laBoratory DiagnoSiS of Dengue viruS infeCtionS … · clinical management of DHF patients needs to be determined in prospective studies. ... Fig 1– Diagnostic pathway in patients

laBoratory DiagnoSiS of Dengue viruS infeCtionS

ananda nisalak

Department of Virology, Armed Forces Research Institute of Medical Sciences(AFRIMS), Bangkok, Thailand

Abstract:Accurateandefficientdiagnosisofdengue is important for clinical care, surveillance support,pathogenesisstudies,andvaccineresearch.Laboratorydiagnosis isalso importantforcaseconfirmation.

Here ispresenteda reviewof thecurrent stateofdenguevirusdiagnosis. Laboratorydenguediagnosis

canbeperformedthroughvirusisolation,genomeandantigendetectionandserologicalstudies.Forvirus

detection,dengueviremiaisshort,usuallyobservedtwoorthreedaysbeforeonsetoffeverandlastsfour

tofivedayslater.Therefore,samplesforvirusdetectionmustbetakeninthefirstfourtofivedaysofdisease

duringthefebrilephase.Inrecentyears,PCR(polymerasechainreaction)hasbecomeanimportanttoolas

aquickmethodfordiagnosisofdengue.AnothermethodofdiagnosisisdetectionofNS1antigen,usinga

commercialELISAkit.AlthoughPCRisthemostsensitiveandrapidmethodforthedetectionofdenguevirus

inearlystageofdisease,classicaldengueviruscultureusingdenguetissuecultureseedsalsohasimportant

benefit,especiallyforgenotypestudies.Inserologicallybaseddiagnosisofprimaryinfection,thedominant

immunoglobulinisotypeisIgM.Anti-IgMmayappearduringthefebrilephasein50%ofcases,intheother

half,itappearswithin2-3daysfollowingdefervescence.Oncedetectable,IgMlevelsrisequicklyandappear

topeakabout2weeksaftertheonsetofsymptoms,andthentheydeclinetoanundetectablelevelover

2-3months.Anti-IgGappearsshortlyafterwardsataverylowlevel.Thephysiologicaldefinitionofaprimary

infectionisthereforecharacterizedbyahighmolarfractionofanti-dengueIgMandlowmolarfractionofIgG.

SecondarydengueinfectionsarecharacterizedbyarapidincreaseinIgGantibodiesandwhenanti-dengue

IgMappearsinmostinstances,thelevelsaredramaticallylower.

Keywords: dengueinfection,denguevirus,laboratorydiagnosis

Correspondence: AnandaNisalak,MD,Department ofVirology,AFRIMS, 420/8 Ratchawithi Road,Bangkok10400,Thailand.Tel:+66(0)26962760;Fax:+66(0)26444760;E-mail:[email protected]

TheviewsexpressedinthisarticlearethoseoftheauthoranddonotreflecttheofficialpolicyoftheDepartmentoftheArmy,DepartmentofDefense,ortheUSGovernment.

IntroDuctIon

Denguevirus(DENV)consistsoffourantigeni-callydistinctserotypes(DENV-1,DENV-2,DENV-3,andDENV-4)thatdisplayahighdegreeofantigeniccross-reactivitywith each other aswell aswithothermosquitoandtick-borneflavivirusessuchasJapaneseencephalitis(JE),yellowfever,WestNile,andtick-borneencephalitisviruses(Calisheret al,1989;Innis,1995).Incountrieswheredengueisnoworisbecomingendemic,thecocirculationof

twoormoreflavivirusessuchasdengueandJEinAsia,dengueandJEandWestNileinIndia,anddengueandyellowfeverintheAmericasmakesthe serologicdiagnosisof acuteDENV infectionadifficulttask.Fromapublichealthandclinicalcareperspective,theaccuratediagnosisofDENVinfectionaswellastheidentificationofcirculatingDENVserotypesaffectspublichealthpolicyandactionforvectorcontrolaswellasindividualcasemanagement.

Vol. 48 (Supplement 1) 2017 SoutheaSt aSian J trop Med public health 83

AW SAJ 2017.indd 83 8/25/2560 BE 12:41 PM

By 1964, dengue fever (DF) and denguehemorrhagic fever (DHF)were recognized as aseriouspublichealthprobleminmostofSoutheastAsia.HammonandSather(1964)summarizedthedifficultiesofmakingaserotype-specificdiagnosisdue to the serologic cross reactivity betweendengueandotherflaviviruses.Threeassayswereavailablein1964todistinguishDENVserotypes:virusneutralizationusingasucklingmousemodel(Sabin,1950),complementfixation(Casals,1949)andhemagglutinationinhibition(HI)(ClarkeandCasals,1958).Thelattertwomethodswereplaguedwithahighdegreeofserologiccross-reactivity,andtheformercomplicatedbytheuseofsucklingmice.By1990,severalmilestonesindenguediagnosiswereachieved,toincludetheuseofmosquitoestopropagateDENV,thedevelopmentofmosquitocelllinesforvirusisolation,monoclonalantibodiestoallfourDENVserotypes,theadaptationoftheenzyme immunoassay (EIA) format to denguediagnosis,andthefirstgenome-baseddiagnosticefforts(Shope,1990).

Thepurposeofthischapteristodetailassayscurrently available to diagnoseDENV infection.Understandingadiagnosticassay’sadvantagesandlimitationsisessentialtoproperinterpretationandapplicationwhetherthatapplicationisatthelevelofthepatientorpopulation.

antIboDy anD vIrus Patterns In Dengue vIrus InfectIon

Upon the bite of an infected mosquito,DENV is introduced intradermally and replicateswithin skin dendritic cells (Wu et al, 2000).From the time of inoculation, DENV replicatesanddisseminates through the lymphatic systemproducingmeasurable viremia approximately3 days after inoculation lasting, approximatelyanother 4 days (Fig 1). The onset of fever andsymptomsoccurs approximately 24 hours afterthe onset of measurable viremia. Fever willlast 4 days on average followed by a suddendefervescence(ChandlerandRice,1923;Sileret al,1926;Simmons,1931;Sabin,1955;Kalayanaroojet al, 1997; Vaughn et al, 1997). The day ofdefervescence is an immunologically important

landmark in the course ofDENV infection as itdefinestheapproximateonsetofplasmaleakageinpatientswithDHF(WHO,1997;Greenet al,1999).

Primary dengue PrimaryDENVinfectionoccurswhenapatientlackingpreviousexposuretoaflavivirusdevelopsanacuteDENVinfectionthatresults indengue-specificantibodyproduction.Thepatientwithaprimary infection rarely developsDHF and anti-dengueantibodyevolvesslowlyduringthecourseoftheclinicalillnesswithhighproductionofIgMantibody (Vaughnet al, 1997). Themolar ratioofIgMtoIgGishigh(≥1.8:1.0)foratleastthreeweeksfollowinginfection(Inniset al,1989).UsingHIandneutralizingantibody,aprimaryinfectionisdefinedbylowtitersofantibodythatdevelopslowly(Russellet al,1967;WHO,1997).

Secondary dengue Secondary dengue implies previous flavivirusexposure (secondary flavivirus infection) or apreviousDENVinfectionwithadifferentserotype(secondary DENV infection). The IgG antibodyresponseoccursearlyandvigorouslyduring theclinicalillness;nearlyallpatientshavediagnosticlevels of antibody by EIAwithin 24 hours ofdefervescence (Inniset al, 1989; Vaughnet al,1997).Therapidincreaseinantidengueantibodytohighlevels indicatesananamnestic(memory)response (Halstead et al, 1983). The dengue-specificIgMresponseismorevariablewithaIgMtoIgGratio≤1.8asmeasuredbyIgMcaptureELISA(Inniset al,1989).

The level of IgM responsemay relate to thenumberofnewepitopespresentonthecurrentinfectingviruscomparedtothepreviousflavivirus.That is,previous infectionwithamoredistantlyrelatedflavivirus(eg,JEvirus)willresultinhigherIgM levels by EIA when infected by a DENVthan if the current infection follows a previousDENV infectionwith another serotype. HI andneutralizingantibodylevelselevatequicklytohightiters in secondary infections (HI titer≥1:2560)(WHO,1997).Again,thedegreeofsharedepitopesmayinfluencethedegreeofanamnesticantibodyresponse.

laboratory dengue diagnoSiS

84 SoutheaSt aSian J trop Med public health Vol. 48 (Supplement 1) 2017

AW SAJ 2017.indd 84 8/25/2560 BE 12:41 PM

Viremia pattern Due to antibody cross-reaction, definitivediagnosis of DENV infection requires isolationof the virus or detection of virus genome. Thepatternofviremia indenguepatientscorrelatescloselywithfeverwithpeaklevels(titersupto10logs/ml)occurring2to3daysfollowingtheonsetof illness,which is typically2 to3daysprior todefervescence(Vaughnet al,2000).ForDENV-1,DENV-2,andDENV-3,theviremiatitercorrelateswithdenguediseaseseverity(Vaughnet al,2000;Libratyet al,2002).Thisfindingisofmorethantheoreticalinterestasitprovidesapotentialtoolforthecliniciantoassesstheriskofseveredisease(DHF)priortotheonsetofplasmaleakage.

Withthedevelopmentofnewandrapidtoolstomeasureviremiatiter, thismayprovetobeapotentially importantmeasurement in definingpersonsatriskforDHF.Viremialevel,asoriginallydeterminedbyVaughnandcolleagues(2000),usedserial titrations of viremic plasma fromdenguepatientsinoculatedintoToxorhynchites splendensmosquitoes.ThetiteratwhichinfectionoccurredinhalfoftheT. splendenswasdeterminedusingprobit analysis resulting in themean infectious

dose50%(MID50).Thisisatimeconsumingand

tediousprocessrequiringserialspecimendilutionandnumerousmosquitoinoculationsfollowedbyatwo-weekincubationperiodandvirusdetectionineachmosquitobyindirectimmunofluorescenceorRT-PCR.

