Rhodes, J; Beale, MA; Vanhove, M; Jarvis, JN; Kannambath, S;Simpson, JA; Ryan, A; Meintjes, G; Harrison, TS; Fisher, MC; Bi-canic, T (2017) A Population Genomics Approach to Assessing theGenetic Basis of Within-Host Microevolution Underlying RecurrentCryptococcal Meningitis Infection. G3 (Bethesda). ISSN 2160-1836DOI: 10.1534/g3.116.037499

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1

Apopulationgenomicsapproachtoassessingthegeneticbasis1

ofwithin-hostmicroevolutionunderlyingrecurrentcryptococcal2

meningitisinfection3

JohannaRhodes1,MathewA.Beale2,MathieuVanhove1,JosephN.Jarvis3,4,5,Shichina4

Kannambath6,JohnA.Simpson7,AntheaRyan7,GraemeMeintjes8,ThomasS.Harrison6,5

MatthewC.Fisher1*,TihanaBicanic6*6

1-DepartmentofInfectiousDiseaseEpidemiology,ImperialCollegeLondon,London,7

UnitedKingdom8

2-DivisionofInfectionandImmunity,FacultyofMedicalSciences,UniversityCollege9

London,London,UnitedKingdom10

3-Botswana-UPennPartnership,Gaborone,Botswana11

4-DivisionofInfectiousDiseases,DepartmentofMedicine,PerelmanSchoolof12

Medicine,UniversityofPennsylvania,Philadelphia,Pennsylvania,UnitedStatesof13

America14

5-DepartmentofClinicalResearch,FacultyofInfectiousandTropicalDiseases,London15

SchoolofHygieneandTropicalMedicine,London,UnitedKingdom16

6-InstituteofInfectionandImmunity,St.George’sUniversityLondon,London,United17

Kingdom18

G3: Genes|Genomes|Genetics Early Online, published on February 10, 2017 as doi:10.1534/g3.116.037499

© The Author(s) 2013. Published by the Genetics Society of America.

2

7-NationalHealthLaboratoryService,Greenpoint,CapeTown,SouthAfrica19

8-ClinicalInfectiousDiseasesResearchInitiative,InstituteofInfectiousDiseaseand20

MolecularMedicineandDepartmentofMedicine,UniversityofCapeTown,CapeTown,21

SouthAfrica22

*Correspondingauthors:matthew.fisher@imperial.ac.uk;tbicanic@sgul.ac.uk23

Abstract24

RecurrenceofmeningitisduetoCryptococcusneoformansaftertreatmentcauses25

substantialmortalityinHIV/AIDSpatientsacrosssub-SaharanAfrica.Inorderto26

determinewhetherrecurrenceoccurredduetorelapseoftheoriginalinfectingisolate27

orreinfectionwithadifferentisolateweeksormonthsafterinitialtreatment,weused28

whole-genomesequencingtoassessthegeneticbasisofinfectionin17HIV-infected29

individualswithrecurrentcryptococcalmeningitis.Comparisonsrevealedaclonal30

relationshipfor15pairsofisolatesrecoveredbeforeandafterrecurrenceshowing31

relapseoftheoriginalinfection.Thetworemainingpairsshowedhighlevelsofgenetic32

heterogeneity;inonepairwefoundthistobearesultofinfectionbymixedgenotypes,33

whilstthesecondwasaresultofnonsensemutationsinthegeneencodingtheDNA34

mismatchrepairproteinsMSH2,MSH5andRAD5.Thesenonsensemutationsledtoa35

hypermutatorstate,leadingtodramaticallyelevatedratesofsynonymousandnon-36

synonymoussubstitutions.Hypermutatorphenotypesowingtononsensemutationsin37

thesegeneshavenotpreviouslybeenreportedinC.neoformansandrepresentanovel38

3

pathwayforrapidwithin-hostadaptationandevolutionofresistancetofirst-line39

antifungaldrugs.40

Introduction41

TheHIV/AIDSpandemichasledtoalargepopulationofprofoundly42

immunocompromisedindividualsthatarevulnerabletoinfectionbytheopportunistic43

funguspathogenCryptococcusneoformans(Hagenetal.2015).Thismycosisposesa44

considerablepublichealthprobleminsub-SaharanAfrica,whichhasthehighest45

estimatedannualincidenceofcryptococcalmeningitis(CM)globally(Parketal.2009),46

withthemajorityofinfectionscausedbyCryptococcusneoformanssensustricto47

(previouslyreferredtoasCryptococcusneoformansvar.grubii)(Jarvis&Harrison2007).48

StandardtreatmentforHIV-associatedCMincludesthelong-termuseofazoledrugs49

suchasfluconazole,followinginitial1-2weekinductiontreatmentwithamphotericinB,50

whichisoftennotavailable(Brouweretal.2004).Microevolutionoccursinresponseto51

drugpressure,leadingtoresistance,aphenomenonpreviouslydescribedinC.52

neoformans(Ormerodetal.2013;Sionovetal.2010).Patientswhoappearsuccessfully53

treated(evidencedbysymptomresolutionandsterilisationofcerebralspinalfluid(CSF))54

canrelapseduetopersistinginfections,whichinsomecasesappeartohaveevolved55

resistancetofirstlineantifungaldrugs.Intheabsenceofcontinuedantifungaltherapy56

andrestorationoftheirimmunesystemthroughantiretroviraltherapy(ART),patients57

withHIV/AIDSalsohaveahighprobabilityofrecurrenceofCM(Bozzetteetal.1991).58

4

Variousmethodsofwithin-hostevolutionareavailabletoeukaryoticpathogens,most59

notablysexualandparasexualreproduction,althoughthesearedifficulttoobservedue60

totheoftencrypticnaturerecombinationoffungi.Aneuploidy,recombinationinthe61

telomeres,andmutatorstates(Roderoetal.2003)alsoprovidemeansofrapidwithin-62

hostevolution,withothermechanismsstilllikelytobediscovered.Theaccumulationof63

SNPsalongsidecopynumbervariationandaneuploidyhasbeenwitnessedduring64

infectionindifferentfungalpathogenspecies,enablingrapidadaptiveevolution(Caloet65

al.2013)andconferringresistancetoantifungaldugs(Hickmanetal.2015).Candidiasis66

iscausedbynumerousCandidaspecies,yetwithin-hostevolutionamongstthese67

speciesdiffers.InfectiousstrainsofCandidaalbicansareusuallysusceptibletoazole68

antifungaldrugs,butresistancecanevolveviatheevolutionofdrug-resistantaneuploid69

isolates,whichcontainanisochromosomeoftheleftarmofchromosome5(Selmeckiet70

al.2009).Theleftarmofchromosome5containstwoimportantgenesinvolvedin71

resistancetoantifungals:ERG11,atargetofazoles,andTAC1,atranscriptionfactorthat72

activatesdrugeffluxpumpexpression.Conversely,Candidaglabrataareintrinsically73

poorlysusceptibletoazoles,andhavemorerecentlyevolvedmulti-drugresistanceto74

bothazolesandechinocandins(Pfaller2012;Panackaletal.2006;Alexanderetal.2013).75

Theoccurrenceofwithin-hostdiversityandrecombinationhasbeenwitnessedin76

eukaryoticpathogens,notablyC.albicans:mutationandrecombinationratescanbe77

increasedunderstressfulconditions,suchasdrugtreatment(Forcheetal.2009;Fordet78

al.2015),resultinginloss-of-heterozygosity(LOH)andaneuploidy(Forcheetal.2009).79

ThesegeneticalterationscontributetothemaintenanceofapopulationofC.albicans80

5

withinthehostenvironment(Forcheetal.2009),anddrugpressurecanresultin81

diverginglevelsoffitness(Cowenetal.2001).82

SimilarresponsestoantifungaldrugshavebeenobservedinC.neoformans;Point83

mutationsintheorthologERG11werealsoshowntoconferfluconazoleresistance,by84

causingtheaminoacidsubstitutionG484S(Roderoetal.2003).Sionovetal.(Sionovet85

al.2010)demonstratedlargescalechromosomalduplications(primarilychromosome1)86

arefundamentaltoovercomingfluconazole(FLC)drugpressureinamousemodel,87

contributingtofailureofFLCtherapy.Theduplicationofchromosome1included88

increasedcopynumberofgenesERG11,thetargetofFLC,andAFR1,atransporterof89

azoles(Sionovetal.2010),althoughothergenesarealsothoughttobeinvolvedinFLC90

resistance(Sionovetal.2013;Pauletal.2015).PreviousstudiesofseriallycollectedC.91

neoformansisolateshaveconfirmedin-hostmicroevolution,includingtheoccurrenceof92

large-scalegenomicrearrangements(Fraseretal.2005;Blasietal.2001;Illnait-Zaragozi93

etal.2010).LikeC.albicans,theC.neoformansgenomeiscapableofundergoing94

chromosomalduplicationandlossunderstressessuchasdrugpressureorinvasionof95

thehumanhost(Friesetal.1996).Thesechromosomalduplicationsareoftenlostwhen96

theselectivepressureisremoved(Sionovetal.2010).97

Thedevelopmentofmutatorstatesviahypermutabilityisarapidlyexpandingareaof98

studyinbacteria,particularlyPseudomonasaeruginosaincysticfibrosis(CF)patients.99

Here,hypermutabilityhasbeenshowntohaveanassociationwithantimicrobial100

resistance(Oliveretal.2000;Maciáetal.2005),causingsignificantimplicationsinthe101

