Zika detection: comparison of methodologies

brazilian journal of microbiology49(2018)144–147

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Clinical

Microbiology

Zika

detection:

comparison

of

methodologies

Tatiana

Elias

Colombo

_,

_Ana

_Carolina

_Bernardes

_Terzian

_,

_João

_Pessoa

_Araújo

_Júnior

_,

Ricardo

Parreira

_,

_Eliana

_Márcia

_Sotello

_Cabrera

_,

_Izalco

_Nuremberg

_Penha

_dos

_Santos

_,

Andréia

Francesli

Negri

Reis

_,

_Fabiana

_Rodrigues

_Costa

_,

Lilian

Elisa

Arão

Antônio

Cruz

_,

_Patrícia

_Lopes

_Rombola

_,

_Maurício

_Lacerda

_Nogueira

a_{FaculdadedeMedicinadeSãoJosédoRioPreto(FAMERP),SãoJosédoRioPreto,SP,Brazil}

b_{UniversidadeEstadualPaulista,InstitutodeBiociênciasdeBotucatu,LaboratóriodeVirologia,Botucatu,SP,Brazil}

c_{UniversidadeNovadeLisboa,GlobalHealthandTropicalMedicine(GHTM),Lisboa,Portugal}

d_{PrefeituradeSãoJosédoRioPreto,DepartamentodeVigilânciaemSaúde,SãoJosédoRioPreto,SP,Brazil}

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Articlehistory:

Received28December2016 Accepted21April2017 Availableonline30August2017 AssociateEditor:RoxanePiazza

Keywords: Zikavirus Detection Sensitivity Specificity

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ManycountriesintheAmericashavedetectedlocaltransmissionofmultiplearboviruses thatcausefebrileillnesses.Therefore,laboratorytestinghasbecomeanimportanttoolfor confirmingtheetiologyofthesediseases.Thepresentstudyaimedtocomparethe sen-sitivityandspecificityofthreedifferentZikavirusdetectionassays.Onehundredserum samplesfrompatientspresentingwithacutefebrilesymptomsweretestedusinga previ-ouslyreportedTaqMan®RT-qPCRassay.WeusedaSYBR®GreenRT-qPCRandaconventional PCRmethodologiestocomparetheresults.Ofthesamplesthatweredeterminedtobe neg-ativebytheTaqMan®RT-qPCRassay,100%(Kappa=0.670)werealsofoundtobenegativeby SYBR®GreenRT-qPCRbasedonTmcomparison;however,14%(Kappa=0.035)werefound tobepositivebyconventionalPCRfollowedbyagarosegelelectrophoresis.Thedifferences betweentheZIKVstrainscirculatingworldwideandthelowviremiaperiodcancompromise diagnosticaccuracyandtherebytheaccuracyofoutbreakdata.Therefore,improvedassays arerequiredtoimprovethediagnosisandsurveillanceofarbovirus.

©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

licenses/by-nc-nd/4.0/).

Introduction

Many countriesinthe Americas havedetectedlocal trans-mission ofmultiplearbovirusesthatcause febrile illnesses accompaniedbyrash,myalgia,orarthralgia.Therefore, labo-ratorytestinghasbecomeanimportanttoolforconfirmingthe

∗ _{Correspondingauthor.}

E-mail:mnogueira@famerp.br(M.L.Nogueira).

etiologyofthesediseases.Patientswiththeabovesymptoms shouldbeconsideredsuspectedcasesofZikafever, chikun-gunya, anddengue virus infections,especiallyif theyhave traveledtowithintheprevioustwoweeksorarelivinginan areawithrecentreportsofarbovirustransmission.1

Laboratoryevidenceofrecentinfectionwithchikungunya (CHIKV), dengue (DENV), or Zika (ZIKV) virus is generally

https://doi.org/10.1016/j.bjm.2017.04.011

1517-8382/©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

brazilian journal of microbiology49(2018)144–147

145

establishedbytesting patientserum todetect viralnucleic acid or virus specific immunoglobulins and neutralizing antibodies.2 _{However,serologicalcross-reactivitymayoccur}

between ZIKV and other flaviviruses (DENV, yellow fever virus,St.Louisencephalitisvirus,Japaneseencephalitisvirus, andWestNilevirus).Hence,emphasisshouldbeplacedon molecular diagnostic assays (e.g., RT-PCR) for acute speci-menscollected from individuals withclinically compatible illnesses.3–6

The current diagnostic tests for ZIKV have several limitations.7_{Therefore,thisworkaimedtocomparethe}

sen-sitivity and specificityof thepreviously reported TaqMan® RT-qPCRforZIKVdetection5_{withthoseofSYBR}® _Green

RT-qPCR,andconventionalPCR.