Newmolecular tools are proving useful tomeasure dengue viral load (Hounget al, 2001;Libratyet al, 2002).However,wheremosquitoorcell culture inoculationmeasuresviablevirus,molecular approaches detect only the presenceof genome. RT-PCRmethodologiesmay allowa virus-based diagnosis extendingmany hourspast the detection of replicating virus due toits detection of viral genome in virus-antibodycomplexes(neutralizedvirus)(Wanget al,2003).HowrapidmoleculardiagnostictoolwillaffecttheclinicalmanagementofDHFpatientsneedstobedeterminedinprospectivestudies.

DIagnostIc PathWay In PatIents WIth susPecteD acute Dengue

Fig1illustratesthediagnosticoptionscurrentlyavailable to diagnose an acuteDENV infection


Fig1–Diagnosticpathwayinpatientswithsuspecteddengue.


AW SAJ 2017.indd 85 8/25/2560 BE 12:41 PM

in patientswith suspected dengue. Diagnosticoptionsaredividedfirstintoassaysthatdetectthepresenceofthevirus(includingvirusantigenandvirusgenome)versusassaysthatdetectthehost’sresponsetothevirus (antibody).Virusdetectionmethodscanbedividedbetweentime-consumingvirusculturemethodsusinganimals,mosquitoes,or cell culture versus rapid antigen or genomedetectionmethods.Likewise,antibodydetectionmethodsincludeassaysthatcanbecompletedinafewminutesorrequireaweekormoretoascertain.

The utilization ofmultiple approacheswithproperly timed specimens assures diagnosis invirtually every case of suspected dengue. Thisdiagnostic rigor is required to conduct researchindenguepathogenesis.Isthistypeofdiagnosticbreadthneededforeverylaboratorytobemadeavailabletoeveryclinician?Ideally,everycountryexperiencing or at risk for dengue epidemicsshouldhaveatleastonecenterofexcellencethatisproficienttoisolatevirus,detectgenome,andtocarefullycharacterizeantibodyresponses.Thiswouldhelptobetterunderstandthedepthandbreadthofthedenguepandemic.

The capacity to diagnose clinically similaroutbreakssuchas leptospirosis isalso important.Serotypespecificvirologicdiagnosesovertimemaysomedayallow thepredictionof the timingandseverityofdengueepidemicinordertoallowvectorcontroleffortstopreventorabatesuchepidemics.

For the clinician, a hospital-based laboratorythat can perform basic dengue serology canquicklyconfirmadengueoutbreak.However,sinceantibody-based assays donot routinely becomepositiveuntilthedangerperiodforDHFhaspassed,thediagnosisisoflessvaluetothepatientwhois recovering.Tohavean impacton thecareofan individual patient, a reliable rapidmoleculardiagnosis is needed to assist in the differentialdiagnosisandtoreducemorbidityandmortality.Forsuspecteddenguepatients,serumspecimensshouldbecollectedearlyinthefebrilephaseandstoredat -70°Cforvirus isolation/detectionandat -20°C for acute phase serology to bepairedwithaspecimendrawnat least7days later for

convalescentserology(WHO,1997).

The practice inmany dengue hyperendemiccountriesistodrawblooduponadmissionandatthetimeofdischargeonetotwodaysfollowingdefervescence. In this setting, most denguepatients are experiencing secondary infectionsand the serology will be positive soon afterdefervescence.Forpatientswithprimaryinfections,a subsequent follow-up specimen is needed toconfirmorruleoutDENVinfectionifattemptsatvirusisolationoridentificationfail.

FortravelersreturningtotheUnitedStateswithsuspecteddengue,acuteandconvalescentsamplescanbesenttothestatehealthdepartmentwitharequesttoforwardthemtotheCDCfortesting.

serologIcal assays

Hemagglutination inhibition (HI) assay The path towards the serologic diagnosisof dengue started in 1950,when Sabin (1950)discoveredthatarbovirusesingeneralanddenguevirus(DENV)inparticular,areabletoagglutinatecertaintypesoferythrocytes.Normalerythrocytesinasuspensionwillsettletothebottomofatesttubeorwellandformacompact,densebuttonofredbloodcellsafter30minutestoonehour.Agglutinationofredcellsusingvirusantigenwillpreventthisnormalsettlingtooccurandresultsinauniformlatticeofcrosslinkedcellscoveringthelowerpartofthetubeorwell(hemagglutination).CasalsandBrown(1954)modifiedthetechniqueusing acetone and ether to extract (purify) thehemagglutinatingantigens(HA)anddevelopedahemagglutinationinhibition(HI)assay.Inthisassay,DENVspecificantibodybindstheHApreventinglatticeformationallowingtheredbloodcells toclumptothebottomofthetestwell(Fig2).

Test serum could be tittered to determinewhenhemagglutinationwasnolongerinhibited.Porterfield (1954, 1957) andClarke andCasals(1955)furtherimprovedthisassaybyusingfemalegooseredbloodcellsratherthanhumancellstoremovenonspecificinhibitorsofhemaglutination.ThetechniquesusedforhemagglutinationandHIasdescribedbyClarkeandCasals in1958have



AW SAJ 2017.indd 86 8/25/2560 BE 12:41 PM

remained largely unchanged today apart fromadaptationtomicrotiterplatesin1980andremainsa fundamental tool to diagnose acute DENVinfection and in seroprevalence studies (WHO,1997).

AgglutinationofredcellsisdependentonpHandtheamountofHApresent.HAisquantifiedas a titerwhere anHAunit of 1 represents thehighestdilutionwhichcauseshemagglutinationofredcells.Anadditional1HAunitisthusgivenforeachsubsequentlowertiterofantigen.Forexample,if thehighest titer of aDENV-2antigen causinghemagglutinationwasfoundtobe1:160andbyconventiongivenanHAunitof1atthistiter,2HAunitswouldbeatiterof1:80;3HAunitsfor1:40;4HAunitsfor1:20and5HAunitsfor1:10.Thehemagglutinationassayallowsquantificationandstandardizationofdengueantigen(HA)producedfromavarietyofsourceswiththemostcommonsourcesbeingsucklingmousebrainandcellculture.

AsuntilrecentlypracticedintheArmedForcesResearch Institute ofMedical Sciences (AFRIMS)in Bangkok, Thailand, 1-to-2 day old sucklingmice(Mus musculus)areintracraniallyinoculatedwith0.02mlofadengueserotypespecificvirussuspension.Mice are observed twice a day for

the first signs of encephalitis (failure to eat asevidencedby lackofmilk in thestomach,colorchange,wasting,ortremors),whichoccurs3-to-10daysafterinoculation.ForDENV-1thisusuallyoccurs at day 5, day 4 for DENV-2, day 7 forDENV-3,day4forDENV-4,andday3forJapaneseencephalitisvirus.Harvestedbrainismadeintoa20%(weight/volume)suspensioninan8%sucrosesolution andhomogenized. The homogenate isthenacetoneextracted,condensed,washedandresuspendedinsterilenormalsaline.AllantigenisassayedforthenumberofHAunits.

Thehemagglutinationassayitselfisperformedbestusinggooseredbloodcells(RBCs)thoughRBCfromotherspeciescanbeused.GooseRBCscanbeobtainedfromadultfemalegeese(Anser cinereus)and should immediately be placed inAlsever’ssolution (glutaraldehyde-fixed goose RBCsmayalso be used) (Wolffet al, 1977).Goose RBC’sare thenwashedwith dextrose-gelatin-veronalsolutionandarebroughtuptoafinal8%solutionjustpriortoassayuse.BuffersofdifferingpHhavebeenfoundtobeoptimalfortheagglutinationofdifferentflavivirusesandspecifictoeachdengueserotype:pHof6.2forDENV-1;6.4forDENV-2;6.6forDENV-3;and6.8forDENV-4.Itisessential

Fig2–Hemagglutinationinhibitionassay.



AW SAJ 2017.indd 87 8/25/2560 BE 12:41 PM

thattheRBCsbesuspendedinabuffersetataspecificpHforeachdengueserotypepriortouseintheassay.

Topreparethetestsera,non-specificinhibitorsmustberemoved(thatis,somehumanserumcaninhibitagglutinationintheabsenceofmeasurabledengue antibody). This is done using acetone,etherorkaolinprior toperformingtheHIassay(Monathet al,1970).Diethylaminoethyl-Sephadex(DEAE-Sephadex)hasalsobeenusedwithsuccess(Altemeieret al,1970).AtAFRIMS,acetoneextrac-tionisthepreferredmethodandisperformedbyaddingheat-inactivatedserumtocoldacetoneanddecantingthemixture.Adropofgooseredbloodcellsisaddedtothesera,mixedandremovedtoeliminatenon-specificagglutinationoferythrocytes.

TheHIassayatAFRIMSlaboratoryisperformedin18x10v-shapedwellLuciteplatesthatareclearlymarkedforeachserumanddilution.Two-foldserialdilutionsofthetestseraandstandardpositiveandnegativecontrolsareplacedintoeachwellusinga0.025mlloopfollowedbytheadditionof0.025mlof8to16HAunitsofspecificdengueserotypeantigenandtheplatesarecoveredandincubatedat4°Covernight.Ideally,allserafromasinglepatientshouldbetestedinthesameassay.Asascreeningtest, twobroadly cross-reactivedengueantigensmaybeused(DENV-2andDENV-3)withonlyslightlossofsensitivity.Thefollowingmorningthetestplatesareallowedtoreachroomtemperatureand0.05mlof an8%goose red cell stock solutiondiluted1:24intheproperpHbufferisaddedtoeachwell.Theplateisallowedtositundisturbedforonehouratroomtemperatureandthewellsthathaveorhavenotagglutinatedarerecorded.TheHItiteristakenasthehighestserumdilutionthatcausescomplete inhibitionof agglutination (Fig2). TheinterpretationofdengueHIantibodytiterisbasedonWHOcriteria(WHO,1997).WorkbyBurkeandcolleaguessuggestagoodcorrelationbetweenHIandPRNT50inpairedspecimenscollected7monthsapart(Burkeet al,1988).