6

earlytreatmentofcysticfibrosispatientstopreventchronicinfection(Burnsetal.2001;102

Maciáetal.2005).Fewstudieshaveexploredhypermutationinpathogenicfungi;103

however,mutationsintheyeastSaccharomycescerevisiaegenesPMS1,MLH1and104

MSH2,whichareallinvolvedinmismatchrepair,havebeenshowntoleadto100-to105

700-foldincreasesinmutationsthroughoutthegenome(Strandetal.1993).106

FrameshiftmutationsinanorthologofthemismatchrepairgeneMSH2havealsobeen107

showntocontributetomicroevolutioninthesisterspeciesofC.neoformans,108

Cryptococcusgattii(Billmyreetal.2014).109

Herewedescribeacomparativegenome-sequencingbasedapproachtoinvestigate110

microevolutioninseriallycollectedisolatesofC.neoformans.Theseisolateswere111

grownandstoredfromfreshCSFofpatientswithCM,priortostartingandduring112

antifungaltherapyusinginductionwithamphotericinB-basedregimens,followedby113

fluconazole.Weusedwhole-genomesequencingtodescribethenatureofinfectionin114

17patientstogaininsightsintothedynamicsofrecurrentinfections.115

MaterialsandMethods116

Samplesandpatients117

SixteenSouthAfricanpatientsandoneUgandanpatientdemonstratingclinicalevidence118

ofcryptococcalmeningitiswerestudied.Allpatientswereeitherpartofobservational119

studiesorclinicaltrial(Bicanicetal.2007;Bicanicetal.2008;Jarvisetal.2012;Longley120

etal.2008;Jarvisetal.2010).EthicalapprovalwasobtainedfromtheWandsworth121

ResearchEthicsCommitteecoveringSt.George’sUniversityofLondon(Longleyetal.122

7

2008;Bicanicetal.2007;Bicanicetal.2008;Jarvisetal.2012).InSouthAfrica123

additionalethicalapprovalwasobtainedfromtheUniversityofCapeTownResearch124

EthicsCommittee;inUganda,fromtheResearchEthicsCommitteeofMbarara125

UniversityofScienceandTechnology.Allpatientsinitiallypresentedwithcryptococcal126

meningitisandweretreatedusinginductiontherapywith7-14days’amphotericinB127

deoxycholate0.7-1mg/kg/d,withorwithout100mg/kg/dofflucytosine(withone128

patient,IFNR63,alsoreceivingadjunctiveinterferongamma),followedbyfluconazole129

consolidationat400mg/dfor8weeksandmaintenancetherapyat200mg/dfor6-12130

months(n=16pairs),untilimmunerestorationonARTwithaCD4countof>200131

cells/µL.ThesinglepatientinUgandareceivedinductiontherapywithfluconazole1600132

mg/dfor2weeksfollowedbyfluconazoleconsolidationandmaintenanceandART,as133

above(n=1pair).Aspartofstudyprocedure,patientsenrolledinclinicaltrialshad134

quantitativecryptococcalculturesperformedonserialCSFsamples.Patientswitha135

recurrenceoftheircryptococcaldiseasefollowinginitialtreatmentandpositiveCSF136

cultureforCryptococcusatthetimeofdiseaserecurrencewereincludedinthestudy.137

Westudiedtheclinicalcryptococcalisolatestakenoninitialdiagnosis(priortoinitiation138

oftreatment)andcomparedeachwiththeCryptococcusisolatedfromCSFon139

recurrenceofdiseaseinthesamepatient(Table1).140

Multi-locussequencetyping141

TodiscernwhethermixedorsinglegenotypeinfectionswereextractedfromCSF,multi-142

locussequencetyping(MLST)wasperformedonthreeindependentcoloniesfora143

8

subsetofthestudyisolates,accordingtothemethodsoutlinesinMeyeretal.(Meyeret144

al.2009),withmodificationsasoutlinedinBealeetal.(Bealeetal.2015).145

Molecularmethods146

C.neoformanswasisolatedfromHIVinfectedindividualsonlocationbyplatingCSFonto147

SabourandDextrose(SD)agar(Oxoid,FisherScientific),andgrowingat30˚Cfor48148

hours.ArepresentativesampleoftheC.neoformanspopulationwastakenbyselecting149

abroad‘sweep’ofallcoloniesontheSDagarplate,whichwasstoredin150

cryopreservativemedium(80%SDbroth,20%glycerol)at-80˚Cuntilfurthertesting.151

Thisapproachensuresallgeneticdiversityismaintainedthroughtheprocess,andsingle152

colonypickingonlyoccursatthefinalstageofliquidcultureandDNAextraction.153

FrozenstockswereplatedontoSDagarandculturedfor72hours.Asinglecolonywas154

inoculatedinto6mlYeastPeptoneDigestbroth(Oxoid)supplementedwith0.5MNaCl155

andculturedat37°Cwithagitation(165rpm)for40hours,followedbygenomicDNA156

extractionusingtheMasterpureYeastDNApurificationkit(Epicentre)modifiedby157

additionoftwocyclesofrapidbeadbeating(45secondsat4.5m/second)usinga158

FastPrep24homogeniser(MPBio).GenomicDNAlibrarieswerepreparedusingthe159

TruSeqDNAv2orTruSeqNanoDNAkit(Illumina),andwholegenomesequencingwas160

performedonanIlluminaHiSeq2500atMedicalResearchCouncilClinicalGenomics161

Centre(ImperialCollegeLondon)aspreviouslydescribed(Rhodesetal.2014).162

Datapolicy163

9

Allrawreadsandinformationonlineagesofisolatesinthisstudyhavebeensubmitted164

totheEuropeanNucleotideArchiveundertheprojectaccessionPRJEB11842.165

Wholegenomesequenceanalysis166

RawIlluminareadswerealignedtotheC.neoformansreferencegenomeH99(Loftuset167

al.2005)usingtheBurrows-WheelerAligner(BWA)v0.75amemalgorithm(Li2013)168

withdefaultparameterstoobtainhighdepthalignments(average104x).Samtools(Li169

etal.2009)version1.2wasusedtosortandindexresultingBAMfiles,andgenerate170

statisticsregardingthequalityofalignment.Picardversion1.72wasusedtoidentify171

duplicatereadsandassigncorrectreadgroupstoBAMfiles.Furthermore,BAMfiles172

werelocallyrealignedaroundinsertionsanddeletions(INDELs)usingGATK(McKennaet173

al.2010)version3.4-46‘RealignerTargetCreator’and‘IndelRealigner’,followingbest174

practiceguidelines(VanderAuweraetal.2013).175

Singlenucleotidepolymorphisms(SNPs)andINDELswerecalledfromallalignments176

usingGATK(McKennaetal.2010)version3.4-46‘HaplotypeCaller’inhaploidmodewith177

arequirementthatallvariantscalledandemittedareaboveaphred-scaleconfidence178

thresholdof30.BothSNPsandINDELswerehardfilteredduetoalackoftrainingsets179

availableforC.neoformansbyrunningVariantFiltrationwithparameters“DP<5||MQ180

<40.0||QD<2.0||FS>60.0”;thisexpressionensuredlowconfidencevariantswere181

filteredoutiftheymetjustoneofthefilterexpressioncriteria.Resultinghigh-182

confidencevariantsweremappedtogenesusingVCF-annotator(BroadInstitute,183

10

Cambridge,MA,USA)andthelatestrelease(CNA3)oftheC.neoformansreference184

genomeH99andgeneontology.185

Someisolatesweresuspectedofhavingnon-haploidgenomesduetothehighnumber186

oflowconfidencevariants.Fortheseisolates,‘HaplotypeCaller’wasrepeatedindiploid187

mode.188

Theaverage(mean)coverageforeachisolateweredeterminedusingGATK(McKenna189

etal.2010)version3.4-46‘DepthOfCoverage’underdefaultsettings.TheC.190

neoformansH99(Loftusetal.2005)wasagainusedasreference.Inordertodetermine191

aneuploidy,whole-genomecoveragedatawasnormalisedandregionsdisplaying192

normalisedcoverageequalto2weredeemeddiploidevents(likewise,normalised193

coverageequalto3weredeemedtriploidevents,andsoon),whereasnormalised194

coverageequaltozerowasdeemedadeletionevent.195

Susceptibilitytesting196

ThesusceptibilitytestingofallrelapseisolateswereperformedwiththeMICRONAUT-197

AMsusceptibilitytestingsystemforyeast(Merlin)asrecommendedbythe198

manufacturer.MICRONAUT-AMallowsthedeterminationofMICsofamphotericinB,199

flucytosine,fluconazole,voriconazole,posaconazole,itraconazole,micafungin,200

anidulafunginandcaspofungin,andcommercialisesthewell-established,butlaborious,201

