+Diagnosing Balamuthia mandrillari Encephalitis With Metagenomic De

RESEARCHARTICLEDiagnosingBalamuthMD,1,agMD,PhD,,lMD,PhD,dMD,MAS,BS,3BarlasBenkliBA,5BrianD.O’DonovanMS,,eatingDVM,ackePhD,,PhD,6AndrewBollenMD,PhD1,2Objective:Identificationofaparticularcauseofmeningoencephalitiscanbechallengingowingtothemyriadbacte-ria,viruses,fungi,andparasitesthatcanproduceoverlappingclinicalphenotypes,nomicdeepsequencing(MDS)approachestoinfectiousdiseasediagnosticsareknownfortheirabil-itytoidentifyunusualornovelvirusesandthusarewes:Wepresentthecaseofa7ercerebrospinalfluid(CSF)s:SequencesaligningtoBalllarisinCSF,braintissue,andvitreousfluidfromthepatient’llarisserologyandimmunohistocretation:ThediagnosiswasmadeusingMDSafterthepatienthadbeenhopowerfuldiagnostictoolwiththepotentialforrapidanduROL2015;00:000–000Meningoencephalitispresentssignificantdiagnosticchal-lengesowing,inpart,ncludeinfectious,inflammatory,paraneoplastic,andtoxic-metabolicetiologies,rmore,confirminganinfectiousetiologycanbechallenging,giventhatmanypathogen-specificserologicalandpolymerasechainreaction(PCR)assaysareexpensive,time-consuming,e-basedapproaches,thoughtheycanoftendetectmultiplespecies,arealsolimited,giventhatmanyinfectiousardcnomicsisthestudyofgeneticmaterialrecov-eredfromanenvironmentalsample,dvancesinsequencingtechnologyandbioinformaticshavemadean:10.1002/ana.24499ReceivedJun3,2015,andinrevisedformJul18,edforpublicationAug2,scorrespondencetoDrMichaelWilson,DepartmentofNeurology,UniversityofCaliforniaSanFrancisco,1500OwensStreet,SanFrancisco,CA94158.E-mail:@mthe1DepartmentofBiochemistryandBiophysics,UniversityofCalifornia,SanFrancisco,CA;2HowardHughesMedicalInstitute,ChevyChase,MD;3DepartmentofNeurology,UniversityofCalifornia,SanFrancisco,CA;4DepartmentofMedicine,DivisionofInfectiousDiseases,UniversityofCalifornia,SanFrancisco,CA;5KocUniversity,Istanbul,Turkey;6DepartmentofPathology,DivisionofNeuropathology,UniversityofCalifornia,SanFrancisco,CA;7CaliforniaDepartmentofPublicHealth,ViralandRickettsialDiseaseLaboratory,Richmond,CA;8Free-LivingandIntestinalAmebasLaboratory,CentersforDiseaseControlandPrevention,Atlanta,GA;and9InfectiousDiseasesPathologyBranch,CentersforDiseaseControlandPrevention,Atlanta,GAThisisanopenaccessarticleunderthetermsoftheCreativeCommonsAttribution-NonCommercial-NoDerivsLicense,whichpermitsuseanddistributioninanymedium,providedtheoriginalworkisproperlycited,theuseisnon-commercialandnomodificationsoradaptationsaremade.1C2015TheAuthorsAnnalsofNeurologypublishedbyWileyPeriodicals,lfofAmericanNeurologicalAssociation.V