Theprimarysourcesofinter-assayvariationfortheHIassayare:1)theamountandqualityofHA(appropriate amount varies by serotype), 2) pH

(variesbyserotype),and3)interoperatorvariationtointerpretagglutinationineachwell.Sourcesofassayvariationcanbereducedby:establishingHAunits for stock dengue antigen andperiodicallycheckingtoensurethatantigenHAactivityisnotlost,strictlyfollowingguidelinesontheproperpHforeachdengueserotypeantigen,runningbothpositiveandnegativeserumcontrolsoneachplate,testingpairedseraonthesameplate,andreadingtheplateonawhitebackgroundora low-lightfluorescentlightbox.

Interpretationof results is relatively straight-forward(Table1).Forsuspectedacutedengue,thetimingofthecollectionofpairedseraisimportant.Sera obtained 7 days or more apart shoulddemonstrateafour-foldriseintiterifthepatientwasacutelyinfectedwithDENV:Serumcollectedaweekapartwilloftenrequirethepatienttobeseeninfollow-up;aproblemformanyclinics.Ifserumis collected less than7days apart and a4-foldrise in titer isnot seen; the resultmustbe readoutasnon-diagnostic.Ifthereisa4-foldincreaseandthetiterrisesto1:2560orhigher,itindicatesanacutesecondaryflavivirusinfection.Iftheendtiteris1:1280orless,theinterpretationisanacuteprimaryDENVinfection.FormanypatientswithasecondaryDENVinfection,a4-fold increasewillbeseenwellbefore7days.Ifasinglespecimenorpairedspecimensshowatiterof1:2560orhigherwithouta4-foldincrease,theinterpretationisarecentDENV infection,possibly acuteorhavingoccurredduringthepreviouscoupleofmonths.

Importantly, theHI test fails to discriminateadequatelybetweenthecloselyrelatedflavivirusessuch as dengue, JE, andWestNile. This couldproduce results that are difficult to interpretin countrieswhere these viruses co-circulate.Despite these limitations, the HI assay is apowerfultechniquethatisstillastandardassayforseroprevalencestudiesaswellasinthediagnosisofacuteprimaryandsecondaryDENVinfections.TheHIassayisarobustassaythatcanbeperformedwithminimallaboratoryequipment,reagentsandexpense.Equallyimportantisthatthereagentscanbemadelocallyandarereadilysustainableforthedevelopingdenguediagnosticlaboratory.



AW SAJ 2017.indd 88 8/25/2560 BE 12:41 PM

Plaque reduction neutralization assay (PRNT) Dulbecco (1952) described a chick embryofibroblastplaqueassayforseveralvirusesincludingWesternequineencephalitisandNewcastlediseaseviruses.Thistechniqueallowedquantificationofviruses in vitroasplaqueformingunits (PFU). In1959,HendersonandTaylordescribedamethodtodetectarbovirusviralplaquesinanagaroverlaystainedwith neutral red and demonstrated itsutility tomeasureserumantibodyneutralizationtiters. The standard neutralization test prior totheavailabilityoftissueculturewasthesucklingorweanlingmouse neutralization test, usuallyperformedusingaconstantserumdilutionmixedwithlogdilutionsofviruspriortoadministrationto the test animals andmonitoring for illness.The ability of the serum to neutralize wascalculated as the log neutralization index. TheDENVneutralizationassayusingsucklingmiceinachallengevirusresistanceassaywasadaptedtoBS-C-1, PS and LLC-MK2 cell lines byHalsteadet al (1964)andSukhavachanaet al (1966).

Itwasnotuntil 1967however, that adirectin vitro assay tomeasure DENV neutralizingantibody and DENV identification by serology

wasdevelopedbyRussellet al(1967).Thisassaybecameknownasthedengueplaquereductionneutralizationtest (PRNT)andutilizedprototypedengueviruses,monkeyseracontrols,LLC-MK2celllinesandanagaroverlaymediawithneutralredstaining.Aprobitanalysiswasusedtomeasureplaque reduction as the serum titer required toreducedengueviralplaquesby50%(PRNT

50).This

techniqueintroducedanefficientandreproducibleassay to measure dengue serotype specificneutralizing antibody andbecame the standardassaytomeasuredengueantibody.

Variationsof this techniquewere introducedthereafter, such as amicrometabolic inhibitiontestusingBHK-21cellsandamicrocultureplaque-reduction test utilizing the LLC-MK2 cell line(Sukhavachanaet al, 1969),microplate culturesusing BHK-21 cells (Fujitaet al, 1975), a focusreductionmethodusingperoxidase-anti-peroxidasestainingof BHK-21 cells (Okunoet al, 1978), asemi-micromethodinLLC-MK2cellsusinga70%plaquereductioncriteria(Morenset al,1985a),andasimplifiedPRNTassayusingBHK-21cells(Morenset al, 1985b). These assays are being used todetermineserologicalresponsestodenguevaccines

Table1. WorldHealthOrganizationcriteriatointerpretdenguehemagglutinationinhibitionassayresults.

Changeinantibodytiter

Sampleinterval Antibodytiterattimeof

convalescence

Interpretation

≥toa4-foldrise Pairedserawith≥7daysofseparation.

≤1:1280 Acuteprimaryflavivirusinfection.

≥toa4-foldrise Withanyspecimen. ≥1:2560 Acutesecondaryflavivirusinfection.

≥toa4-foldrise Pairedserawith<7daysofseparation.

≤1:1280 Acuteflavivirusinfection,indeterminateprimary/secondary.

Nochangeintiter Withanyspecimen. ≥1:2560 Recentsecondaryflavivirusinfection.

Nochangeintiter Pairedserawith≥7daysofseparation.

≤1:1280 Notdengue.

Nochangeintiter Pairedserawith<7daysofseparation.

≤1:1280 None

Uncertain Oneseraspecimen. ≤1:1280 None

Source:WHO,1997.



AW SAJ 2017.indd 89 8/25/2560 BE 12:41 PM

(Jacobs andYoung, 2003), in seroepidemiologicstudiesdefiningoutbreaksofDENV(Halsteadet al,2001)andinpathogenesisstudiesofdengueshocksyndrome(Greenet al,1999).

Themicroneutralization assay (VorndamandBeltran,2002)isbasedonthesameprincipleasPRNT;however, insteadofcountingthenumberofplaquesperwell,thisassayusesacolorimetricmeasurement of virus-induced cell lysis todeterminetheend-pointdilution.

Enzyme immunosorbent assays (EIA) Innisandcolleaguesin1989appliedtheanti-JEIgMantibodycaptureEIAofBurkeandNisalak(1982) to dengue and developed a serologicalassay to diagnose acute dengue in countrieswhere JE anddengue viruses co-circulate (Inniset al,1989).Thisassayallowstherapid(within48hours)diagnosisofacuteDENVinfection.Equallyimportant,thisassaywasoptimizedtodistinguishprimary fromsecondarydengueonthebasisofIgM/IgGratiosandbyrunningaconcomitantIgMcapture JE ELISA, eliminating the false positivesthatmightoccur fromacute JE virus infections.This assayhas servedas a standard tomeasurenewerassays(Vaughnet al,1999).

TheELISAassayiswidelyusedinthediagnosisofviralpathogensduetotherelativeeasetosetuptheassayina96-wellformat,itshighdegreeofreproducibilityandfortheoptiontouseautomaticplatewashers and scanners. In the Innis assay(Innis et al, 1989), anti-dengue isotypecaptureenzymeimmunoassaysmeasuretheproportionofimmunoglobulinisotypereactivewithdengueorJEantigen.Briefly,themethoduses96wellplatessensitizedovernightwitheithergoatanti-humanIgMor IgG antibody.Control and test sera arediluted1:100andplacedinthewellsovernight.IgMor IgGisthencapturedontotherespectiveplatesandfollowedbytetravalentdengueantigen(16HA units each ofDENV-1,DENV-2,DENV-3, and 8HA units of DENV-4) for the dengueEIA or JE antigen (50HAunits) for the JE EIA.This is followedbyananti-flavivirushorseradishperoxidaseconjugatewithsubstrateproducingaquantitativecolorimetricresultreadbyanEIAplate

reader.Abinding index (81) is calculatedusingtheopticaldensity(0D)ofthetestsampleminustheODofthenegativecontrolalldividedbytheODoftheweakpositivecontrolminustheODofthe negative control. The 81multiplied by 100generatesEIAunitswhere≥40unitsisconsideredpositivefortheIgMcaptureassay.Thesensitivityofdiagnosingacutedengueis78%onadmissionseraand97%inpairedserawithaspecificityof100%.ThevalueofthisassayinadditiontodiagnosingacutedengueisindistinguishingbetweenacutedengueversusacuteJE.Aratioofanti-dengueIgMtoanti-JEIgMof≥1.0istypicalofacutedenguewhereasaratio≤1.0 isconsistentwithacuteJEvirusinfection.

Theaddedvalueofthisassayisthatitdistin-guishesprimaryfromsecondaryDENVinfection.Seradefinedasconsistentwithprimaryorsecond-arydenguebyHItitersestablishedratiocut-offsfor IgM/IgGwhere a ratio of IgM to IgG units≥1.8 is consistentwith primaryDENV infectionandaratioof<1.8isconsistentwithsecondarydengue.DengueEIA,whencomparedtoHIusingWHOstandards,demonstratedahighcoefficientofcorrelationandagreementrate(Chungueet al,1989;WHO,1997).