Material

and

methods

Atotalof100serumsamplesfrompatientspresentingwith acutefebriledisease for≤5daysfrom JanuarytoMay 2016 weretestedforZIKVinfectionbyTaqMan®RT-qPCR,5_SYBR®

Greenreal-timeRT-PCR,andconventionalPCR(unpub.data). Thepatients were examined by the São José do RioPreto HealthService,andthe illnesswasdiagnosedasZikafever basedonlyonclinical-epidemiologicaldata(signs compati-blewithZIKVinfection),providedbytheBrazilianMinistry ofHealth.Informationontheclinicalpresentationofthe dis-easewascollectedandregisteredintheNationalSystemof InjuryofNotifications(SINAN).Allpatientswereexaminedby thePublicHealthOfficeandwereconsideredautochthonous cases.Thisworkwaspartofanongoingsurveillanceproject approved by the FAMERP’s Ethical Review Board (CEP n◦ 02078812.8.0000.5415).

Samplepreparation

Bloodsampleswerecollected,and theserawere separated andstoredat−80◦_{C.ViralRNAwasextractedfrom140␮Lof}

eachserum usingtheQIAamp ViralRNAMinikit(QIAGEN, Germany)accordingtothemanufacturer’sinstructions,and theRNAsweretestedbythevariousPCR-basedassays. ZIKVdetectionbyTaqMan®

The RNA was initially tested for ZIKV by a TaqMan® RT-qPCR, as previously described,5 using the GoTaq® Probe 1-StepRT-qPCRSystem (Promega).Thesame sampleswere alsoscreenedforDENVandCHIKVbyMultiplex-nested-PCR8

andRT-qPCR9_{respectively.TheqPCRswereperformedusing}

the ThermocyclerStepOne Real-TimePCRSystem (Applied Biosystems) and the multiplex-nested-PCR was performed usingtheVeriti96wellThermalCycler(AppliedBiosystem). ZIKVdetectionbySYBR®

ToidentifyZIKVbySYBRGreen® RT-qPCR,primersdescribed byLanciottietal.,5_{thattargettheFlavivirusenvelopeprotein}

region(ZIKV1086andZIKV1162c)wereused.Thereactions

wereperformedusingtheSuperScript® IIIPlatinum®SYBR® GreenOne-StepqRT-PCRKit(Invitrogen).TheRT-PCRmixture contained10␮Lof2× SybrGreenbuffer,0.3␮Lof10␮Mof each primer,5 _{0.4␮LofROXReferenceDye(25␮M),0.4␮Lof}

SuperScript® III/Platinum® TaqMix,5␮LoftheRNAsample andwatertomakethevolumeupto20␮L.TheRT-PCR mix-turewasincubatedfor3minat42◦C,5minat95◦C,followed by40 cycles of15sat95◦Cand 1minat60◦C,and afinal singlecyclefor1minat42◦C.Theamplificationstepwas, fol-lowedbyameltingcurveanalysis.Thethermalcyclingwas performedinthethermocyclerStepOneReal-TimePCR Sys-tem(Applied Biosystems).Positivitywasdetermined byTm comparisonbetweenthepositivecontrolsandthesamples.

ZIKVdetectionbyconventionalPCR

ToperformtheconventionalPCR,fourprimerstargetingthe ZIKV NS5 protein were designed. Theprimers were based on infectious clones from the African ZIKV strains (Gen-BankaccessionsLC002520.1,KF268948.1andKF268950.1)and were kindly provided by Prof. Ricardo Parreira, the Tropi-cal Medicineand HygieneInstitute, Universidade NOVAde Lisboa,Portugal.Themethodologywasbasedonnested RT-PCR. The RT mixture contained8␮L ofthe RNA template, 4␮L of 5× first strand buffer (250mM Tris–HCl [pH 8.3], 375mMKCl,15mMMgCl2),1.4␮Lofdithiothreitol(0.1M),1␮L

of reverse primer (510097CATGTCCTCAGTRGTCATCC101163;