CLSIbrothmicrodilutiontechnique.Briefly,foreachisolatefivecolonieswereusedto202

preparea0.5-McFarland-standardsuspensionin0.9%NaCl.1:20dilutionwasprepared203

in0.9%NaCland1:5dilutionwaspreparedin11mlRPMIbrothprovidedwiththekit.204

11

100µlASTindicatorand50µlMethylenebluesolutionsweremixedwiththebrothfor205

manualsusceptibilitytesting.ThebrothwastheninoculatedontoMerlinMICRONAUT206

96-welltestingplates(100ul/well)andincubatedat30°Cfor72hours.Thelowest207

concentrationofanantifungalagentwithnodetectablegrowth(MIC)wasdetermined208

foreachisolatebasedonfungalgrowth(pink)ornogrowth(blue).ObtainedMICswere209

interpretedaccordingtoC.albicansEUCAST(Vers.7.0/12-08-2014)values(Rodríguez-210

Tudelaetal.2010;Alastruey-Izquierdo&Cuenca-Estrella2012).211

Phylogeneticanalysis212

Whole-genomeSNPswereconvertedintorelaxedinterleavedPhylipformat.Rapid213

bootstrapphylogeneticanalysisusing500bootstrapreplicateswascarriedouton62214

isolatesintotal(Table1)usingRAxML-HPCversion7.3.0(Stamatakis2006)asdescribed215

inAbdolrasoulietal.(Abdolrasoulietal.2015):35isolatesfromthisstudyinadditionto216

27isolates(‘non-study’)wereincludedtoshowthephylogeneticcontextoftruerelapse217

infections.Thesenon-studyisolates,whilstfromaclinicalsource,werenotrecurrent218

isolatesandwerenotisolatedaspartoftheclinicaltrialsdescribedintheearlier219

Methodssection.ResultingphylogenieswerevisualisedinFigTreeversion1.4.2220

(http://tree.bio.ed.ac.uk/software/figtree/).Thesameprocesswascompletedforeach221

chromosomeindividuallyforall62isolates,using250replicatesintherapidbootstrap222

analysis.223

GeneOntologyandKEGGpathwayanalysis224

12

Non-synonymous(nsSNPs)mutationsuniquetoeachtimepointforeachpairwere225

assessedforsignificantlyoverrepresentedgeneontology(GO)annotationsand226

metabolicpathways.Briefly,genesfoundtocontainansSNPmutationwere227

interrogatedforoverrepresentedBiologicalProcessOntologyintheC.neoformansH99228

database.GOtermsthatwerefoundtobeassociatedwithgenesmappingtothe229

InterProdomaindatabaseweretransferredtoGOassociations,usingap-valuecut-off230

ofp<0.05.FormetabolicpathwayenrichmentingenescontainingnsSNPs,geneswere231

interrogatedagainsttheKEGG(Kanehisaetal.2016)pathwaysourceforC.neoformans232

H99,usingap-valuecut-offofp<0.05.233

Identifyingsitesunderselection234

BayeScan2.01(Foll&Gaggiotti2008)usesanoutlierapproachtoidentifycandidateloci235

undernaturalselection.Themethodusestheallelefrequenciesthatarecharacteristic236

ofeachpopulationandestimatestheposteriorprobabilitiesofagivenlocusundera237

modelthatincludesselectionandaneutralmodel.Theprogrammethendetermines238

whetherthemodelthatincludesselectionbetterfitsthedata.Thisapproachallowsthe239

simultaneousassessmentoftheinfluenceofbothbalancingandpurifyingselection.240

LociunderbalancingselectionwillpresentlowFSTvalueswhereashighFSTvaluesreflect241

patternsoflocaladaptation(purifyingselection)(Excoffieretal.2009).Analysiswasnot242