Astheformatofthisassayhasbecomewidelyused in the diagnosis of acute dengue, it isworthwhiletodiscusssomeofthequalitycontrolaspectsofthisassayandthemethodsemployedto decrease inter and intra-assay variation. Theprimary sources of inter-assay variation for thisassayare:1)thedilutionoftheanti-flavivirusIgGenzymeconjugate;2)durationofthechromogen-substrate reaction; and 3) the plate coatingsensitizationstep(amountofanti-isotypeantibodybound).Samplesandantigensusedinthisassayareused inquantities that saturate the capturesystemthereforethekeylimitingcomponentsoftheassayaretheboundanti-isotypeantibodyandtheIgG-enzymeconjugate.Theamountofboundanti-isotypeantibody isaffectedbythequantityused,thedurationofsensitization,andthetypeofplatesused.

Reductionofvariationinthesensitizationstepcanbeaccomplishedbyconsistentlyusingplates



AW SAJ 2017.indd 90 8/25/2560 BE 12:41 PM

thatwereusedtooptimizetheassayandtopre-sensitizeplatesinbatchesandstoreat-20°C.Platescanbekeptat-20°Cforuptoonemonthwithoutsignificantlossofboundanti-isotypeantibody.Itisalsoimportanttodeterminetheassaydilutionof the anti-flavivirus IgGHRP conjugate. This isaccomplishedby determining the assay dilutionthatyieldsanODat492nmof0.4(establishedcut-offforapositivetest)whena1:100dilutionoftheweakpositivecontrolisusedwithtestsera.Intra-assayvariationshouldbe10%orless.Withinone complete assay, all four positive standardsshouldbeintheODrangeof0.25to0.55.Valuesabovethisrangeresultinadecreaseintheassay’ssensitivityandvaluesbelowthisrangewillresultinadecreaseofspecificity.

EIAsusingcellculturederiveddengueantigensand thoseusingdenguemonoclonal antibodiesrather than patient-derived control serumhavebeenfoundtobeassensitiveandspecificastheInnis assay (Lamet al, 1987;Kunoet al, 1991;Cardosa et al, 1992). Additionally, commercialalternativesarenowavailableandcomparefavor-ablywiththeoriginalEIAdescribedbyInnisandcolleagues(Lamet al,1996;Cuzzubboet al,1997;Sanget al,1998;Vaughnet al,1999;Wuet al,1999;Cuzzubboet al,2000;Groenet al,2000;Lamet al,2000).ThedengueIgMandIgGcaptureEIAassay (PanBioDengueDuo)demonstratedasensitivityof99%andspecificityof92%comparedtotheInnisassay(Vaughnet al,1999).

AvariationofthetraditionalserumbasedformoftheEIAhasrecentlybeendevelopedusingsalivaforthediagnosisofacuteinfection(Cuzzubboet al,1998;ArtimosdeOliveiraet al,1999).Salivacontainsbothdengue-specificIgMandIgGthatcanbemeasuredinatraditionalIgMcaptureEIAformat.OneprospectivestudyusingthePanBioDengueDuo ELISA determined a sensitivity of92%andaspecificityof100%comparedtotheserumbasedInnisEIA(Cuzzubboet al,1998).

ThedengueIgM/IgGEIAallowshighthroughputand reagents are commercially available orreadily available from reference laboratories.Theadvantagesoftheanti-dengueIgMandIgG

isotype-capture enzyme immunoassay are itsabilitytodetectelevatedlevelsofIgMinsamplescollectedduringtheacuteperiod,anintervalwhenmostDHFpatientsarestillhospitalized;thatis,asinglespecimenpositivefordengueIgMindicatesacutedengue.However,itmaytakeuntil3dayspost-defervescence before all dengue patientstest-positivebyEIA.SerumcontainsnoinhibitoroftheEIA,thereforenopre-treatmentisneeded;andtheassaycanbeoptimizedtodistinguishbetweendifferentflaviviruses.

AfurtheradvantageoftheEIAisitsabilitytodetectIgMintheCSFincaseswhereencephalitisissuspectedorsalivatominimizevenipunctureinprospectivefield studies.Commercially availableEIAkitshavedemonstratedhighlevelsofsensitivityandspecificity foracuteDENV infectionmakingthis diagnostic assay a key tool for the denguediagnosticlaboratory.

Indirect fluorescent antibody (IFA) test ThisassaytodetectdenguespecificIgMandIgGusingfluorescentantibodyisusedprimarilyinresearchlaboratories.Vathanophasandcolleaguesdevelopedit in1973(Vathanophaset al,1973).The assayutilizes a solid phase (usually denguevirus infected cells that are cold acetone fixedontoslides)thatbindshumandengueantibody,whichinturnisdetectedbyfluorescein-conjugatedanti-human antibody. Visible fluorescence seenon the slides constitutes a positive antibodytest. Serial dilutions of test serum are used tomeasure the amountof antibodypresent as anantibody titer. Thismethod is limiteddue to itstime-intensiveness,subjectivereading,relianceoninfectedcellsastheantibodycaptureagentandlackofautomation.Themajoradvantageofthisassayisitsrelativelowcosttoperformwithafewsamples(Boonpucknaviget al,1975).

Rapid antibody assays In 1988,Cardosa and colleagues reported asimplifieddengueassaythatcouldbeusedinlessdeveloped laboratorieswith the potential to beused in thefield (Cardosaet al,1988).Thedotenzymeimmunoassay(DEIA)wasmoresensitivethantheHItestandhadtheadvantageofusinga



AW SAJ 2017.indd 91 8/25/2560 BE 12:41 PM

singleserumdilutionasacutoffpointtodiagnoseacuteDENVinfection.ThisandsimilarassaysareanextensionofthewesternblotwhereDENVproteinsaretransferredontoasolidphasepaperordipstickand upon exposure to DENV-specific antibodyand an anti-human antibody detection system,wherebyabandappearsindicatingaserologicalresponsetoDENV.

These dot enzyme immunoassays (DEIA) ordot-blottestsofferadiagnostictoolthat israpid(usually4-6hoursorless),abletodetectbothIgMandIgGdengueantibodyandhenceacuteprimaryorsecondaryDENVinfection,andrequireminimalexpertiseorlaboratoryequipment(justacentrifugeandwaterbath).InthedotblotassayofCardosaand colleagues, strips containingDENV antigenwereincubatedwithhumanserawhichallowsthebindingofdengue-specificantibodytotheDENVantigenontheteststrip.Abiotinylatedanti-humanIgG, or IgM, depending on the assay, detectedthebounddengueantibodyandafterasignalingreaction,displayedacolorbandontheteststrip.ApositiveIgMalonesignifiedanacuteprimaryDENVinfectionandbothapositiveIgMandIgGindicatedanacutesecondaryDENVinfection.

Another example of this approachwas theDengueRapidTestdevelopedbyPanBio,LTD(LamandDevine, 1998;Vaughnet al, 1998). In lessthan7minutes,thisimmunochromatographictestdetecteddengue-specificIgMandIgGantibodies.Comparedtostandarddiagnostictechniquesandusinghospitaladmissionanddischargesera,thistestdemonstrated100%sensitivity todiagnoseDENV infection and reliably distinguish primaryfrom secondaryDENV. This assay hasmigratedtowards the use of recombinant antigens anda dipstick format (Wu et al, 1999; Cuzzubboet al,2001;Paridaet al,2001).Additionalrapidassays are being developed and coming to themarket (Groen et al, 2000). The limitations ofrapid antibody-based assays are that specificityisdecreaseddue to the cross-reactivenatureofantibodytootherflavivirusessuchasJEvirusanditsdependenceontheappearanceofIgM,whichcanappear late inthecourseof infectionorbebluntedduringsecondaryDENVinfections.

vIrus DetectIon

Intracerebral inoculation of suckling mice DENVwasfirstisolatedinAugust1942duringan epidemic of dengue that occurred in theNagasaki-Sasebo regionof Japan (Hotta,1952).Bloodtakenfrompatientswithin48hoursafterthe first rise of temperaturewere inoculatedintracerebrally (IC) into sucklingwhitemice.Infectedmice developed debility, tremors, andparalysisoccurringinthehindlegs.Sincethisfirstdescription, IC inoculationof sucklingmice hasbecomeastandardmethodtogeneratedengueserotype-specific antigen (for description oftechniqueseesectiononHAandHIassays).Forroutine viral isolation however, the technique isslowandcumbersomeandrequiresasteadysupplyofsucklingmice.ForthesereasonsICofsucklingmicehasbeensupplantedwithmorereliableandsensitivemethodsdiscussedbelow.

Mosquito inoculation Mosquito inoculation of patient sera as atechniquetoisolateDENVwasdevelopedinthe1970’s taking advantage of the natural vectorof DENV as a biological amplification system(RosenandGubler,1974).AvarietyofmosquitovectorshavebeenusedincludingToxorhynchites splendens,Aedes albopictus, orAedes aegypti (Gubleret al, 1979; Rosen and Shroyer, 1985;Rosen et al, 1985). Toxorhynchites splendensmosquitoeshavetheadvantageofbeinglargerandeasierto inoculatewithhumansera (RosenandShroyer,1985).Thereisnoriskofspreadingthevirusthroughthesemosquitoes,asToxorhynchites splendensarenon-bloodfeeders.

At the AFRIMS laboratory, Toxorhynchites splendens isusedbyinjecting0.02μlofhumansera intrathoracically into themosquito. Afteran incubationperiodof10-to-14days,DENV isdetected inthemosquitoheadusinganti-DENVimmunofluorescence antibody. If positive, themosquitobodyistrituratedandinoculatedontoC6/36cellsforvirusexpansionandtyping.Isolationrates using thismethod are higher than directinoculation onto cell lineswith isolation ratesnearlyequivalenttomoleculardetectionofvirus



AW SAJ 2017.indd 92 8/25/2560 BE 12:41 PM

byRT-PCR(Vaughnet al,2000).Therequirementforinsectariesrestrictstheuseofthisapproach.