15␮M), 1.6␮L of dNTP mixture (250␮M each dNTP), 200U ofRNaseinhibitor (RNaseOUT;Invitrogen), 200U ofreverse transcriptase (Superscript; Invitrogen), and water to make the volume up to 20␮L. The mixture was incubated at 50◦C for 50min and at 70◦C for 5min to inactivate the reverse transcriptase. The PCR mixture contained 8␮L of cDNA, 5␮L of 10× PCR buffer (200mM Tris–HCl [pH 8.4], 500mMKCl), 2␮LofMgCl2(50mM), 1␮Lofforwardprimer

(59104CCATCTGGTACATGTGG91203) at 15␮M, 4␮L of dNTP

mixture(250␮MeachdNTP),1UofTaqDNApolymerase (Plat-inumTaqDNApolymerase;Invitrogen),andwatertomakethe volumeupto50␮L.Themixturewassubjectedto30cyclesat 94◦Cfor1min,53◦Cfor1min,and72◦Cfor2min,followedby afinalextensionstepat72◦Cfor5min.

The nested-PCR mixture contained 1␮L ofthe mixture from the first amplification, 5␮L of PCR buffer (200mM Tris–HCl[pH8.4],500mMKCl),2␮LofMgCl2(50mM),1␮Lof

each primer (59671GTGGAGATGACTGCGTTGTGAAGCC96953

and 59990CCATCAGTCGAAGGTCTCTTCTGTGG100153; 15␮M),

4␮LofdNTPmixture(250␮MeachdNTP),1UofTaqDNA poly-merase(PlatinumTaqDNApolymerase;Invitrogen)andwater tomakethevolumeupto50␮L.Themixturewassubjectedto 25cyclesat94◦Cfor1min,53◦Cfor1min,and72◦Cfor2min. Afinalextensionstepwascarriedoutat72◦Cfor5min.The nestedRT-PCRwasperformedintheVeriti96wellThermal Cycler(AppliedBiosystems).

PCR products were loaded onto a 1.5%agarose gel and visualizedunderultravioletlight.Ampliconsizeswere deter-minedbycomparisonwitha100bpDNAladder(Invitrogen). Precautionstoavoidcontaminationweretaken;positive(Zika virusstrainMR766)andnegative(water+allPCRcomponents)

146

controlswere used inall reactions/amplification protocols, andtheprocedurewasrepeated3times.10

Statisticalanalyses

Theaccuracyofany diagnostic methodcanbedetermined bycalculatingitspositivepredictivevalue(PPV)andnegative predictivevalue(NPV).Theeffectofprevalenceonpredictive valueswascalculatedaccordingtoBayes’stheorem.11

Differ-encesintheresultsofthetestswereassessedusingFisher’s exact test12,13 _{and Kappatest.}14 _{Theconfidenceinterval of}

theestimateswascomputedusingthe‘exactmethod’ devel-opedbyClopperandPearson(GraphPadPrismpackage2.01; Graph-Pad).

Results

Weselected100serumsamplescollectedbetweenJanuaryand May2016frompatientswithsuspectedcasesofZIKVdisease, basedonthepresenceofmacularorpapularrashwithtwoor moreofthefollowingsignsandsymptoms:feveror conjuncti-valhyperemiawithoutsecretionandpruritus,polyarthralgia orjointedema.Ofthese,59samples(59%)weredetermined

Table1–PerformanceofTaqMan®,SYBR-green®and conventionalPCRassayforZIKVdetectioninone hundredserasamples.

Laboratorymethods Numberofsamples TaqMan®RT-qPCR

Positive Negative Total

SYBR®GreenRT-qPCR Positive 42 0 42 Negative 17 41 58 Total 59 41 100 ConventionalPCR Positive 11 6 17 Negative 48 35 83 Total 59 41 100

Fig.1–AgarosegeleletrophoresisofampliconsfromPCR assayforZIKV.MW,molecularweightmarker(100bp); positivesamples,1,3,6,9,14,24;negativesamples:2,4,7, 8;P,positivecontrolforZIKV(ZikavirusstrainMR766);N, negativecontrol.PCRassayshowingtheampliconof345bp ofZIKV.

positive(Ct<38.5)and41samples(41%)weredetermined neg-ative(Ct>38.5)usingTaqMan®RT-qPCR.5