undertakenfortheVNIIandVNBlineagesduetolownumbersofisolates,whichwould243

beinsufficienttoovercomethestrongpopulationstructure.VNIIsolatesatday0were244

assignedtoapopulationandtheirassociatedrelapseisolatesconstitutedthesecond245

13

population.Analyseswereconductedusingthestandardparametersincludinga50,000246

burninperiodand100,000iterations.Severalanalyseswereconductedvaryingthe247

priorodds(from10,100to1,000)fortheneutralmodel.248

Results249

Clinicalanddemographicinformation250

Thestudyincludedpairedisolatesfrom17patients,withamedianageof32years(IQR251

26-36)andmedianCD4countatCMdiagnosisof22(IQR9-71)cells/µL.Sixpatients252

weremale,9werefemale,withthegenderoftwopatientsunrecorded.Themedian253

timebetweeninitialandrecurrenceisolateswas115days(minimum55days,maximum254

409days).InthoseforwhomARTstatuswasknown,2of16(13%)patientswere255

alreadyonARTattheinitialCMepisode;6outof15(40%)patientshadnotstartedART256

priortoCMrecurrence.257

DetailedclinicalnoteswereavailablefortherecurrentCMepisodein7patients:two258

(CCTP52andRCT9)hadnotattendedfollowupandneverstartedARTpriortoadmission259

withrecurrence–bothdiedoftherecurrentCM.Onepatient(CCTP32)hadnotbeen260

takingfluconazolefor2weekspriortorecurrence.Infourpatients(CCTP27,CCTP50,261

RCT24,IFNR63)whowereadherenttobothARTandfluconazoleatrecurrencewere262

assessedashavingCMimmunereconstitutioninflammatorysyndrome(CM-IRIS).263

SequencingofpairedsamplesisolatedfrompatientsinfectedwithC.neoformans264

14

Priortosequencing,multiplecoloniesfromasubsetofisolatesincludedinthisstudy265

wereanalysedusingMLSTtoinvestigatewhetheramixedinfectionwaspresentinthe266

originalCSFextract.Theresultsshowthatmixedinfectionswerenotpresentin12out267

of17Pairsincludedinthisstudy.OnePair(Pair7)wasonlytestedonce,andallele268

types(AT)werenotsufficienttoconcludewhethersequencetype(ST)100or196was269

presentinbothoriginalandrecurrentisolate.Ontwoseparateattempts,STsforPairs3270

and17couldnotbedetermined,reflectinganeedforwhole-genomesequencing(WGS)271

tocharacterisethesePairs.STsforPairs1and6wereinconclusive,andsuggestiveofa272

mixedinfectionpresent.273

Werecoveredanaverageof23.9millionreadsfromeachisolate,withanaverageof274

98.8%ofreadsmappedtotheC.neoformansH99referencegenome(Loftusetal.2005),275

andanaveragecoverageof104+/-31.2(standarddeviation).Toenablecomparative276

studiesanddetectmicro-evolutionarychanges,precisevariant-callingwasneeded;277

variantswereidentifiedandfalsepositivelow-confidencevariantswerefilteredoutto278

provideasetofhigh-confidenceSNPs(seeMaterialsandMethods).Fullalignment,279

coverageandvariantcallingstatisticsareprovidedinSupplementaryMaterialsTable5.280

Duetoahighnumberoflow-confidenceSNPsfilteredoutinsomeisolates,whichis281

suggestiveofheterozygousSNPs,variantcallingwasre-runindiploidmode(see282

Methods)forallisolatesinPairs3,4,5and17(resultsinSupplementaryMaterialsTable283

6).284

15

AhighlevelofdiversitywasobservedwithintheVNBlineage,resultinginlongbranch285

lengthsamongstisolateswithinthisclade(Figure1).AlthoughallVNBisolateswere286

mappedtotheC.neoformansH99reference(Loftusetal.2005),whichisaVNIlineage287

isolate,wedonotbelieveSNPnumbersobservedintheVNBlineageareinflatedbythe288

largephylogeneticdistancetothereferencegenome.ThisisbecauseSNP289

determinationrevealedonly21.6%ofSNPsthatwediscoveredweresharedbythe290

threeVNBPairsincludedinthisstudy(Pairs3,12and17),highlightingthelarge291

amountsofgeneticdiversityseenwithinthislineageaswehavepreviouslynoted(58).292

Phylogeneticanalysisshowedthatofthe17pairsofrelapseisolates,threepairswere293

lineageVNB,whilstfourandtenbelongedtolineagesVNIIandVNIrespectively(Figure294

1).TheaveragepairwiseSNPdiversitywasfarhigheramongstisolatesfromtheVNB295

lineage(140,835SNPs)comparedtoisolatesintheVNI(17,808SNPs)andVNII(938296

SNPs)lineages,showingthattheVNIandVNIIlineagesarelessdiversethanVNBacross297

ourcohort.Onaverage,isolatesoftheVNB,VNIIandVNIlineagesaccumulated365,12298

and3uniqueSNPsperdaybetweenthetimeoftheoriginalisolationandtherecurrence299

ofinfection.IsolatesintheVNBlineageweremorelikelytoexperienceaploidyevent,300

withanaverageof1.6changesinploidyperisolate.LessthanoneisolateintheVNII301

andVNIlineageswould,onaverage,experienceploidyevents(0.375and0.26302

respectively).303

Allpairs,withtheexceptionofPair7,wereisolatedfrompatientsinSouthAfrica;Pair7304

wasisolatedfromapatientinUganda.Weclassifiedthesecondisolateasarelapseof305

16

theoriginalinfectionifmorethan97%ofSNPswereincommonbetweenoriginaland306

recurrentisolates.Themajorityofpairshad>99%SNPsimilarity(Table2)between307

originalandrecurrentisolates,withPairs6,7and14displaying97%,98%and97%308

similarityrespectively.Therefore,allpairs,withtheexceptionofPairs3and17(SNP309

similarity44%and56%)couldbeclassifiedasrelapsedinfectionsonthisbasis.This310

confirmspreviousresultsobtainedbyMLST,andthattheoriginalandrecurrentisolates311

sequencedofPairs1and7(whichhadpreviouslydemonstratedapotentialmixed312

infection)wereindeedtruerelapseinfections.313

WithintheVNBpairs(3,12and17),theaccumulationofSNPsbetweenoriginaland314

recurrentinfectionvariedwidely.Weobserved178and304SNPs/dayforCCTP50-d257315

andCCTP50-d409respectively(Pair3),8SNPs/dayforPair12,and968SNPs/dayforPair316

17.DuetothevariationinSNPaccumulationbetweenPair12andPairs3and17,we317

hypothesisedthatPair12wasatruerelapseoftheoriginalinfection,whilstPairs3and318

17areshowinginflatedSNPnumbersduetoreinfectionorananomalousrateof319

evolution.320

Antifungalsusceptibilitytesting321

Fluconazolesusceptibilitytesting(Table1)usingtheEtest®(bioMerieux)wascarriedout322

for12isolates(includingthreepairedisolates)inthisstudybytheaccreditedcentral323

MicrobiologylaboratoryinCapeTownatthetimeoftheclinicalepisode;fiveofthese324

(CCTP27-d121inPair1;CCTP50andCCTP50-d257inPair3;RCT24-d154inPair6;325

IFNR11-d203inPair15)hadMICsabovetheestablishedepidemiologicalcut-offvalue(≥326

17

8μg/ml)forfluconazole(FLC).Allpairswereretestedfollowing5-10yearsfrozen327

storageinglycerolusingtheMICRONAUT-AMsystemforyeastsusceptibility(Methods):328

allwerefoundtobesensitivetoFLC.329

ThefourfoldincreaseinFLCMICobservedinPairs1and3initialandrecurrent330

infectionsprovideasoundbasisforrelapseofinfectionduetodrugresistance:inPair1331

(patientCCTP27),theinitialisolatehadasusceptibleFLCMICof4ug/ml,whilstthe332

recurrentisolatewasresistantatanMICof64ug/ml;inPair3(CCTP50),theinitial333

isolateMICwas16ug/ml(intermediate),whilstahighlyresistantMICof256ug/mlwas334

foundonrecurrenceatday257.335

Serialisolatessharearecentcommonancestor,suggestingrelapseofinfection336

ToinvestigatewhethertheC.neoformansisolatedfromthesamepatientwererelapse337

infectionoftheoriginalisolate,orinfectionwithanewisolate,weundertook338

phylogeneticanalysestodeterminetheirrelationships.339

Asdescribedabove,thehighlevelofcommonSNPs,andsubsequentlowlevelofunique340

SNPs,betweenrecurrentisolatesindicatedthatallpairs,withtheexceptionofPairs3341

and17,wererelapseoftheoriginalinfections(Table2).Phylogeneticanalysis(Figure1)342

confirmedthatallpairs(exceptingPairs3and17)clusteredtogetherwithshortbranch343

lengths,confirmingthelowlevelofdivergencebetweenoriginalandrecurrentisolates,344

thusconfirmingthattheywererelapseoftheoriginalinfections.However,only46%and345

56%ofSNPswerefoundtobeincommonbetweeninitialandrelapseinfectioninPairs346

3and17respectively(Figure1andTable2).TheseVNBpairs(Pair3;CCTP50,CCTP50-347

18

d257andCCTP50-d409andPair17;IFNR23andIFNR23-d179)showedmarkedlylonger348

branchlengths,suggestingeitherreinfectionorelevatedratesofwithin-hostevolution.349

Furtheranalysiswasundertakentoconfirmorrefutethatreinfectionbyadifferent350

isolatewasresponsibleforPairs3and17.Phylogeneticanalysisforallisolatesincluded351

inFigure1wererepeatedforeachofthe14C.neoformanschromosomesindividually352

(SupplementaryFigure1).353

PhylogeneticanalysisofPair3showedthattheoriginalinfectinggenotypeofCCTP50354

washighlyrelatedtotheisolateIFN26(notincludedinthisstudy,butincludedinthe355

phylogenytoassistwithdefininglineages-seeMaterialsandMethods).Allthree356

genotypesfromPair3werefoundtobephylogeneticallyclusteredtogether,butwith357

longbranches(Figure1).Chromosome-by-chromosomeanalysisindicatedthatPair3358

seriallyisolatedgenotypesdisplayeddifferingrelationshipsforeachchromosome,and359

allthreeserialgenotypeswereclusteredtogetheronlyinthephylogenyfor360

chromosome1(SupplementaryFigure1).Allthreegenotypeswerephylogenetically361

similarforthreeotherchromosomes,howeverlongbranchesandclusteringwith362

additionalnon-studyisolatessuggesteddifferingevolutionaryrelationships.Thethree363

seriallyisolatedgenotypesofPair3werecompletelyphylogeneticallydissimilarinthree364

chromosomes;theremainingchromosomessaweithertheday1isolate(CCTP50)and365

day409isolate(CCTP50-d409),ortheday257isolate(CCTP50-d257)andday409366

isolate(CCTP50-d409)phylogeneticallymorerelated.367

19

Pair17isolates(IDIFNR23)clusteredtogetherinonlytwoofthe14chromosomal368

phylogeniesexplored(Chromosomes10and12);intheremainingchromosomal369

phylogenies,thePair17isolateseitherdisplayedaclosephylogeneticrelationship,but370