Cell culture inoculation AvarietyofcelllinesarepermissiveforDENVinfectionandcangenerallybedividedintoinsectormammaliancelllines.Insectcelllinesincommonuse for propagating DENV are themosquitoderivedcelllines:AP-61fromAe. pseudoscutellaris,C6/36fromAe. albopictusandTRA-284fromTx. amboinensis (Varmaet al,1974; Igarashi,1978;Kuno, 1982).Of the three, C6/36 is themostcommonlyusedduetoitseaseofuse,availability,andminimalbackgroundwhenusingadenguetypingEIAordirectfluorescentantibodystaining.

Mammaliancells incommonusearederivedfromhamsters,BHK-21,orprimatessuchasLLC-MK2orVero.Mammaliancellsoffertheadvantageofcytopathicchangesandplaqueformationwheninfected(Yuillet al,1968).Therefore,mammaliancellsarecommonlyusedintheplaquereductionneutralizationassay.Forviralisolation,mosquitocelllinesaremoresensitivethanmammaliancells(RosenandGubler,1974).Isolatingvirusininsectcellsandplaquequantifyingasecondspecimenonmammaliancellscancombineuseof insectandmammaliancelltypes.

One approach to quantify DENV by plaqueformationonmonolayersofmammalian cells incultureistodilutesamplescontainingvirusseriallyten-fold and then to inoculate 0.2ml of eachdilutionontoduplicatewellsofcellmonolayersin6-wellplates.Aftera1-hourvirusadsorption,cellsareoverlayedwithagaroseinmaintenancemedium.Afteranappropriateincubationperiod,usuallyfiveto sixdays,plaquesaredetectedby staining themonolayerswithneutral red,which stains livingcells.Therefore,plaquesarevisualizedasclearareasinaredbackground.Thevirustiter,reportedasPFUper0.2mliscalculatedastheaveragenumberofvirusplaquescountedatagivendilution(containingabout30-100PFU)multipliedbythedilutionfactor(Putnaket al,1996).

Dengue virus serotype identification Methods to identify the infecting DENVserotypeintheserumofadenguepatientbased

ontheantibodyresponsearelimitedduetothehighdegreeofantibodycross-reactivityamongtheDENVserotypes(Kunoet al,1993).WhilemolecularapproachesusingRT-PCRhaveevolvedsignificantlyin recentyears, thestandardforDENVserotypeidentification remains the isolation of the virusby animal, cell culture ormosquito inoculation,further expansion in cell culture, and serotypeidentification using DENV serotype-specificmonoclonalantibodiesinanimmunofluorescenceassayoranantigencaptureEIAformat(Henchalet al, 1983). Commonmonoclonal antibodiesusedforserotype identification include1F1 forDENV-1,3H5forDENV-2,5D4forDENV-3and1H10forDENV-4(Kunoet al,1987;MalergueandChungue,1995).

Antigen capture EIA for the identificationofDENV has been demonstrated to be a simpleand reliable technique (Kuno et al, 1985). AttheAFRIMS laboratory, an antigen capture EIAutilizesvirusisolatedinToxorhynchites splendensmosquitoes after expansion usingC6/36 cells.ImmulonUplatesare sensitizedwithgoatanti-mouseIgGineachwell.Dengueserotypespecificmousemonoclonalantibodies(4G2anti-flavivirus,1F1anti-DENV-1,3H5anti-DENV-2,10C10anti-DENV-3,1H10anti-DENV-4,and2H2anti-denguegroup) are bound onto the plate followed bycapture of the unknown DENV serotype. Acolorimetricreactionisformedaftertheadditionof anti-flavivirus-horse-radish-peroxidase andits substrate. DENV serotype specific mousebrainedderivedantigen(DENV-1Hawaii,DENV-2NGC,DENV-3H87,DENV-4914669)areusedinparallelaspositivecontrols.Opticaldensity(OD)isreadatawavelengthof492nmandtheresultsare interpreted by comparingwith positive andnegative controlswhere positive controlOD isalways≥0.20andanegativecontrolODis<0.20.MatchingthehighestODreadingtothepositivedengue control serotype identifies the DENVserotype(Kunoet al,1985).

ImmunohIstochemIstry

Immunohistochemistry is the staining oftissue specimens for the presence of specific



AW SAJ 2017.indd 93 8/25/2560 BE 12:41 PM

proteins.Dengueantigenstainingisapowerfultechniquetodiagnosedengueinfatalcaseswhenserology is non-diagnostic and virus isolationisnotavailableornotsuccessful.Anumberoftechniques have been used to detect dengueantigen in tissue specimens to include bothdirectandindirectfluorescentantibodystaining,and enzyme conjugates using peroxidase andphosphatase conjugates (Boonpucknavig et al,1975;Boonpucknaviget al,1976;Boonpucknaviget al,1981;Hallet al,1991).

AttheAFRIMSlaboratory,tissuespecimensarefixedinMillonig’sformalinfor2hours,irradiatedin amicrowave oven and then embedded inparaffin.Viralantigenisdetectedbyperformingamodificationoftheimmunoalkalinephosphatasemethod as described byHallet al (1991). Thestainingmethod of the tissue section involvesdeparaffinization inabsolutealcoholandwater,immersion ina0.05%solutionofproteaseVIII,application ofHistoMark Blue® (Kirkegaard andPerry Laboratories: Gaithersburg, MD), andblockingwith normal horse serum and bovineserum albumin. The specimen is incubatedovernightwithpolyclonalmousedengueantibodyfollowedbyasecondarybiotinylatedhorseanti-mouseIgGthenstreptavidin-alkalinephosphatase,AS-B1phosphate,hexazotizednewfuchsinandlevamisol,asthechromogenicsubstrate.Thetissuespecimen is then counterstainedwithMayer’shematoxylin.

genome baseD assays

Hybridization probes and polymerase chain reaction (PCR) In 1987, Henchal and colleagues describedmoleculartechniquestodiagnoseacutedengueusing slot-blot nucleic acid hybridizationwith aradiolabeledeDNAprobe (Henchalet al,1987).In 1991, reverse transcription (RT) of viral RNAfollowed by polymerase chain reaction (PCR)allowedtherapid(lessthan12hours)detectionofDENVinpatientsera.Anestedtechniqueallowedfor the serotype specific diagnosis of dengue(Henchalet al,1991;Moritaet al,1991;Lanciottiet al,1992).

Henchal’s slot-blot nucleic acid hybridizationtechnique used a radiolabeled eDNAprobe todetect as little as 11 plaque forming units ofeachofthefourDENVserotypes(Henchalet al,1987).Unlikeantibodybasedassaysthatrelyonthe appearance of dengue-specific IgMor IgG,molecular techniques offer the advantage ofdetecting the virus directly, early in the courseofaDENVinfection.Nucleicacidhybridizationisnotaffectedbyantibodyandtheappearanceofvirus-specifiedRNAcoincidedwiththedetectionofantigenininfectedcells.

AmodificationofthistechniquebyRuizandcolleagues utilized amicroplate hybridizationmethod(Ruizet al,1995).DENVRNAwasisolatedfrom serumor tissue samples and immobilizedontowellsfollowedbyhybridizationwithabiotin-labeled eDNA-probewith signal detection byperoxidaseconjugation.Thisassaywasfoundtohaveasensitivityof95%andspecificityof100%forallfourDENVserotypes.

Henchalandcolleaguesdevelopedauniversalsetofsenseandanti-senseoligomericDNAprimersthatmatchedallknownDENVsequences(Henchalet al,1991).ThisRT-PCRwasfoundtobe80%sensitive and 100% specific for acute denguecompared to virus isolation in livemosquitoes.ModificationstothisassaybyLanciottiet al(1992),usingnestedRT-PCR techniques have increasedsensitivityandreducedassaytimetolessthan6hours.

The dengue RT-PCR assay provides a rapid,sensitive,diagnostictooltodetectDENVinpatientspecimensaswellasinthemosquitovector(Chanet al, 1994). The primary limitation for patientdiagnosisisthatDENVviremiaoccursearlyinthecourseof infectionanddrops tonon-diagnosticlevels soon after defervescence (Vaughn et al,2000). Other limitations include the need fora laboratory equippedwith a ultra-centrifuge,thermocycler,andelectrophoresisequipment.Theuseofpositiveandnegativecontrolsisessentialandstrictadherencetospecifiedtechniquesarerequiredto eliminate cross-contaminationwith RNAandDNAtoproducefalsepositiveresults.Despitethese



AW SAJ 2017.indd 94 8/25/2560 BE 12:41 PM

limitations,dengueRT-PCR is apowerful tool todiagnosedengueserotype-specificviremia.NewerRT-PCRtechniquesarebeingdevelopedthatmaybemorepracticalforthedevelopingdenguediagnosticlaboratory including pocket thermocyclerswithgelcartridgescontainingalltheessentialreagentsthatcanbeusedinthefieldandrequireminimaltechnicalexpertise.

Nucleic acid sequence-based amplification (NASBA) NASBA is an isothermal RNA amplificationmethod that uses electrochemiluminescence todetectmRNAutilizingtheNuclisensTMbasickitand the Nuclisens Reader (Organon Teknika).UnlikeRTPCR,whichreliesontheconversionofRNA intoeDNAand thenamplification,NASBAdirectlyamplifiesRNAusingprimersandcaptureprobes at isothermal temperatures. NASBAhas been successfully used in other pathogenssuch asmalaria, cytomegalovirus and humanimmunodeficiency virus (Berndt et al, 2000;Bloket al,2000;Schooneet al,2000;Wittet al,2000).Recently,NASBAhasbeenappliedtothediagnosisofdengue(Wuet al,2001).Usingspikedsera,NASBAhadadetectionthresholdof1to10PFU/ml.When testedagainst clinical samples, athreshold of 25 PFU/mlwas observed, a 100%serotype concordancewith viral isolation, anda sensitivity of 98.3%and specificity of 100%.NASBAthoughpreliminaryinresults,mayprovetobeausefuldiagnostictoolintheearlyviremicphaseofacuteDENVinfection.

