Alere-i-Isothermal-Amplification

AlereiIsothermalAmplificationNormanMoore,[e-mail]...“it’sjusttheflu”AbitofhistoryTherearefluepidemicsevery1to3yearsforatleastthelast400years.Pandemics(worldwide)occuraroundevery10to20years.HistoryHippocratesdescribedflubackinthe5thcentury.Columbusbroughtadevastatingfluonhissecondvoyagetothenewworld.Spanishfluof1918-1919wasthesinglegreatestepidemicinhistory.•50to100millionpeoplewerekilled(3-6%oftheworld’spopulation!)•Another500millionwereinfected(1/3rdoftheworld’spopulation)InfluenzaDiseaseBurdenEstimatedaverageglobalburdenofseasonalinfluenza•600millioncasesperyear•3millioncasesofsevereillness•250,000–500,000deathsConcernishighestin•Theveryyoung,theelderly•personswithunderlyinghealthconditions•pregnantwomenWHOReport:AcuteRespiratoryInfections(UpdateSeptember2009)BurdenonHealthcare&IndustryFlucauses30-50%increaseinprimarycareconsultations2-3foldincreaseinhospitaladmissionsduringepidemics10%ofallsicknessabsencesfromworkImpactinindustrialisedcountries:•US$10-60million/millionpopulationLostproductivity:•US$12billion/annuminUSaloneInfluenzaAversusInfluenzaBInfluenzaA•MoreseverediseasethanB•CancausediseaseinawidevarietyofanimalsInfluenzaB•Causesamilderflu,usuallyinthespringmonthsTreatingRespiratoryDiseasesintheEmergencyDepartmentIsthepathogenbacterialorviral?InfluenzaandpneumoniasymptomscanoverlapdramaticallyWhodoyoutest?Ifitisfluseason,doyoutestforotherpathogens?Whatdoyoutestthemfor?Differentagegroupsarelinkedtodifferentpathogens.Cantreatmentbeimpactediftheappropriatetestingisdone?Stopindiscriminateusebroadspectrumantibiotics.MisuseofAntibioticsCanLeadtoOtherMedicalIssuesRespiratoryissuescantreatedwithfluoroquinoloneDisruptsnormalintestinalfloraO27strainofC.difficileisspecificallyresistanttofluoroquinoloneDiagnosticMethodsforInfluenzaCultureDFAPCRRapidTestsIssueswithClinicalSamplesViraltiterishighestinfirst48hoursPropersamplecollectionisnecessaryDilutionintransportmediaRapidTestsPro•Teststakeminimaltime•SometestsaresosimplethattheycanbeCLIA-waived•Canbeusedtotriagepatients•Positiveresultscanbeusedtoruleoutotherissueslikepneumoniasodon’tgiveunnecessarychestx-ray,antibiotics,etc.Con•Performanceisnotasgoodasculture,PCR,orDFAMolecularAssaysPro•Forrespiratoryspecimens,highperformance•SamedayresultsCon•Turnaroundtimefromlabmaybeextensive,especiallyifbatchingspecimens•Expensive•Mayrequireexperiencedtechnicians,labs,dedicatedequipment,etc.Theneedisatestrapidenoughtotriagepatientslikearapidtestwithmolecularresults.WhatisAlerei?AlereTMiisamoleculardiagnostictestusingisothermalamplificationIsothermalamplificationisnotPCRbutofferssimilarsensitivityandspecificityUsesaconstantandlowtemperaturesothereisnoneedforthermalcyclerVeryrapidamplification(manytargetsamplifiedinonly5-10minutes)DoesnotrequireDNApurificationstep,whichaddstime,complexityandcostAbilitytodeployanincrediblysensitivetestforinfectiousdiseasesatthePOCWhatisIsothermalAmplification?DefinitionNEAR,orNickingEnzymeAmplificationReaction,enablestheisothermalunwindingandsubsequentamplificationofverysmallampliconsusingasetoftarget-specifictemplates(primers),anickingendonucleaseandastranddisplacingDNApolymerase.Amplicondetectioncanbeperformedusingavarietyofprobeformatsincludingmolecularbeaconsorbylateralflowsandwich.InitialReaction•InfluenzaisRNAvirus•Performreverse-transcriptaseamplification•NopurificationofthenucleicacidTheprimed,duplexamplicon:•bluerecognizesasiteononestrandofthetarget•brownrecognizesanearlyadjacenttargetontheotherstrand•orangeisanextrapieceoneachprimerwitha“nicking”site(N)andother“stabilizing”sequences•thereisa10-12bp“gap”betweenthe“templates”(arrows);thisenablesuseofatarget-specificprobeforproductdetection.•afterduplexsynthesis,nickingatornear“N”enablesstranddisplacementamplification,providingadditionalproductthatcanbere-amplifiedusingadditionaltemplates(primers).Amplification19iNATFluA/BWorkflowInfluenzaA/B:FirstPublications|21:FirstPublications|22:FirstPublications|23Conclusions:ComparedwelltoviralcellcultureDemonstratesimpressivesensitivityCombinesspeedofaRATwithsensitivityofaNATinPOCsettingOfferstheopportunitytoimprovepatientmanagement:100%/97.9%FluB:97.7%/100%VTMvsProFlu+PCRFluA:88.8%/98.3%FluB:100%/100%AdvantagesofRapidFluTests•AntiviralmedicationANDantibiotics•Avoidexacerbationofbacterial&viralresistance•Inpandemic,canhelpqualifywhogetsmedicationAccuratedeterminationofwhoneedsmedication•Reducecomplications•Reducespread•ReducehealthcareburdenEarlytreatmentofhighriskpatients,to•Directedtherapy•ReducedmorbidityandmortalityreduceshospitalcostsCostbenefits