withlongbranches(6chromosomes),orwerephylogeneticallydistinctfromone371

another,andweremorephylogeneticallyrelatedwithotherstudyoradditionalisolates372

(6chromosomes).373

Microevolutionwithinthehumanhost374

Ourdatapresentauniqueopportunitytoobservemicroevolutionofallthreelineagesof375

C.neoformansinthehumanhost.Althoughmultiplefactorsdetermineevolutionary376

rates,identifyingnon-synonymousSNPs(nsSNPs)thatcauseaminoacidchangeisa377

standardmethodforinferringgeneticdiversityandobservingnaturalselectionon378

codons.379

Lessthan3%ofnsSNPswereuniquetorecurrentisolatesinallpairs,furthersuggesting380

thatallpairsarerelapseoftheoriginalinfection,withtheexceptionoftheVNBPairs3381

and17(15.2%and59.8%ofallnsSNPsareunique,respectively).382

SNPsuniquetoeachtimepointforeachpairwereidentified.AllSNPsatDay1inall383

pairs,andallSNPsattimeofisolateofrecurrentinfectioninallpairs,werecompared.384

NoSNPswerefoundtobecommontoall17pairsateitherDay1oratpointof385

recurrentinfection;however,therewereVNIIandVNBlineage-specific,andtimepoint-386

specific,commonSNPs.387

20

FiveSNPs,allintergenic,werefoundtobecommonatDay1,alongwithfivedifferent388

SNPs,alsointergenic,withinVNBpairs(Pairs3,12and17).ThreeintergenicSNPswere389

commontoallVNIIpairs(Pairs2,4,5and9)attimepointDay1,whilst14SNPswere390

commontoallVNIIpairsatthepointofrecurrentinfection,fiveofwhichwere391

intergenic.Theremaining9SNPswerelocatedinthe5’untranslatedregion(UTR)gene392

SMF1(CNAG_05640),ametaliontransporterwithanaturalresistance-associated393

macrophageprotein.Selectionanalysisindicatedthatthisgenewasnotunderselection394

pressure,however.395

Toevaluatethegeneticdivergence,Wright’sfixationindexes(FST)werecalculatedto396

identifySNPsunderselectioninVNIoriginalandrecurrentinfectionpopulations397

investigating96,856loci(seeMethods).Noputativelociundereitherdiversifyingor398

balancingselectioncouldbedetectedusingafalsediscoveryrate(FDR)of0.05.FST399

valueswerelimitedtonotexceed3.47x10-5.400

Aneuploidyasageneratorofdiversityinrecurrentinfection401

Normalisedwhole-genomecoveragewasplottedtoobservepossibleaneuploidy402

(increaseordecreaseincopiesofchromosomes)andcopynumbervariation(CNV)403

events.Aneuploidyeventswereobservedin7genomepairs,suggestingeither404

interspersedortandemduplicationsoflargesegmentsofthegenome.405

Ormerodetal.(Ormerodetal.2013)previouslypublishedastudyshowingrelapse406

isolatesexhibitinganeuploidiesofchromosome12.Weobservedaneuploidyof407

chromosome12infourpairs(Pairs1,5,10and14)includedinthisstudy.The408

21

aneuploidyspanneddifferentregionsofchromosome12inallpairs,butallaneuploidies409

werepresentintherightarmofthechromosome:inPair5theaneuploidywas410

restrictedto392kbpofonechromosomearm;inPair1,theaneuploidyspannedan411

entirearmofchromosome12(603kbp).Pair14displayedthisaneuploidyinboththe412

initialandrecurrentinfection,andtheaneuploidyspannedthewholechromosome;the413

recurrentinfectionisolateofPair10alsodisplayedaneuploidyalongthewhole414

chromosome.Evaluationofthereaddepthalongchromosome12revealedtriplication415

ofthechromosome12arminthePair1recurrentinfectionisolate,aphenomenonalso416

seeninOrmerodetal.(Ormerodetal.2013).FurtheranalysisofreaddepthinthePair417

5recurrentisolaterevealedadiploidgenome,andchromosome12wasalso418

experiencingtriploidy.CurrentannotationoftheC.neoformansH99genomereveals419

thepresenceof327genesinchromosome12;therightarmofchromosome12has260420

genespresent.Wescannedthegenespresentinthisarmofchromosome12forgenes421

potentiallyinvolvedinvirulence,whichmightproveadvantageoustotheprogressionof422

infectionordrugresistance.OnesuchgenewasSFB2(CNAG_06093),whichisinvolved423

intheconservationofthesterolregulatoryelement-bindingproteinpathway(SREBP)424

(Changetal.2009).Analcoholdehydrogenase(GNO1–CNAG_06168)wasalsopresent,425

whichisthoughttobeinvolvedinthedefenceagainsthostresponse(deJesús-Berríos426

etal.2003).Analysisforenrichmentofmetabolicpathwaysalsorevealedthatthe427

genespresentinthischromosomearmaresignificantlyinvolvedinthemetabolismof428

drugs(correctedp-valuep<3.81e-2).429

22

Wesearchedforcopynumbervariationingenesknowntobeinvolvedindrug430

resistanceandvirulence.CAP10appearedtobehaploidinallisolates,withthe431

exceptionofPairs4and5,wheretheinitialinfection(CCTP52)andrecurrentinfection432

(RCT9-d99)werefoundtobediploid,respectively.However,oncloserinspection(see433

Methods),webelievethattheisolatesinPairs4and5(CCTP52andRCT9-d99)have434

diploidgenomes,implyingthattheCAP10geneisactuallytetraploid.Whetherthe435

remainingisolatesinthesetwopairs(CCTP52-d55andRCT9)havediploidgenomes436

couldnotbedistinguished,however,itisclearthatCAP10losesploidyfrominitial437

infectiontorelapseforPair4,withnoevidenceoflossofheterozygosity(LoH),whilst438

thereverseistrueforPair5.CAP10wasalsofoundtobetetraploid(asthegenomesof439

theseisolateswerefoundtobediploid)inbothinitialandrecurrentinfectionsforPairs440

3and17.441

TheERG11geneonchromosome1wasfoundtohaveincreasedcopynumberin442

numerouspairs(2,3,4,5,9,12,and17),andwasnotfoundtobelineage-associated.443

HoweverthisCNVwasmaintainedthroughoutinfectiontorecurrenceinallpairs,with444

theexceptiontoPair4;sincePair4initialinfection(CCTP52)wasfoundtohaveadiploid445

genome,ERG11wastetraploid,andlostthisploidytobediploidwithrespecttotherest446

ofthegenomeintherecurrentinfection(CCTP52-d55).Whilstchromosome1was447

duplicatedintheinitialinfectionisolateofPair15(IFNR11),ERG11wasfoundtobe448

haploid;theploidyofchromosomewassubsequentlylostintherecurrentinfection449

isolateofPair15(IFNR11-d203).450

23

ERG11inPair4(IDCCTP52)didnothaveanynsSNPsintheoriginalinfection(CCTP52),451

butonensSNPswaspresentinERG11intherecurrentinfection(CCTP52-d55).Clinical452

notesshowthatthepatientfromwhichPair4wasisolatedwasgivenfluconazole(400453

mg/d)oninitialinfection,didnotattendfollowuporreceiveARTorfurtherfluconazole,454

andwasthenre-admittedanddiedfromCMrecurrenceatday55.MICvalueswere455

unfortunatelynotavailableforeitheroriginalorrecurrentisolates.456

NonsensemutationsinDNAmismatchrepairgenescausehypermutatorstates457

Phylogeneticanalysisonachromosome-by-chromosomebasisrevealedthatPair3458

isolatesonlyclusteredtogetherintwoofthefourteenchromosomes;thethreeisolates459

werephylogeneticallydissimilarin4chromosomes,whilstday257and409isolates(Pair460

3CCTP50-d257andCCTP50-d409)werephylogeneticallymoresimilartoeachother461

thantotheday1isolate(CCTP50)infivechromosomes.Day1andday409isolates462

weremorephylogeneticallysimilarthantotheday257isolateinthreechromosomes.463