Fluorogenic probe-based 5’ exonuclease assay (Taqman) The fluorogenic probe-based 5’ exonucleaseassay (Taqman) using the PerkinElmer AppliedBiosystemsautomatedsequencedetectionsystem7700hasbeensuccessfullyusedtodiagnoseandquantifyanumberofhumanpathogensincludingmany viruses (Morriset al, 1996;Hawrami andBreuer,1999;Jordenset al,2000;Lanciottiet al,2000;Loebet al,2000;Schuttenet al,2000).Thistechnique isbasedon theuseofafluorescent-taggedprobethathybridizeswiththetargeteDNAsequence (following theRT step).Afluorescentsignal is released through the5’-3’exonuclease

activity ofDNA Taq polymerase (Hollandet al,1991). This allows real-timemonitoring of thetargetedPCRproductand,withaninternalcontrol,a quantitativemeasurement. Taqmanhas beensuccessfully used to detect and quantifyDENVinfection(Laueet al,1999;Callahanet al,2001;Hounget al,2001;Warrilowet al,2002).Taqmanmay prove to be a useful technique to rapidlydiagnosis DENV infection and in particular torapidlyquantifyviremiaanditscorrelateindenguediseaseseverity.

Detection of dengue virus NS1 antigen using enzyme immunoassay Alcon et al (2002) reported that the NS1antigenwasfoundcirculatingfromthefirstdayafter theonsetof feveruptoday9:NS1 levelsranged from 0.04 to 2 μg/ml in acute-phaseserumsamples(fromdays0-to-7),andthe levelforaconvalescentphaseserum(day8andlater)was0.04μg/ml.Insecondaryinfection,theNS1level rangedfrom0.01to2μg/mlandwasnotdetectable in convalescent-phase sera (Alconet al, 2002). Shuet al (2002) reported data fromacute-phaseserawitheitherprimaryorsecondaryinfection thatwere in agreementwith thoseofAlconet al(2002).Moreover,theirdatasuggestedthattheNS1antigenwasdetectableduringdays1-to-8ofillness(Shuet al,2002).

Dengue genotypes studies AlthoughPCRisthemostsensitiveandrapidmethodforthedetectionofdenguevirusinearlystageofdisease,classicaldenguevirusculturehasimportant benefits: Longitudinal collections ofdenguevirusprovidematerial formany studies,such as studies of pathogenesis, phylogeniccharacterization,aswellasantigenicdrift.Withfurthersequencingusingdengueserotypesseedculturesoneisabletotellthegenotypesineachserotype including fromdifferent years. Studiesthatattempttoassociatevirulencewithgenotypesclearlyplayanimportantroleintheselectionofparentstrainsforattenuatedvaccines.

Dengue1-4 genotypes (Klungthong,Chonticha,Molecularsection,VirologyDepartment,AFRIMS,personnelcommunication)(Figs3-6):



AW SAJ 2017.indd 95 8/25/2560 BE 12:41 PM

Fig3–DENV-1inThailand.




AW SAJ 2017.indd 96 8/25/2560 BE 12:41 PM





AW SAJ 2017.indd 97 8/25/2560 BE 12:41 PM

DENV-1inThailand:MostoftheDENVserotype1inThailandisgenotypeI,circulatingsince1980tothepresent.AfewDENVserotype1specimensofgenotypeIIIwerefoundduring1980-1983,andthendisappearedfromcirculation.

DENV-2inThailand:MostoftheDENVserotype2inThailandareAsianIgenotype,circulatingsince1991tothepresenttime.AfewDENVserotype2 specimens ofAsian/American genotypewerefoundduringthe1980to1990periodandthendisappearedfromcirculation.

DENV-3inThailand:MostoftheDENVserotype3 circulating since 1990 to the present aregenotype II.Genotype IIIwasalsofoundduring2009to2010(Thisgenotypeisbeinginvestigationuntilpresent).

DENV-4inThailand:MostoftheDENVserotype4inThailandaregenotypeI,circulatingsince1998tothepresent.OnegenotypeIIwasfoundintheyear 2000.A fewgenotype III specimenswerefound during the 1997 to2001 timeframe andthendisappearedfromcirculation.

conclusIon

Understandingthepatternofimmuneresponsesto first or subsequent DENV infections in thecontextoftheclinicalillnessisessentialtoidentifytheappropriatediagnostictoolstodiagnoseacuteDENV infection.Antibody-based assayswill notbepositiveearlyinthecourseofdisease;patientswithsuspecteddengueshouldnotbesenthomebelievingthefeverisnotduetodengue.Theymustbewarnedaboutthesignsofplasmaleakageandappropriatelyfollowed.Serotypespecificdiagnosisisdifficultpost-defervescenceandthelimitationsoftheantibodyresponseinapatientwithapreviousDENVinfectionmustbetakenintoaccount.Withtheseconceptsinmind,whatwouldbetheidealassaytodiagnoseDENVinfection?First,theassaymustbebothsensitiveandspecific,withahighpredictivevaluedespitealowincidenceofdisease.Thiswillbeimportantincountrieswheredengueisanemergingdisease. Itmusthave lowcross-reactivitywith other co-circulating flavivirusessuchas JE, yellow feverorWestNile virus. The

assaymust be reproduciblewith low inter andintra-assayvariabilityanditmustbeinexpensivesothatdevelopingcountrieswithdengueepidemicsmightbeabletoutilizetheconsidereddiagnostictechnique.Likewisetheassaymustbesimpletoperformwithminimal training and diagnosticequipment.Suchanassaywouldidentifyserotype-specificdengueantigenduringtheviremicperiodand IgMand IgGduring the lateacuteorearlyconvalescentperiod.

Giventhesecriteria,onecanvisualizearapiddiagnostictestthatwilltakeminutestoperformusing a finger-prick ofwhole blood or salivaplacedontoacardwithonespaceforadengueserotype-specific antigen capture and anotherfor the detection of dengue specific IgM andIgGduringthelateacuteorconvalescentperiod.Futureassaywillgobeyondconfirmingorrefutingdengue as the etiology to distinguishmultipleetiologies of fever. Etiologic diagnosis basedongeneexpressioninresponsetoinfectionneedstobeevaluated.Thesechallengesforflavivirologistsandcommercialcompaniespresentanopportunitytoaidinthecontrolandtreatmentofthisglobalhealthproblem.

references

Alcon S, Talarmin A, DebruyneM, FalconarA,Deubel V, Flamand M. Enzymed-linkedimmunosorbent assay specific to denguevirus type1nonstruturalproteinNS1revealscirculationoftheantigeninthebloodduringtheacutephaseofdiseaseinpatientsexperiencingprimaryorsecondaryinfections.J Clin Microbiol 2002;40:376-81.

AltemeierWAIll,MundonFK,TopFHJr,RussellPK.Methodofextractingviralhemagglutination-inhibiting antibodies from the nonspecificinhibitorsofserum.Appl Microbiol1970;19:785-90.

Artimos deOliveira S, RodriguesCV,CamachoLA,MiagostovichMP,ArayoES,NogueiraRM.Diagnosis of dengue infection by detectingspecificimmunoglobulinMantibodiesinsalivasamples.J Virol Methods1999;77:81-6.



AW SAJ 2017.indd 98 8/25/2560 BE 12:41 PM

BerndtC,MullerU,BergmannF,SchmittU,KaiserR,MullerC.Comparisonbetweenanucleicacidsequence-based amplification and branchedDNAtestforquantifyingHIVRNAloadinbloodplasma.J Virol Methods2000;89:177-81.

BlokMJ,LautenschlagerI,GoossensVJ,et al.Diag-nosticimplicationsofhumancytomegalovirusimmediateearly-1andpp67mRNAdetectioninwhole-bloodsamplesfromliver transplantpatientsusingnucleicacidsequence-basedam-plification.J Clin Microbiol 2000;38:4485-91.

BoonpucknavigS,BhamarapravatiN,NimmannityaS,PhalavadhtanaA,SiripontJ.Immunofluores-centstainingofthesurfacesoflymphocytesinsuspensionfrompatientswithdenguehemor-rhagicfever.Am J Pathol1976;85:37-47.

BoonpucknavigS,Vuttiviroj0,BoonpucknavigV.Infection of young adultmicewith denguevirustype2.Trans R Soc Trop Med Hyg1981;75:647-53.

Boonpucknavig S, Vuttivirojana 0, Siripont J,FutrakulP,NimmannityaS.Indirectfluorescentantibodytechnicfordemonstrationofserumantibodyindenguehemorrhagicfevercases.Am J Clin Pathol1975;64:365-71.

Burke DS, Nisalak A. Detection of JapaneseencephalitisvirusimmunoglobulinMantibodiesinserumbyantibodycaptureradioimmunoassay.J Clin Microbiol1982;15:353-61.

BurkeDS,NisalakA, JohnsonDE, Scott RM.Aprospective study of dengue infections inBangkok.Am J Trop Med Hyg 1988;38:172-80.

CalisherCH,KarabatsosN,Dalrymple JM,et al.Antigenicrelationshipsbetweenflavivirusesasdeterminedbycross-neutralizationtestswithpolyclonalantisera. J Gen Viral1989;70:37-43.

Callahan JD, Wu SJ, Dian-Schultz A, et al.Developmentandevaluationofserotypeandgroup-specificfluorogenicreversetranscriptasePCR(TaqMan)assaysfordenguevirus.J Clin Microbiol2001;39:4119-24.

CardosaMJ,HooiTP,ShaariNS.Developmentof

adot enzyme immunoassay fordengue3: asensitivemethodforthedetectionofantidengueantibodies.J Viral Methods1988;22:81-8.

CardosaMJ,TioPH,NimmannityaS,NisalakA,InnisB.IgMcaptureELISAfordetectionofIgMantibodies todenguevirus: comparisonof2formatsusinghemagglutininsandcellculturederivedantigens.Southeast Asian J Trop Med Public Health1992;23:726-9.

