian isolate in media containing 50% human serum and 50%
patientswhenP.falciparummalariawasdiagnosedbymicroscopic GIBCO AlbuMAX II (Invitrogen) were dispensed into the plates,
examination.Atotalof600mLofbloodwastakenfromeachtube andcompoundsweredispensedusingaPinToolwitha50nLhead,
understerileconditionsinalaminar-flowhoodfordrugsuscepti- sothatthefinalvolumeofeachwellwas50mL.Thefinalhemat-
bilitytesting.Onemilliliterwasusedforthecryopreservationof ocrit was 2.5% with a 0.075% parasitemia, and the final DMSO
parasites.Oftheremainder,somewasspottedontoWhatman3M concentrationwas0.1%.Theassayplateswereincubatedat37°C
chromatographypaper(Whatman,Inc.)forPCR;somewasusedto for96hinairtightincubationunitswhichweregasseddailywith
prepare thick and thin smears for parasite confirmation and 93%nitrogen,4%carbondioxide,and3%oxygen.Atotalof10mL
quantification; and the rest was frozen at –80°C. Microsatellite of detectionreagent, 103 SYBR Green I (Invitrogen; supplied in
testingshowedaverylowfrequencyofpolyclonalinfections(<1%) 10,0003 concentration) in lysis buffer (20 mM Tris-HCl, 5 mM
(Baconetal.2009)intheregion,andsamplesfrompolyclonalin- EDTA,0.16%Saponinw/v,1.6%TritonX-100v/v),wasdispensed
fectionswerenotusedinthesestudies. intoeachwell.Theplateswereincubatedatroomtempfor24h.
ThefluorescenceintensityfromtheplatewasreadusinganAnalyst
GT multimode reader (Molecular Devices); the reader required
DNAmethods
a505dichroicmirrorwith485-nmexcitationand530-nmemis-
3D7(MRA-151)wasobtainedfromtheMalariaResearchandRef- sionsettings,andtheplatewasreadfromthebottom.
erence Reagent Resource Center (MR4; American Type Culture Forthelonger144hEC assayswithclindamycin(‘‘delayed-
50
Collection).3D7andPeruvianP.falciparumpatientisolateswere death’’),amodifiedprotocolwasrequired.The144-htimepoint
propagatedinhumanerythrocytesaspreviouslydescribed(Trager waschosenbytakingintoaccounttheslowergrowthrate,onav-
and Jenson 1978) with medium for 3D7 containing 5% human erage,ofthePeruvianisolatescomparedwithstandardlaboratory
serumand1.2%GIBCOAlbuMAXII(Invitrogen),whiletheme- strains.Froma10-mMcompoundstock,a1:5dilutionwithPBSwas
diumforpatientisolatescontained10%humanserum.Genomic made.Then,1:3serialdilutionswereperformedina1:5dilution
DNAwasisolatedbystandardphenol-chloroformextraction.Fif- solutionofDMSOandPBSacrossa96-wellcompoundplate.A150-
teen microgramsofgenomicDNAfromeach isolateand 2.5ng mLsuspensionofeachPeruvianisolatewasaliquotedintoaseparate
eachofBioB,BioC,BioD,andCreAffymetrixcontrolplasmids Costar96-wellroundbottomplate(CorningInc.)inmediumthat
(AffymetrixInc.)werefragmentedwithDNaseIandend-labeled containedhumanserumwithnoAlbuMAX.Thefinalhematocrit
withbiotin(Winzeleretal.1998).Thesampleswerehybridizedto was2.5%with0.15%parasitemia.Usingamatrixpipette,0.75mLof
GenomeResearch 1541
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Dharia et al.
compoundfromthecompoundplatewastransferredintotheassay waspreparedaspartoftheirofficialduties.E.A.W.wassupported
plate.The finalDMSOconcentrationwas0.1%. The assayplates by grants from the WM Keck Foundation and the National In-
wereincubatedat37°C for 144 h inairtight incubation units as stitutesofHealth(R01AI059472).MalariaworkatGNFispartially
describedabove.Parasitegrowthwas monitored bytakingblood supportedbygrantsfromtheMedicinesforMalariaVentureand
smears from untreated wells. The medium was removed with a theWellcomeTrust.Theviewsexpressedinthisarticlearethoseof
multichannelpipetteand150mLofmediumcontainingnohuman theauthorsanddonotnecessarilyreflecttheofficialpolicyofthe
serum,orAlbuMAXwasaddedtoeachwell.Thecontentsofthe Department of the Navy, the Department of Defense, the De-
wellsweretransferredtoaCostar96-wellwhiteclearbottomplate partmentofHealthandHumanServices,ortheU.S.government.
(CorningInc.).Atotalof30mLof103SYBRGreenIdetectionre- ThestudyprotocolwasapprovedbytheNavalMedicalResearch
agentdescribedabovewasdispensedintoeachwell.Theplateswere Center Institutional Review Board in compliance with all appli-
incubatedatroomtempfor24h.Thefluorescenceintensityfrom cable Federal regulations governing the protection of human
thebottomoftheplatewasreadusinganAnalystGTmultimode subjects.
reader(MolecularDevices). Author contributions: N.V.D. conceived, designed, and per-
formedexperimentsanddataanalysisandwrotethemanuscript.
D.P., S.E.R.B., G.E.G., C.L., T.J.K., C.S., and V.S. conceived, de-
Assemblyof23SrRNAcodingsequencefromotherisolates
signed, and performed experiments. B.B. performed homology
WedownloadedallP.falciparum,P.vivaxSalI,P.ovaleNigeria1CDC, modeling. D.J.B. and E.A.W. conceived, designed, and directed
P. reichenowi, P. knowlesi, and P. berghei ANKA reads from NCBI theprojectandwrotethemanuscript.
TraceDB (ftp://ftp.ncbi.nih.gov/pub/TraceDB/) (downloaded Feb
2009),BLASTedthereadsforeachisolateagainstPFC10_API0010
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