ANNALSofNeurologyapproachtometagenomicsanincreasinglyfeasiblemethoiouslyreportedontheabilityofmeta-genomicdeepsequencing(MDS)ofcerebrospinalfluid(CSF)toidentifythecauseofinfectiousmeningoencephali-tisinrealtime,leadingtoadramaticimprovementinapatient’smedicalcondition.1Here,wedescribeacaseofmeningoencephalitisina74-year-oldwomanthatillus-tratestheabilityofaienthadhadanextensivediagnosticworkup,includingtwobrainbiopsies,ybeforeherdeath,asampleofCSFwassub-mittedforMDS,whichledtotheidentificationofBala-muthiamandrillaris,agnosiswaslatercon-firmedbytheCentersforDiseaseControlandPrevention(CDC),theCaliforniaDepartmentofPublicHealth(CADPH),andanalysisofadditionalimvidesanefficientandunbiaseddiagnosticapproachtoidentifyculpritcentralnervoussystem(CNS)infectionsandmaygeneratelargecostsavingswhenusedtoimprovediagnosisratesinpatientswithcostlyconditionssuchasmeningoencephalitis.2EarlytreatmentguidedbyrapidandaccuratediagnosismayalsoimproveoutcomesincasessuchastheseinwhichportA74-year-oldChinesewomanwithahistoryofrheuma-toidarthritistreatedwithhydroxycimmigratedtotheUnitedStatesfromChinaapprox-notusetobacco,alcohol,4weeksprecedinghospitalizationatourinstitution,occasion,shecondadmission,shewasdiagnosedwithendophthalmi-tisandtreatedempiricallywithintravitrealvancomycin,ceftazidime,andvoriconazole,thoughvitrealandbloodcultureseventuallyshowednogrowth,andPCRsonthevitreousfluidforherpessimplexvirus,cytomegalovirus,-tion,shewasstartedontherapyforToxoplasmagondiiinthesettingofapositiveserumimmunoglobulinM(IgM)titer(seeTable).Althoughhervisiondidnotimprove,shewasbrieflydischarged,butthenbroughtbacktothehospitalforathirdadmissionwhenacervicalspinemag-2neticresonanceimaging(MRI)studyperformedtoeval-uatespinaquentbrainMRIshowedsmallareasofrestricteddiffusioninmultiplevas-cularterritoriesconsistentwithinfarcts(seeFigA).Astrokeworkup,includingatransesophagealechocardio-gram,ientwasdischargedathirdtime,ghshehadbeenruledoutforacid-fastbacilliduringhersecondhospitalization,thepatientpre-sentedtoourinstitution2dayslaterforanevaluationatthecity’stuberculosis(TB)cliBclinic,itwasfoundthatherneurologicalstattransferreddirectlytotheemergencydepartmentwherehervitalsignswere:temperature,1038F;heartrate,111beatsperminute;bloodpressure,123/81mmHg;andoxygensaturation,93%edallfourextremitiesspontaneously,butwasunabletofollowcommandsorverbalize,point,shewasintubatedforairwayprotectionandadmittedtotheintensivecareunit(ICU).Initiallaboratoryevaluationrevealedawhitebloodcellcountof12.63103cells/lL,hemoglobinof13.7g/dL,serumsodiumof129mEq/L,andserumglucoseof135mg/lysisdemonstrated13redbloodcells/mm3,347whitebloodcells/mm3(57%neutrophils,11%monocytes,31%lymphocytes,and1%eosino-phils),glucose30mg/dL,andprotein71mg/RIdemonstratedinnumerableringenhancinganddiffusion-restrictedsupra-andinfratentoriallesionsthatwerefelttobemonherinitialclinicalandradiographicpre-sentation,shewasinitiatedonbroadspectrumantimicro-bialandanti-toxoplasmosiscoverageincludingvancomycin,cefepime,metronidazole,pyrimethamine,sulfadiazine,,urine,andCSFbac-terialandfungalcultureswereeventuallynegative,aswereCSFtestsforneurosyphilis,TB,andCryptococcus(seeTable).ebroadantibioticcoverage,shecontinuedtodeterioratewithprogressiitalday10,arepeatlumbarpunctureshowedworseningpleocytosiswithamoreprominenteosinophilia(seeTable).Giventhesefindings,broad-spectrumantibioticswerestoppedonday10,Volume00,No.00