Casals J. Aectone-ether extracted antigens forcomplement-fixationwithcertainneurotropicviruses.Prac Soc Exp Biol1949;70:339-43.

Casals J, Brown LV. Hemagglutination witharthropod-borneviruses.J Exp Med1954;99:429-49.

Chan SY, Kautner I, Lam SK. Detection andserotypingofdenguevirusesbyPCR:asimple,rapidmethodfortheisolationofviralRNAfrominfectedmosquito larvae.Southeast Asian J Trop Med Public Health1994;25:258-61.

ChandlerA,RiceL.Observationsontheetiologyofdenguefever.Am J Trop Med Hyg1923;3:233-62.

ChungueE,MarcheG,PlichartR,BoutinJP,RouxJ.ComparisonofimmunoglobulinGenzyme-linked immunosorbentassay (IgG-ELISA)andhaemagglutinationinhibition(HI)testforthedetectionofdengueantibodies.PrevalenceofdengueIgGELISAantibodiesinTahiti.Trans R Soc Trop Med Hyg1989;83:708-11.

Clarke DH, Casals J. Improvedmethods forhemagglutinationstudieswitharthropodborneviruses.Prac Soc Exp Biol Med1955;88:96-9.

Clarke DH, Casals J. Techniques for hemag-glutination and hemagglutination inhibitionwitharthropod-borneviruses.Am J Trop Med Hyg1958;7:561-73.

CuzzubboAJ, Endy TP,NisalakA,et al. Use ofrecombinantenvelopeproteinsforserologicaldiagnosis of dengue virus infection in animmunochromatographicassay.Clin Diagn Lab Immunol2001;8:1150-5.



AW SAJ 2017.indd 99 8/25/2560 BE 12:41 PM

CuzzubboA,VaughnDW,NisalakA,McBrideJ,AaskovJ,Devine,P.Commercialassaysfortheserological diagnosis of dengue infections.Arbovirus Res Austr1997;7:56-60.

Cuzzubbo AJ, Vaughn DW, Nisalak A, et al.ComparisonofPanBioDengueDuoIgMandIgG capture ELISA and venture technologiesdengueIgMandIgGdotblot.J Clin Virol2000;16:135-44.

CuzzubboAJ,VaughnDW,NisalakA,SuntayakornS,Aaskov J,DevinePL.Detectionof specificantibodiesinsalivaduringdengueinfection.J Clin Microbiol1998;36:3737-9.

DulbeccoR.Productionofplaquesinmonolayertissueculturesbysingleparticlesofananimalvirus.Proc Natl Acad Sci USA1952;38:747-52.

FujitaN,TamuraM,HottaS.Denguevirusplaqueformation onmicroplate cultures and itsapplicationtovirusneutralization(38564).Proc Soc Exp Bioi Med1975;148:472-5.

GreenS,VaughnDW,KalayanaroojS,et al.Earlyimmuneactivation inacutedengue illness isrelatedtodevelopmentofplasmaleakageanddiseaseseverity.J Infect Dis1999;179:755-62.

GroenJ,KorakaP,VelzingJ,CopraC,OsterhausADME. Evaluation of six immunoassays fordetection of dengue virus-specific immuno-globulinMandGantibodies.Clin Diagn Lab Immunol 2000;7:867-71.

GublerOJ,NalimS,TanR,SaipanH,SuliantiSarosoJ. Variation in susceptibility to oral infectionwithdenguevirusesamonggeographicstrainsofAedesaegypti.Am J Trop Med Hyg 1979;28:1045-52.

HallWC,CrowellTP,WattsOM,et al.Demonstrationof yellow fever and dengue antigens informalin-fixedparaffin-embeddedhumanliverby immunohistochemicalanalysis.Am J Trop Med Hyg 1991;45:408-17.

Halstead SB, Rojanasuphot S, SangkawibhaN.Original antigenic sin in dengue.Am J Trop Med Hyg 1983;32:154-6.

HalsteadSB,StreitTG,LafontantJG,et al.Haiti:absenceofdenguehemorrhagicfeverdespitehyperendemicdenguevirustransmission.Am J Trop Med Hyg 2001;65:180-3.

HalsteadSB,SukhavachanaP,NisalakA.Assayofmouseadapteddenguevirusesinmammaliancellculturesbyaninterferencemethod.Proc Soc Exp Bioi Med 1964;115:1062-8.

HammonW,SatherG.Problemsoftypingdengueviruses.Mil Med 1964;129:130-5.

HawramiK,BreuerJ.Developmentofafluorogenicpolymerasechainreactionassay(TaqMan)forthe detection and quantitation of varicellazostervirus.J Viral Methods 1999;79:33-40.

HenchalEA,McCownJM,SeguinMC,GentryMK,BrandtWE.Rapididentificationofdenguevirusisolatesbyusingmonoclonalantibodiesinanindirectimmunofluorescenceassay.Am J Trop Med Hyg 1983;32:164-9.

HenchalEA,NarupitiS,FeighnyR,PadmanabhanR,VakhariaV.DetectionofdenguevirusRNAusingnucleicacidhybridization.J Viral Methods 1987;15:187-200.

HenchalEA,PoloSL,VorndamV,YaemsiriC,InnisBL,HokeCH. Sensitivity and specificity of auniversalprimersetfortherapiddiagnosisofdengue virus infections by polymerase chainreactionandnucleicacidhybridization.Am J Trop Med Hyg 1991;45:418-28.

Henderson J, Taylor R. Arthropod-borne virusplaquesinagaroverlaidtubecultures.Proc Soc Exp Bioi Med 1959:257-9.

HollandPM,AbramsonRD,WatsonR,GelfandDH.Detectionofspecificpolymerasechainreactionproduct by utilizing the 5’----3’ exonucleaseactivityofThermus aquaticusDNApolymerase.Proc Natl Acad Sci USA 1991;88:7276-80.

HottaS.ExperimentalstudiesindengueI.Isolation,identificationandmodificationofthevirus.J Infect Dis 1952;90:1-9.

HoungHH,Chung-MingChenR, VaughnOW,Kanesa-thasanN.Developmentofafluorogenic



AW SAJ 2017.indd 100 8/25/2560 BE 12:41 PM

RT-PCR system for quantitative identificationofdenguevirusserotypes1-4usingconservedandserotype-specific3’noncodingsequences.J Viral Methods 2001;95:19-32.

IgarashiA.IsolationofaSingh’sAedes albopictuscellclonesensitivetodengueandChikungunyaviruses.J Gen Viral 1978;40:531-44.

Innis BL. Dengue and dengue hemorrhagicfever. In: Porterfield JS, ed. Kass handbookof infectiousdiseases:exoticvirus infections.London: Chapman & Hall Medical, 1995:103-46.

Innis BL, Nisalak A, Nimmannitya S, et al. Anenzyme-linked immunosorbent assay tocharacterizedengueinfectionswheredengueand Japanese encephalitis cocirculate.Am J Trop Med Hyg 1989;40:418-27.

JacobsM,YoungP.Denguevaccines:preparingtorollbackdengue.Curr Opin lnvestig Drugs2003;4:168-71.

JordensJZ,LanhamS,PickettMA,AmarasekaraS, Abeywickrema I,Watt PJ. Amplificationwithmolecular beacon primers and reverselineblotting for thedetection and typingofhumanpapillomaviruses.J Viral Methods 2000;89:29-37.

KalayanaroojS,VaughnOW,NimmannityaS,et al.Earlyclinicalandlaboratoryindicatorsofacutedengueillness.J Infect Dis1997;176:313-21.

Kuno G. Persistent infection of a nonvectormosquito cell line (TRA-171)with dengueviruses.lntervirology1982;18:45-55.

KunoG,GomezI,GublerDJ.Detectingartificialanti-dengueIgMimmunecomplexesusinganenzyme-linkedimmunosorbentassay.Am J Trop Med Hyg1987;36:153-9.

KunoG,GomezI,GublerDJ.AnELISAprocedureforthediagnosisofdengueinfections.J Viral Methods 1991;33:101-13.

KunoG, Gubler OJ, Oliver A. Use of originalantigenicsintheorytodeterminetheserotypesofpreviousdengueinfections.Trans R Soc Trop

Med Hyg1993;87:103-5.

KunoG,GublerDJ,SantiagodeWeilNS.AntigencaptureELISAfortheidentificationofdengueviruses.J Viral Methods 1985;12:93-103.

LamSK,DevinePL. Evaluationof captureELISAand rapid immunochromatographic test forthedeterminationofIgMandIgGantibodiesproducedduringdengueinfection.Clin Diagn Virol1998;10:75-81.

LamSK,DeviS,PangT.DetectionofspecificIgMindengueinfection.Southeast Asian J Trop Med Public Health1987;18:532-8.

LamSK,FongMY,ChungueE,et al.Multicentreevaluation of dengue IgM dot enzymeimmunoassay.Clin Diagn Viral1996;7:93-8.

LamSK,LanEwC,MitchellJL,CuzzubboAJ,DevineP. Evaluationof a capture screeningenzyme-linked immunosorbent assay for combineddetermination of immunoglobulinM andGantibodiesproducedduringdengueinfection.Clin Diagn Lab lmmunol2000;7:850-2.

Lanciotti RS, Calisher CH, Gubler DJ, ChangGJ, VorndamAV. Rapid detection and typingof dengue viruses from clinical samples byusing reverse transcriptase-polymerase chainreaction.J Clin Microbiol1992;30:545-51.

Lanciotti RS, Kerst AJ, Nasci RS, et al. Rapiddetection ofWest Nile virus from humanclinicalspecimens,field-collectedmosquitoes,and avian samples by a TaqMan reversetranscriptase-PCRassay.J Clin Microbiol 2000;38:4066-71.

Laue T, Emmerich P, Schmitz H. Detection ofdengue virus RNA in patients after primaryor secondary dengue infection by using theTaqManautomatedamplificationsystem.J Clin Microbiol1999;37:2543-7.