Thelackofphylogeneticsimilarity(12outof14chromosomes)showninthethree464

isolatesofPair3indicatedthatthesethreeisolatesdonotshowarecentcommon465

ancestor,andprovidesevidenceforreinfectionwithanewisolate,ratherthanrelapse.466

Incontrast,thetwoisolatesinPair17wereonlyphylogeneticallyrelatedforfiveoutof467

the14chromosomes(SupplementaryFigure1),suggestingthatonthisbasisthe468

recurrentinfectionwasdistinctenoughastobedefinedasanon-relapseinfectionin469

thisPair.However,onfurtherinvestigationthiswasfoundnottobethecase.470

24

Weanalysedthecoverageprofilesandsynonymous/non-synonymousratiosofboth471

isolatesinPair17(Figure2c).Althoughtheaneuploidiesobservedwereextensive472

throughoutthegenome,theincreasesinploidyappearedonsimilarchromosomesin473

bothisolates.Asimilarobservationwasseenforthestrikinglyincreasednumberof474

nsSNPsinbothisolatesinPair17:41,549and46,622nsSNPsforIFNR23andIFNR23-475

d179respectively;anevenlargerincreaseinsynonymousSNPswasalsoobserved476

(68,094and82,172synonymousSNPsforIFNR23andIFNR23-d179respectively).We477

thereforesoughttoidentifyamechanismresponsibleforthehighnumberof478

synonymousandnsSNPs,andploidy.479

PreviousstudieshavereportedthatmutationsintheDNAmismatchrepairgeneMSH2480

haveresultedhypermutatoreffectsinbacteriaandtheyeastS.cerevisiae(Drotschmann481

etal.1999).BothPair17isolateswerefoundtoharbourtwononsense(i.e.point482

mutationsintheDNAsequencethatresultinaprematurestopcodon)mutationswithin483

thecodingregionofthegeneencodingMSH2,theDNAmismatchrepairprotein.484

NonsensemutationsinMSH2werenotobservedinanyotherpairsincludedinthis485

study.Thesemutationswereinthesamepositionsinboththeoriginalandrecurrent486

isolates(Ser-888-STOPandSer-86-STOP).487

Wethenperformedagenome-widesearchinbothPair17isolatestoidentifyfurther488

nonsensemutationsinDNAmismatchrepairgenes.BothPair17isolateswerefoundto489

harbourasinglenonsensemutationwithinthecodingregionsofgenesencodingMSH5490

andRAD5.Again,nonsensemutationsinthesegeneswerenotobservedinanyother491

25

pairsincludedinthisstudy.ThesenonsensemutationscausedGln-1066-STOPinRAD5,492

andGln-709-STOPinMSH5inbothoriginalandrecurrentisolates.493

Sincethelikelihoodofsuchmutationsoccurringbychanceinindependentgenomes494

lackingacommonancestorisverysmall,thissuggestthatratherthanbeinga495

reinfection,thiswasindeedarelapseoftheoriginalinfection,andthephylogenetic496

dissimilaritybetweenthetwoisolateswasduetohypermutation.Atotalof293SNPs497

werelocatedinMSH2inbothPair17isolates,comparedtoameanof30SNPsper498

isolateintheremainingPairsthatwestudied(SupplementaryTable2).MoreSNPs499

overallwereobservedintherecurrentisolate(IFNR23-d179)inbothRAD5andMSH5500

(361and357respectively)whencomparedtotheoriginalisolate(IFNR23–320for501

RAD5,305forMSH5).Thesenumbersareconsiderablyhigherthantheaverageof46502

SNPsand37SNPsperisolateintheremainingPairsincludedinthisstudyforRAD5and503

MSH5respectively.504

Discussion505

RelapseofCMcausedbyC.neoformansisusuallyduetothepersistenceandrecurrence506

oftheoriginalinfectingisolate(Spitzeretal.1993),andstudiesoftenfocusonratesof507

within-hostmicroevolutionbetweenseriallycollectedisolates.However,recentstudies508

haveshownthataninfectionofapopulationofdissimilargenotypesisresponsiblefor509

20%ofrelapsecases(Desnos-Ollivieretal.2010).Weusedwhole-genomesequencing510

todistinguishco-infectionsofapopulationofgenotypesfromarelapseofasingle511

genotypeowingtotreatmentfailure(Figure3).UsingWGSwecandistinguishbetween512

26

relapsesofinfectionofthesamegenotype,whichdifferbyonlyafewSNPs,whilstinitial513

infectionbyapopulationofdissimilargenotypeswillseeadifferenceofmanySNPs514

betweeninitialandrelapseinfectionasgeneticdriftoccurs.OurresultsshowthatC.515

neoformansincursnumerousuniquesmallandlarge-scalechangesduringinfection,and516

thatasubsetofthesemayhaveadaptivevalue.Whilstthisstudyisconcernedwiththe517

genomicsofrecurrentinfectionsbyidentifyingSNPchangesandploidypotentially518

involvedinthepersistenceofC.neoformansinfection,futureworkshouldinvestigate519

thepotentialroleofgeneexpressionchangesandgenenetworksinvolvedinchangesin520

fitnessamongstthepopulationsofinfectinggenotypesthatunderpintherecurrenceof521

infection.PreviousstudiesinBrucellainfectionandTBhavehighlightedthemeritsof522

usingtranscriptomicstoidentifypatientsrequiringmoreintensivetreatment(Dufortet523

al.2016)anddifferentiatingbetweendormancyandreactivation(Kondratievaetal.524

2014)respectively.GiventhehighergeneticvariationobservedintheVNBlineage,the525

genomicdatacouldalsobeexploitedtoinvestigategenomecontentvariation,asthisis526

knowntobeamajordeterminantinyeastphenotypicvariation(56).Suchapproaches527

arelikelytoincreaseourunderstandingofclinicalcasesofrecurrentC.neoformans528

infectionsthroughidentifyingthegeneticbasisofphenotypicswitching(D’Souza&529

Heitman2001)andthegeneregulatorynetworksinvolvedinlatency,virulenceand530

resistancetoantifungaltherapies.531

Onepair(Pair3)includedinthisstudydidnotdisplayarelapseoftheinitialinfecting532

isolate.Analysisofthispairshowedthatonly46%ofSNPswereincommonbetween533

theinitialandrecurrentinfection,suggestingthatrelapsewascausedbyanew,albeit534

27

similar,genotype.Theseco-infectioneventsarerarelyreportedinliterature;however,535

Hagenetal.didfindevidenceofco-infectioninasinglepatientusingAFLP(58).The536

extensivechromosomalcopynumbervariations,oraneuploidies,observedinPair3537

(Figure2)alsoshowthatdifferentgenotypeswereisolatedatsubsequenttimepoints538

(days257and409).PhylogeneticanalysesshowedthatthispairbelongstotheVNB539

lineage;itisknownthatapopulationofVNBgenotypescanbefoundinonelocation,540

suchasonthesametree(Vanhoveetal.2016).Therefore,itispossibleforasingle541

immunocompromisedindividualtoinhaleaclusterofbasidiosporesfromasingle542

matingpopulation,whichwouldleadtoaclusterofrelated,butrecombinedgenotypes543

thatthencometodominatetheinfectionatdifferenttimepoints.Althougha544

populationcanresideinanenvironmentalreservoir,recombinationbetweengenotypes545

canoccur,generatingcloselyrelated,yetdistinct,genotypes(Figure3b).Thislatter546

hypothesissupportsourobservationsofdifferingnumbersofnsSNPsbetweenday257547

(CCTP50-d257)andtheoriginal(CCTP50)andday409(CCTP50-d409)isolates,aswellas548

theploidiesandMICvalues(Table1)seenatthetimeofsampleisolation:theday1549

isolate(CCTP50)initiallyhadanintermediateFLCMICof16ug/ml,whilsttherecurrent550

isolateatday257hadahighlyresistantFLCMICof256ug/ml.TheseMICvaluesare551

suggestiveofdrug-resistantgenotypesbeingpresentandselectedforwithinthispatient552