iousDiseaseDiagnosticTestResultsMonth2015TestSiteFirstHospNegative11.4IU/mL(<7.9IU/mL)NegativeNegativeNegativeNegativeNegativeNegativeNegativeNegativeNegativeNegative(32)Negative(32)NegativeNegative(4)NegativeNegative(39)NegativeNegativeNegativeNegativeNegativeNegative(33)NegativeNegativeNegative(32)Negative(32)Negative(33)NegativeNegative(35)NegatIgGAbSerumSerumSerumTrachealaspirateVitreousVitreousVitreousVitreousVitreousVitrIgMELISA(1-3)-PCRHSV-1,-PCRFungalcultureFungalcultureBacterialcultureHIV-1AbHIV-1viralloadHepatitisBsurfaceAgHepatitisBcoreIgMAbHepatitisCAbGramstainVZVPCRandIgGVDRLCryptococcalantigenAFBcultureWilsonetal:AmoebicEncephalitisIdentifiedbyMetagenomicsBacterialculture3

4TABLE:ContinuedANNALSTestCSFCSFCSFCSFUrineUrineSerumTrachealaspirateTrachealaspirateBloodSerumSputum,inducedSputum,inducedBrainBrainBrainBrainBrainBrainSerumNegativeNegativeNegativeNegativeNegativeNegativeNegativeNegativeMildgliosisVasculitis,encephalitisNegative(32)Negative(32)Negative(32)Negative(32)NegativeOralflora(32)Negative(31);Oralflora(31)Negative>gativeNegativeNegative(34);MixedGram1bacteria(31)NegativeNegativeNegativeNegative(32)SiteFirstHospSeconPCRRPRBacterialcultureGramstainFirstpathologySecondpathologyBacterialcultureFungalcultureAFBcultureGramstainVolume00,llarisIgG(CADPH)Borderlinepositive(1:32)

Wilsonetal:AmoebicEncephalitisIdentifiedbyMetagenomicsMonth2015)ANas)D;missdeidousaveeeeeeeuevetrlivvvvvvliivprntviiiiiiftcegaitttttttaaiiiiiixditagiefiessssssswgrtlpADNaePoPoPoPoPoPoPorpa(Nxe(mris5sRepPrReh;y.5roAtVaNrSoRHblaal;mnohocsitrocabaeiesrhrer5tnerpiusasAoohaeNcsFHiRedrs;alrayeermrtesiynlemovep55hcoLtrdpRRsiishoCDhoPTHVn;us;unmleligmciiatbna5tsa5CfgHIdA;ilc;asourd5trinnBvoopsFyCcAcneseoSH;eayiaceissfiseadDtornnoeufbrmsrosmetoinneunaCmm5muihCtpssdroe5Dk1CFiHn;ilV-ht-IleamHe;ysHzunrcieilvbssuu5rueoAtPsFnnnnoeSfoeiiiirIotSaaaaFFertLz-tiiSCrrrrSStiBBBBCCVVEalnle;eycmdirtoarabvpiten5aD))bb5VZaiaabVnrLLA;osfi;l)udRR)iiassissisnrieRCCCC8Cs8CoivCu1Dtoal5nCPPHHII1D81R8e1ReszaiPsssssiiiiiCsDsiCigtrrrrr(ir(iDr(r(leHniiaaaaaaatPonllllliml)ll)lllRallRallRlRCaiiiiiipoDlrrrrrrstldddCirdCirCdCoyA:e)PHd)PHd)PPdPnPhcEtsDnA55C;.C.C.C.C.R..RsoMga(B(B(B(B(HCer5