LibratyDH, Endy TP,HoungHS,et al.Differinginfluences of virus burden and immuneactivation on disease severity in secondarydengue-3 virus infections. J Infect Dis 2002;185:1213-21.



AW SAJ 2017.indd 101 8/25/2560 BE 12:41 PM

LoebKR,JeromeKR,GoddardJ,HuangM,CentA, Corey L. High-throughput quantitativeanalysisofhepatitisBvirusDNAinserumusingthe TaqMan fluorogenic detection system.Hepatology2000;32:626-9.

Malergue F, Chungue E. Rapid and sensitivestreptavidin-biotin amplified fluorogenicenzyme-linkedimmunosorbent-assayfordirectdetection and identification of dengue viralantigensinserum.J Med Virol1995;47:43-7.

MonathTP,LindseyHS,NuckollsJG,ChappellWA,Henderson BE. Comparison ofmethods forremovalofnonspecificinhibitorsofarbovirushemagglutination.Appl Microbiol 1970; 20:748-53.

MorensDM,HalsteadSB,LarsenLK.Comparisonofdenguevirusplaquereductionneutralizationbymacroand“semi-micro’methods inLLC-MK2 cells.Microbiol Immunol 1985a; 29:1197-205.

MorensDM,Halstead SB, Repik PM, PutvatanaR,RaybourneN. Simplifiedplaque reductionneutralization assay for dengue viruses bysemimicromethodsinBHK-21cells:comparisonof the BHK suspension test with standardplaquereductionneutralization.J Clin Microbiol 1985b;22:250-4.

MoritaK,TanakaM,IgarashiA.Rapididentificationofdenguevirusserotypesbyusingpolymerasechainreaction.J Clin Microbiol 1991;29:2107-10.

MorrisT,RobertsonB,GallagherM.Rapidreversetranscription-PeRdetectionofhepatitisCvirusRNAinserumbyusingtheTaqManfluorogenicdetection system. J Clin Microbiol 1996; 34:2933-6.

OkunoY,IgarashiA,FukaiK.NeutralizationtestsfordengueandJapaneseencephalitisvirusesbythefocusreductionmethodusingperoxidase-anti-peroxidase staining.Biken J 1978; 21:137-47.

ParidaMM,UpadhyayC,SaxenaP,DashPK,JanaAM, Seth P. Evaluation of a dipstick ELISAandarapidimmunochromatographictestfor

diagnosisofDenguevirusinfection.Acta Virol 2001;45:299-304.

PorterfieldJ.Thehaemagglutination-inhibitiontestinthediagnosisofyellowfeverinman.Trans R Soc Trop Med Hyg 1954;48:261-6.

PorterfieldJ.Useofgoosecellsinhaemagglutinationtestswith arthropod-borne viruses.Nature1957;180:1201-2.

PutnakR,BarvirDA,BurrousJM,et al.Developmentof a purified, inactivated, dengue-2 virusvaccineprototypeinVerocells:immunogenicityandprotectioninmiceandrhesusmonkeys.J Infect Dis 1996;174:1176-84.

RosenL,GublerOJ.Theuseofmosquitoestode-tectandpropagatedengueviruses.Am J Trop Med Hyg 1974;23:1153-60.

RosenL,RoseboomLE,GublerOJ,LienJC,Chani-otisBN.Comparativesusceptibilityofmosquitospeciesandstrainstooralandparenteralinfec-tionwith dengue and Japanese encephalitisviruses.Am J Trop Med Hyg 1985;34:603-15.

RosenL,ShroyerDA.Comparative susceptibilityoffivespeciesofToxorhynchitesmosquitoestoparenteralinfectionwithdengueandotherfla-viviruses.Am J Trop Med Hyg1985;34:805-9.

RuizBH,ZamoraMP,LiuSG.DetectionofdengueviralRNAbymicroplatehybridization. J Viral Methods1995;54:97-108.

Russell PK, NisalakA, Sukhavachana P, VivonaS.A plaque reduction test for dengue virusneutralization antibodies. J Immunol 1967;99:285-90.

SabinAB. The denguegroupof viruses and itsfamilyrelationship.Bact Rev1950;14:225-32.

SabinAB.Recentadvancesinourknowledgeofdengueandsandflyfever.Am J Trop Med Hyg1955;4:198-207.

SangCT,CuzzubboAJ,DevinePL.Evaluationofacommercialcaptureenzyme-linkedimmunosor-bentassayfordetectionofimmunoglobulinMandGantibodiesproducedduringdenguein-



AW SAJ 2017.indd 102 8/25/2560 BE 12:41 PM

fection.Clin Diagn Lab lmmunol 1998;5:7-10.

SchooneGJ,Oskam L, KroonNC, SchalligHD,Omar SA. Detection and quantification ofPlasmodium falciparum in blood samplesusingquantitativenucleicacidsequencebasedamplification. J Clin Microbiol 2000;38:4072-5.

SchuttenM, van denHoogen B, van der EndeME,GrutersRA,OsterhausAD,NiestersHG.Developmentofareal-timequantitativeRT-PCRforthedetectionofHIV-2RNAinplasma.J Virol Methods 2000;88:81-7.

ShopeRE.Antigen and antibodydetection andupdateonthediagnosisofdengue.Southeast Asian J Trop Med Public Health1990;21:642-5.

ShuPY,ChenLK,ChangSF,et al.Potentialappli-cationofnonstructuralproteinNS1serotype-specific immunoglobulin G enzyme-linkedimmunosorbentassayintheseroepidemiologicstudy of dengue virus infection: correlationofresultswiththoseoftheplaquereductionneutralizationtest.J Clin Microbiol 2002;40:1840-4.

SilerJF,HallMW,HitchensAP.Dengue:itshistory,epidemiology,mechanism of transmission,etiology,clinicalmanifestations,immunity,andprevention.Philip J Sci1926;29:1-304.

SimmonsJS.Denguefever.Am J Trop Med Hyg 1931;11:77-102.

Sukhavachana P,NisalakA,Halstead SB. Tissueculture techniques for the study of dengueviruses.Bull World Health Organ1966;35:65-6.

Sukhavachana P, Yuill TM, Russell PK.Assay ofarbovirus neutralizing antibody by micromethods. Trans R Soc Trop Med Hyg 1969;63:446-55.

VarmaMGR,PudneyM,LeakeCJ.CelllinesfromlarvaeofAedes(Stegomyia)malayensiscollessandAedes(S)pseudoscutellaris(Theobald)andtheirinfectionwithsomearboviruses.Trans R Soc Trop Med Hyg1974;68:374-82.

Vathanophas K, HammonWM, Atchison RW,SatherGE.Attemptedtypespecificdiagnosis

of dengue virus infection by the indirectfluorescent antibody method directed atdifferentiatingIgMandIgGresponses.Proc Soc Exp Bioi Med1973;142:697-702.

VaughnDW,GreenS,KalayanaroojS,et al.Den-gueintheearlyfebrilephase:viremiaandanti-bodyresponses.J Infect Dis1997;176:322-30.

VaughnOW,GreenS,KalayanaroojS,et al.Dengueviremia titer, antibody responsepattern, andvirusserotypecorrelatewithdiseaseseverity.J Infect Dis2000;181:2-9.

VaughnOW,Nisalak A, Kalayanarooj S, et al.Evaluationofarapidimmunochromatographictestforthediagnosisofdengueinfection.J Clin Microbiol1998;36:234-8.

VaughnOW,NisalakA, Solomon T,et al. Rapidserologicdiagnosisofdenguevirus infectionusing a commercial capture ELISA thatdistinguishesprimaryandsecondaryinfections.Am J Trop Med Hyg 1999;60:693-8.

Vorndam V, BeltranM. Enzyme-linked immu-nosorbent assay-formatmicroneutralizationtest fordengueviruses.Am J Trop Med Hyg 2002;66:208-12.

WangWK,ChaoDY,KaoCL,et al.Highlevelsofplasmadengueviralloadduringdefervescencein patientswith dengue hemorrhagic fever:implicationsforpathogenesis.Virology2003;305:330-8.

WarrilowD,NorthillJA,PykeA,SmithGA.Singlerapid TaqManfluorogenic probe based PCRassaythatdetectsallfourdengueserotypes.J Med Virol2002;66:524-8.

WittOJ, KemperM, SteadA, et al. Analyticalperformance and clinical utility of a nucleicacid sequence-based amplification assay fordetectionofcytomegalovirusinfection.J Clin Microbiol 2000;38:3994-9.

WolffKL,TrentOW,KarabatsosN,HudsonBW.Useofglutaraldehyde-fixedgooseerythrocytesin arbovirus serology. J Clin Microbiol 1977;6:55-7.



AW SAJ 2017.indd 103 8/25/2560 BE 12:41 PM

WorldHealthOrganization(WHO).Denguehemor-rhagicfever:diagnosis,treatment,preventionandcontrol.2nded.Geneva:WHO,1997.

WuSJ,Grouard-VogelG,SunW,et al.HumanskinLangerhans cells are targets of dengue virusinfection.Nat Med2000;6:816-20.

WuSJ,KungCG,ChenTB,et al.Comparisonoftworapiddiagnosticassaysforthedetectionof immunoglobulinMantibodies to dengue

virus.Clin Diagn Lab lmmunol1999;7:106-10.

WuSJ,LeeEM,PutvatanaR,et al.DetectionofdengueviralRNAusinganucleicacidsequence-based amplification assay. J Clin Microbiol 2001;39:2794-8.

YuillTM,SukhavachanaP,NisalakA,RussellPK.Dengue-virus recovery by direct anddelayedplaquesinLLC-MK2cells.Am J Trop Med Hyg 1968;17:441-8.



AW SAJ 2017.indd 104 8/25/2560 BE 12:41 PM

Documents

laBoratory DiagnoSiS of Dengue viruS infeCtionS … · clinical management of DHF patients needs to be determined in prospective studies. ... Fig 1– Diagnostic pathway in patients