bytheprolongedmaintenanceonFLCmonotherapyfollowinginductiontherapywith553

amphotericinB.ItisalsolikelythatthepopulationofVNBisolatescirculatinginthe554

patientwerenotsufficientlysampledbysequencingonlyonecolonyateachtimepoint,555

andthatdeepersequencingwouldhaveuncoveredgreatergenomicdiversity.556

28

Theoccurrenceofaneuploidy,whereanabnormalnumberofchromosomesisobserved,557

isseenasanevolutionaryprocessthatrapidlyaltersfitness,andhasbeendescribedin558

multiplehumanfungalpathogensasameansofgeneratingdrugresistance(Selmeckiet559

al.2006;Sionovetal.2010).Sionovetal.(Sionovetal.2010)reportedtheduplication560

ofmultiplechromosomesinresponsetohighconcentrationsofFLC,whichresultedin561

genotypesdevelopingFLCdrugresistance.AssociatedgeneduplicationsinC.562

neoformanschromosome1includedERG11andAFR1,whicharebothtransportersof563

azoledrugs.WhilstduplicationsofERG11wereseeninsevenpairs(2,3,4,5,9,12and564

17),thesewerenotnecessarilyassociatedwithanentireduplicationofchromosome1.565

Sionovetal.suggestedthatERG11contributedtotheduplicationofchromosome1566

(Sionovetal.2010);weobservedonlyoneisolate(IFNR11ofPair15)displayinga567

duplicationofchromosome1,butasinglecopyofERG11,suggestingploidywasnot568

completethroughoutthechromosome.Sincethisisolatewastheinitialinfection,we569

canassumethattheduplicationofchromosome1wasnotsolelyduetostressofazole570

drugtreatment,suggestingthatploidycanbeactivatedunderdifferentconditions,such571

asthestressassociatedwithadaptationtothehost.Apossiblelimitationisthatthe572

observedduplicationmaybeduetoprolongedfrozenstorage.573

Ormerodetal.(Ormerodetal.2013)showedananeuploidy(duplication)in574

chromosome12betweenseriallycollectedisolates.Fourpairsincludedinthisstudy575

(Pair1,5,10and14)allshowedaneuploidyinchromosome12;however,Pair14(ID576

IFNR19)displayedthisaneuploidyinboththeinitialandrelapseinfections.Since577

aneuploidiesaretypicallylostuponremovalofdrugpressure(Sionovetal.2010),one578

29

canassumethatthisaneuploidywasmaintainedduetopreviousdrugexposure579

potentiallynotreportedbythepatient,orthataneuploidyhelpsC.neoformansadaptto580

thehostenvironment(Morrow&Fraser2013).Chromosome12experiencedtriploidy581

inthePair1recurrentisolate(CCTP27-d121);thispairalsodemonstrateddrug582

resistancetofluconazole,withaFLCMICof4atinitialinfection,andaFLCMICof64at583

recurrentinfection.Ormerodetal.(Ormerodetal.2013)hypothesisethatthelarge584

numberofgenesaffectedbytheincreasedcopynumberofchromosome12contributes585

tometabolomedifferences;however,wehypothesisecopynumbervariationof586

chromosome12isaresponsetoFLCstress,resultinginincreasedMIC,andthatsome587

genespresentonchromosome12,suchasERG8andCAP6,maybetargetsofazole588

drugsorinvolvedinC.neoformansvirulence.589

Antimicrobialdrugsimposestrongselectionpressureonpathogens,withmayleadto590

theevolutionofdrugresistance(Muetal.2010;Didelotetal.2016);thereare,however,591

fitnesscostsassociatedwiththeevolutionofresistancetoantifungaldrugsthatmay592

impactfitness(Cowenetal.2001).Genome-widescansforsitesunderselectionleads593

totheidentificationofpossiblesitesofdrugresistance.Wedidnotidentifyany594

significantsiteswhencomparingVNIoriginalinfectionversusrecurrentinfection,and595

thenumberofVNBandVNIIisolatesweretoolowforanalysis.Whilsttheseresults596

couldbeinterpretedastherebeingnositesunderselectionintheVNIisolatessampled597

inthisstudy,itismorelikelythatsimilarpatternswouldnotbeseenamongst598

individualsduetostochasticityandclonalinterference(Didelotetal.2016).Itisalso599

likelythatasthereislittlerecombinationinVNIisolatescomparedtoVNBandVNII600

30

isolates(Khayhanetal.2013;Litvintsevaetal.2011;Litvintseva2005)andtherefore601

linkageiscompleteacrossthegenome,furtherhamperingselectionanalysis.We602

thereforefoundnoevidenceforgeneticallydeterminedalterationsindrugresistancein603

thestudyisolates.604

MICvalueswereonlyobtainedfor9outof35isolatesinthisstudyatthetimeof605

sampling.Susceptibilitytestingatalaterdaterevealedalltheisolatestobesusceptible606

toantifungaldrugsincludingFLC,suggestingthatanyresistantphenotypeshadbeen607

lostintheabsenceofdrugselectivepressure.Itisthereforeimportantforcliniciansto608

requestsusceptibilitytestinginrealtime,attheveryleastinallcasesorrecurrentCM.609

WhilstPair17didnotexhibitahighpercentageofcommonSNPsbetweentheoriginal610

andrecurrentisolatesindicativeofarelapseinfection,theelevatedrateofSNPs611

observedinallchromosomesofbothisolatessuggestedthiswasnotare-infectionas612

seeninPair3(Figure3c).Rather,ourresultssuggestthattheisolatesinthispairwere613

exhibitingahypermutatorphenotype,asaresultoftwononsensemutationsintheDNA614

mismatchrepairgeneMSH2,andonenonsensemutationineachoftheDNAmismatch615

repairgenesRAD5andMSH5.Whilstpreviousstudieshaveshownhypermutator616

phenotypesaidadaptationtostress(Magditchetal.2012),andwehypothesisethat617

hypermutationmayleadtoadaptationofdrugresistanceunderthestressofantifungal618

treatment.Theseresultsarethefirsttotheauthors’knowledgetoreportonnonsense619

mutationsinMSH2,RAD5andMSH5inC.neoformans.Furtherinvestigationisrequired620

todeterminewhetherthesenonsensemutationshavearoleindrugresistance621

31

phenotypesusingtranscriptomicapproachesandcreatingsingle-geneknockout622

mutantsofMSH2,RAD5andMSH5.Itisalsonecessarytotestthevirulenceof623

hypermutatorisolatesinthemousemodelandtodescribetheimpactoftheincreasein624

mutationratethatoccuraresultofthishypermutation.Ourstudyonlyincludesone625

pairofhypermutatorgenotypes,sofurthersamplingisrequiredtoidentifywhetherthis626

phenomenonisspecifictotheVNBlineage,whetherhypermutatorsoccurintheVNII627

andVNIlineages,andwhethertheyareclinicallyrelevant.628

Thisworkrepresentsthemostextensivecomparativegenome-sequencingbasedstudy629

toinvestigatemicroevolutioninseriallycollectedisolatesofC.neoformanstodate.The630

observationofaninfectionofasinglepatientwithapopulationofVNBisolatesis631

clinicallyrelevant,aswidelyuseddrugregimenswithazolemonotherapymaynotbe632

effectiveagainstsuchageneticallydiverseinfection.Itisalsolikelythattheextensive633

geneticdiversityseeninclinicallyisolatedVNBisolatesmaybeduetomixedinfection.634

HypermutationduetononsensemutationsintheDNAmismatchrepairgenesMSH2,635

RAD5andMSH5causeanincreasedmutationandrateofaneuploidyinC.neoformans,636

whichmayconferanincreasedabilitytoadapttodrugpressure.Furthersamplingis637

requiredtoidentifywhetherhypermutationisaphenomenononlyobservedintheVNB638

lineage,andhowthesemutationsimpactthefitnessofC.neoformansbyimposinga639

highgeneticload.640

Acknowledgements641

SpecialthankstoMr.IanWrightforhisfinancialcontributiontothisproject,andDr.642

32

WinnieWuandStarLabUKforsupplyingfilterpipettetipsfreeofcharge.Thisworkwas643

fundedbyaMedicalResearchCouncilgrant(MRCMR/K000373/1)awardedtoMCFand644

agrantfromImperialCollegeLondon(WPIA/F24085)awardedtoJR.JJwassupported645

bytheWellcomeTrust(trainingfellowship,WT081794)andissupportedbyagrantfrom646

thePennCenterforAIDSResearch(CFAR),anNIH-fundedprogram(P30AI045008).647

ThankyoutoSeanLoboandAhsanAwaisBokhariforinitialworkonMICsandMLST,648

respectively. 649

Authorcontributions650

TB,JJ,JS,AR,GMandTSHconductedtheclinicalstudiesandprovidedisolatesincluded651

inthisstudy.TB,JJandMCFconceivedtheresearchquestion.JRandMBconceivedthe652

experiments;MBperformedlibrarypreparationsforsequencing,withassistancefrom653

MV.JRperformedalignments,variantcallinganddownstreamanalysis,andwrotethe654

paper.MVperformedBayeScananalysis.Allauthorsreadandcontributedtothispaper.655

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830

831

832

38

Figurelegends833

834

Figure1–PhylogeneticanalysisofC.neoformansvar.grubiiisolatesinthisstudy835

(coloured),withadditionalisolates(showninblack)addedtodistinguishtruerelapse836

infections,orrecurrentinfections,andassociatedlineages.Wehypothesisethat837

isolatesresultingfromtruerelapseinfectionswouldbecloselyrelatedphylogenetically.838

Bootstrapanalysisover500replicateswasperformedonWGSSNPdatafrom62isolates,839

includingthe35isolatesincludedinthisstudy,togenerateanunrootedmaximum-840

likelihoodphylogeny,withallbranchessupportedto69%orhigher(withtheexception841

toaparticularlyclonalVNIclade,includingPair15only,whichonlyhad47%branch842

support).BranchlengthsrepresentthenumberofSNPsbetweentaxa.843

39

844

Figure2–Extensivechromosomalcopynumbervariationwasobservedinallisolatesin845

Pairs3and17,whencomparedtoH99.Pair2isincludedtoillustrateisolateswithout846

ploidyandextensivensSNPs.Here,normalisedwhole-genomedepthofcoverageis847

shown,averagedover10,000-bpbins,inscatterplots.Barplotsrepresenttheposition848

ofnsSNPs.Thepurpletrackrepresentstheoriginalisolate,orangetherecurrentisolate,849

andgreen(inthecaseofPair3)forthefinalrecurrentisolate.a)Noincreaseinploidyis850

40

observedineithertheoriginalorrecurrentisolateofPair2,andasmallnumberof851

nsSNPsareseen.b)IncreaseinploidyisobservedinmanychromosomesintheDay1852

isolateforPair3,someofwhicharelostovertime.AlargenumberofnsSNPsare853

observedinallchromosomesinisolatesofPair3,withchromosome6beingthe854

exception:veryfewnsSNPsarelocatedinchromosome6inCCTP50andCCTP50-d409,855

whereasover2000nsSNPsareobservedinchromosome6inCCTP50-d257.c)againin856

ploidyisobservedforChromsomes2,4,6and9comparedtotheDay1isolateinPair17,857

whereasploidyremainsunchangedforChromosomes1and12.858

41

859

42

Figure3–HypothesesofroutesofinfectionofthehumanhostbyC.neoformansvar.860

grubii.a)Inhalationofasinglepopulationofbasidiosporesintoanewhost.Duetolow861

within-hostdiversityandbeingdrugnaïve,therewillbeabottleneckinpopulationsize862

duetoantifungaldrugtreatment.However,iftheinitialdrugregimenisinsufficientto863

steriletheCSF,resistancemaydeveloponFLCmaintenancetherapyduetoselection864

pressure,resultinginrelapsedinfectionfromproliferationofadrug-resistantisolate.b)865