ANNALSofNeurologyFIGURE:Neuroimagingandneuropathology.(A)AxialT2-weighted/fluid-attenuatedinversionrecovery(FLAIR)brainmagneticresonanceimaging(MRI)demonstratingmultiplehyperintensities(arrows),whichcorrespondtoareasofrestricteddiffusionondiffusionweightedimaging(notshown).(B)AxialT2-weighted/FLAIRbrainMRIdemonstratingprogressioninthesizeandnumberofhyperintensities(arrows)visualizedintheMRIin(A).(C)PostcontrastaxialT1-weightedbrainMRIdemonstratingmultipleringenhancinglesions(whitearrow)andbasilarmeningitis(yellowarrow)aswellasseverehydrocephalus.(D)Periodicacid-Schiffstainedsectionofbrainparenchymashowsnecrotizingvasculitis,chronicinflammatorycells,andapopulationofamoebictrophozoitesthatcloselyresemblemacrophages(arrows);orityofthetissueshowedarobustnecrotiz-ingvasculitiswithmixedinflammation,includingmacrophagesandnumerouseosinophils.(E)llaristrophozoites;4003(courtesyofM.K.K.,CDC).biopsyperformedonday4wasnondiagnostic,reveatbiopsy1weeklater(day11)demon-stratedaseverenecrotizingvasculitisofunclearetiology,withaprominentcomponentofeosinophils,chronicinflammatorycells,andneutrophils,brainMRIonhospitalday14demonstratedanincreaseinthenumberandsizeofherbrainlesionsaswellasintervaldevelop-mentofbasilarmeningitis(seeFigB,C).Herworseningcerebraledemawasrefractorytomanagementwithhyper-osmolartherapy,aswellastoientultimatelybecamehemodynamicallyunstable,likelyowingtoelevatedintracranialpressure,ilyoptedforcomfortcaremeasurestobeinstituted,ntificationofpotentialpathogensapprovedbytheinsti-tutionalreviewboardsofSanFranciscoGeneralHospital(SFGH)andtheUniversityofCaliforniaSanFrancisco(UCSF).SampleswereprocessedforMDSanalysis,asprevi-ouslydescribed.1Briefly,swererandomlyamplifiedtodouble-strandedcDNAusingtheNuGENOvationv.2kit(NuGEN,SanCarlos,CA)forlownucleicacidcontentsamples,andMDSlibrarieswereconstructedusingtheNexteraprotocol(Illumina,SanDiego,CA),aspreviouslydescribed.1LibrarysizeandconcentrationweredeterminedusingtheBluePippin(SageScience,Beverly,MA)andKapaUniversalquantitativePCRkit(KapaBiosys-tems,Woburn,MA),sweresequencedonanIlluminaHiSeq2500instrumentusing135/135-base-pair(bp)red-endsequenceswereanalyzedforpathogensusingarapidcomputationalpipelineforpathogendetection,sIdentificationofBalamuthiaSequencesinCSFTheMDSoftotalRNAfromthepatientsampleyielded19,642,awereanalyzedusingarapidcomputationalpipelineVolume00,No.00MaterialsandMethodsOnday11ofthepatient’shospitalization,asampleofCSFwassubmittedforunbiasedMDSunderaresearchprotocolfor6

Wilsonetal:AmoebicEncephalitisIdentifiedbyMetagenomicsdevelopedatourinstitutiontoclassifyMDSreadsandiden-tifypotentialpathogensbycomparisontotheentireNationalCenterforBiotechnologyInformation(NCBI)nucleotide(nt)y,paired-endreadswerequalityfilteredusingPriceSeqFilter,acomponentofthePRICEassembler,followedbyalignmenttothehumanreferencegenome(hg19)usingbowtie2v2.2.4.3,4Unalignedreadsthatwereatleast95%identicalwerecompressedbycd-hit(v4.6.1),resultinginatotalof33,093remainingreads,repre-sentingapproximately0.1%oftheoriginaldata.5,6ThesereadswerethenusedasqueriestosearchtheNCBIntdata-baseusinggsnapl(v2014-10-16).7Over97%ofthesereadsyieldedgsnaplalignmentstovertebratesequences(humanreadsnotfilteredbybowtie),iltering,lla-risasthemosthighlyrepresentedorganism,-nineotherindivid-uallybarcodedpatientCSFsamples(includingawatercontrol)wereincludedinthesamesequencingrun,andnonecontainedreadsthatalignedtoBalamuthia,nesultsinformedaretrospectivebioinformaticanalysisoftheoriginaldatagnmentyielded

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