The sensitivityand specificity ofthe methodology were compared with those of other laboratory methods (SYBR® GreenRT-qPCRandconventionalPCR)(Table1).Fromthe sam-ples that were foundtobe negativebyTaqMan® RT-qPCR, 41 (100%)(Kappa=0.670)were alsofoundtobenegativeby SYBR®GreenRT-qPCRbasedonTmcomparison (Supplemen-taryTableS1),but6(14%)(Kappa=0.035,p<0.0001)werefound to bepositivebyconventional PCRfollowedbyagarose gel electrophoresis(Fig.1).Toconfirmtheresults,the6positive sampleswereinoculatedonVerocells.RNAwasextractedand ZIKVidentificationwasperformedbyaTaqMan®RT-qPCR,as previouslydescribed.5

Discussion

ZIKV,whichemergedafterDENVandCHIKVinthecityofSão JosédoRioPreto,representsanewpublichealthconcernin thisregion,andcontinuestobemisdiagnosed.15

Laboratory diagnosis of acute ZIKV infection currently relies upon the detection of ZIKV RNA in biological fluids includeserum,plasma,urine,saliva,andamnioticfluid.16_The

abilityofRT-PCRtodetectedofZIKVRNAinbloodislimited, becauseZIKVviremiaisusuallylowandlimitedtothethird andfourthdayafterdiseaseonset.5_{Futurestudieswithlarger}

numbers ofpositive specimens willbe required tofurther characterizethedynamicsofZIKVlevelsinbloodandto deter-mine whethervirusloadiscorrelatedwithdiseaseseverity andimmuneresponses.16

Accurate diagnosis of acute ZIKVinfection may require testing ofmultiplespecimentypes.Accordingtothe litera-ture,themeandaysofillnesswerenotsignificantlydifferent betweenpatientswithZIKVRNAdetectableonlyinthesaliva andthosewithZIKVRNAdetectableonlyintheserum.16,17

HigherZIKVRNAloadsaredetectableintheurineandsemen afterlongerperiods(10–20and27–62daysafteronsetof symp-toms,respectively),suggestingthatthedetectionoftheZIKV RNAinotherspecimentypescouldbeapotentialalternative diagnostictechnique.4,18

ThecurrentdiagnostictestsforZIKVincludesthe conven-tionalone-stepRT-PCRassaytargetingtheEgene19,20_andthe

nonstructuralproteins1(NS1),20_3(NS3)21_and5(NS5),5

respec-tively. Aprevious geneticstudy usingnucleotidesequences derived from the NS5 gene indicated three ZIKV lineages: East African, West African, and Asian.5 _{Genetic analyses}

haverevealed closerelationshipsbetweenthevirus strains intheSouthAmericanandPacificIslandoutbreaks, suggest-ing thatthe Pacificislandsmay havebeenthe springboard fortheSouthAmericanepidemic.22,23_{Thesequencesofthese}

strains are different from those used for the detection of ZIKV,which weredesignedbythe CDC usingprimer/probe setsbasedonZIKVMR766GenBankaccessionno.AY632535 (country: Uganda).5 _{Interestingly,in the present study, the}

Braziliansamples(Asianlineage)werenegativeforZIKVinthe TaqMan®RT-qPCRandSYBR®GreenRT-qPCRassay,whereas 12%ofthesampleswerepositiveforZIKVintheconventional PCRassay;forbothmethodologies,primersbasedonAfrican ZIKVisolateswereused.

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InadditiontothebenefitscommontoanyqPCRreaction, the statistical parameters (Kappa=0.670) prove optimized SYBR Green method has similar performances to TaqMan methodanddataanalysiswasshowwiththehelpofhigh per-formanceprimerdesigningsoftwareandbyutilizationproper protocolsandmaterialwecanachievehighqualityandprecise databySYBRGreenastheTaqManmethod.24

Futureworkwillinvolvereducingthelimitationsofcurrent diagnostictestsforZIKV.Asthispandemicevolves,further development,evaluation,andwidespreadimplementationof ZIKVdiagnosticswillbecriticaltomonitoring,preventing,and treatingZikafever.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

WethanktheFAPESP(grant#2013/21719-3toMLN)for finan-cialsupport.

Appendix

A.

Supplementary

data

Supplementarydataassociatedwiththisarticlecanbefound, intheonlineversion,atdoi:10.1016/j.bjm.2017.04.011.

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