VNBlineageC.neoformansexiststheenvironmentasapopulation,whichcanundergo866

recombinationtoproducegeneticallysimilarisolates,butwithsignificantlydiversity.867

Duetotransmissionbottlenecks,onlyasampleofthepathogendiversitywillbe868

transferredtothehost,inthiscase,byinhalation,butitispossibleforapopulationofC.869

neoformanstoinfectasingleimmunocompromisedindividual.Someisolatesmaybe870

susceptibletoantifungaldrugsandarethusbecomingremoved,whilstotherisolates871

maybeinherentlyresistantandhencecausearelapseinfection.c)Mutationsinthe872

DNAmismatchrepairgeneMSH2causeanisolatetobecomeahypermutator.Some873

genotypesmaybesusceptibletoantifungaldrugs,butthehighmutationrateallowsthe874

infectiontoadaptrapidlytothehostandevolvedrugresistance.Thesegenotype875

proliferateinthehost,thuscausingrelapseinfection.876

Tables877

Table1:DetailsofC.neoformansisolatesandMICs(ifavailable)attimeofisolationfrom878

SouthAfricaandUganda(Pair7only)usedinthisstudy.AmB=amphotericinB1879

43

mg/kg/d,asperhospitalguidelinesatthattime,unlessotherwisestated;VOR=880

voriconazole300mg/d;5FC=flucytosine;FLC=fluconazole.881

Pair# IsolateID Dayofisolation FluconazoleMIC

atisolation

TreatmentofCM

episode(ifknown)

1 CCTP27 1 4 AmB3days

1 CCTP27-

d121

121 64 VORuntilCD4>200

cells/uL

2 CCTP32 1 4 AmB7days

2 CCTP32-

d132

132 6 AmB7days

3 CCTP50 1 16 AmB14days

3 CCTP50-

d257

257 256 AmB14days

3 CCTP50-

d409

409 n/a AmB7days

4 CCTP52 1 n/a FLC400mg/d

4 CCTP52-d55 55 n/a FLC400mg/d

5 RCT9 1 2 AmB0.7mg/kg/dplus

5FCfor14days

5 RCT9-d99 99 n/a AmBuntildeath

6 RCT24 1 4 AmBplus5FCfor14

44

days

6 RCT24-d154 154 12 FLC800mg/d

7 1600-1 1 n/a FLC16000mg/dfor14

days

7 1600-1-d106 106 n/a

8 IFNR63 1 n/a AmBplus5FC

8 IFNR63-d128 128 n/a

9 IFNR24 1 n/a AmB7days

9 IFNR24-d101 101 n/a

10 IFNR18 1 n/a AmB7days

10 IFNR18-d134 134 n/a

11 IFNR14 1 n/a AmB7days

11 IFNR14-d97 97 n/a

12 IFNR13 1 n/a AmB7days

12 IFNR13-d95 95 1

13 IFNR6 1 n/a AmB7days

13 IFNR6-d73 73 1

14 IFNR19 1 n/a AmB7days

14 IFNR19-d111 111 n/a

15 IFNR11 1 n/a AmB7days

15 IFNR11-d203 203 12

45

16 IFNR27 1 n/a AmB7days

16 IFNR27-d204 204 n/a

17 IFNR23 1 n/a AmB7days

17 IFNR23-d179 179 n/a

882

Table2:AhighnumberofsharedSNPsinmostpairsindicateasharedcommonancestor.883

NumberofSNPscommontobothinitialandrecurrentinfection,alongwithnumberof884

SNPsandnon-synonymousSNPsuniquetoeachtimepoint.Percentagesgivento2d.p.885

Pair CommonSNPs

(%total)

Day1SNPs

(%total)

No.Day

1

nsSNPs

(genes

mapped

)

RelapseSNPs

(%total)

No.Relapse

nsSNPs

(genes

mapped)

Relapse#2

SNPs(%total)

No.Relapse

#2nsSNPs

(genes

mapped)

1 13490(98.49) 97(0.71) 9(8) 110(0.81) 19(8)

2 289557

(99.29)

1080(0.37) 110(55) 991(0.34) 108(45)

3 261718

(46.77)

127631

(32.78)

25331

(2741)

45769(14.88) 8342(2099) 124498

(32.24)

23957

(2541)

4 289574 1169(0.40) 145(58) 1096(0.38) 145(56)

46

886

Supplementarymaterial887

TableS1-MLSTresultsofindependentlytestingthreecoloniesperstudyisolate.888

(99.22)

5 289367

(99.20)

1261(0.43) 127(54) 1080(0.37) 127(59)

6 13122(95.78) 133(1.00) 5(2) 445(3.28) 82(56)

7 47833(97.83) 522(1.08) 81(65) 537(1.11) 94(72)

8 46883(98.76) 294(0.62) 29(9) 296(0.63) 26(16)

9 289458

(99.27)

1033(0.36) 141(58) 1109(0.38) 105(55)

10 12687(98.29) 104(0.81) 4(3) 117(0.91) 11(5)

11 12732(98.38) 110(0.86) 10(2) 99(0.77) 6(5)

12 221973

(99.37)

636(0.29) 55(27) 778(0.35) 61(30)

13 48062(98.58) 396(0.82) 31(11) 294(0.61) 28(15)

14 28960(95.66) 288(0.98) 44(19) 1026(3.42) 105(57)

15 29736(98.40) 254(0.85) 21(14) 228(0.76) 17(13)

16 45437(98.56) 325(0.71) 30(18) 341(0.74) 30(14)

17 376568

(56.43)

117436

(23.77)

22567

(4153)

173359

(31.52)

33543

(4427)

47

TableS2-NumberofSNPsinMSH2ineachisolate,categorisedbytypeorlocation.SYN889

=synonymousSNP,NSY=non-synonymousSNP,p5UTR=5’UTR,p3UTR=3’UTR,890

NON=nonsensemutation.891

TableS3-Non-synonymousSNPsinPair3isolates,perchromosome892

TableS4-Numberofnon-synonymousandsynonymousSNPsperisolate893

TableS5-Detailsofalignmentandvariantcallingforallpairedisolatesincludedinthis894

study895

TableS6-NumberofhomozygousandheterozygousSNPsinisolatesofPairssuspected896

ofdiploidy.897

FigureS1–PhylogeneticanalysiseachchromosomeofC.neoformansiisolatesinthis898

study(coloured)withadditionalisolates(showninblack),toidentifywhetherPair3899

consistsofdifferentisolatesorisarelapsedinfection.WehypothesisedthatifPair3900

wasarelapsedinfection,allisolateswouldsharethesamephylogeneticrelationshipin901

all14chromosomes.Rapidbootstrapanalysisover250replicateswasperformedon902

SNPdatafrom62isolatestogenerateanunrootedmaximum-likelihoodphylogeny(only903

branchesnotsupportedto100%areindicatedabovebranches).Branchlengths904

representthenumberofSNPsbetweentaxa.905

FigureS2–Chromosomalcopynumbervariationcanbeobservedinsomeisolatesin906

Pairs1,4,5,10,14and15,whencomparedtotheC.neoformansreferencegenome,907

H99.Normalisedwhole-genomedepthofcoverageisillustratedhere,averagedover908

48

10,000-bpbinsandrepresentedasascatterplot.Barplotsrepresentthepositionof909

nsSNPs,wherethepurpletrackrepresentstheoriginalisolate,andorangerepresents910

therecurrentisolate.NotethatPairs2,3and17arenotpresentinthisFigure,asthey911

arerepresentedinFigure2.912