A H1N1 assay for rapid diagnosis ofinﬂuenza Evaluation of Wondfo inﬂuenza A&B fast test based onimmunochromatography INFECTIOUS DISEASES

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braz j infect dis.2013;17(2):247–250

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

w w w . e l s e v i e r . c o m / l o c a t e / b j i d

Brief

communication

Evaluation

of

Wondfo

inﬂuenza

A&B

fast

test

based

on

immunochromatography

assay

for

rapid

diagnosis

of

inﬂuenza

A

H1N1

Yunping

Peng

a,b

_,

_Junlin

_Wu

c,∗

_,

_Xiaoyun

_Liu

b

_,

_Jihua

_Wang

b

_,

_Wenmei

_Li

a,b

a_School_of_Food_and_{Bioengineering,}_South_China_University_of_Technology,_Guangzhou,_Guangdong,_China b_Guangzhou_Wondfo_Biotech,_Guangzhou,_Guangdong,_China

c_Guangdong_Institute_of_{Microbiology,}_Guangzhou,_Guangdong,_China

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received16February2012

Accepted28September2012

Availableonline1March2013

Keywords:

InﬂuenzaA(H1N1)

InﬂuenzaB

Fastteststrip

a

b

s

t

r

a

c

t

Inﬂuenzavirusescausesigniﬁcantmorbidityandmortalityinbothchildrenandadults

dur-inglocaloutbreaksorepidemics.Therefore,arapidtestforinﬂuenzaA&Bwouldbeuseful.

ThisstudywasconductedtoevaluatetheclinicalperformanceoftheWondfoinﬂuenzaA&B

testforrapiddiagnosisofinﬂuenzaAH1N1Infection.Therapidtestingassaycould

distin-guishinfectionofinﬂuenzaAandBvirus.ThereferenceviralstrainswereculturedinMDCK

cellswhileTCID50ifthevirusesweredetermined.TheanalyticalsensitivityoftheWondfo

kitwas100TCID50/ml.TheWondfokitdidnotshowcrossreactivitywithothercommon

viruses.1928suspectedcasesofinﬂuenzaA(H1N1)virusinfectionwereanalyzedinthe

WondfoinﬂuenzaA&Btestandothercommerciallyavailableproducts.Inconsistentresults

werefurtherconﬁrmedbyvirusisolationincellculture.Thesensitivity,speciﬁcity,positive

predictivevalue(PPV)andnegativepredictivevalue(NPV)were100%,98.23%,92.45%,and

100%forﬂuA,and96.39%,99.95%,98.77%,and99.84%forﬂuBrespectively.766suspected

casesofinﬂuenzaA(H1N1)virusinfectionwereanalyzedintheWondfoinﬂuenzaA&Btest

andRT-PCR.Thesensitivity,speciﬁcity,PPVandNPVwere56.5%,99.75%,99.52%and71.04%

forﬂuA,25.45%,99.86%,93.33%and94.54%forﬂuBrespectively.Theseresultsindicate

thattheWondfoinﬂuenzaA&Btesthashighpositiveandnegativedetectionrates.One

hundredfifty-sixspecimensofinfluenzaA(H1N1)confirmedbyRT-PCRwereanalyzedby

theWondfoinﬂuenzaA&Btestand66.67%werepositivewhileonly18.59%werepositiveby

thereferencekit.Theseresultsindicatethatourrapiddiagnosticassaymaybeusefulfor

analyzinginﬂuenzaAH1N1infectionsinpatientspecimen.

Introduction

Inﬂuenza, or ﬂu, is an acute respiratory infection caused

bya variety ofinﬂuenza viruses. Inﬂuenza epidemics and

∗ _{Corresponding}_author_at:_No._788,_Shenzhou_Road,_Guangzhou,_China.

E-mailaddress:microwjl@yahoo.com(J.Wu).

pandemics canresult insubstantialhealth(clinical illness,

hospitalization, deaths) and socioeconomic (absenteeism,

decrease in productivity, decrease in travel and trade)

impacts.1_A_novel_inﬂuenza_A_(H1N1)_virus_emerged_in

Mex-icoinearly2009andthenspreadtomorethan170countries

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braz j infect dis.2013;17(2):247–250

andregions.2_Inﬂuenza_are_highly_infectious,_mainly_through

coughing and sneezing and other respiratory pathways of

communication.Inﬂuenzaisasigniﬁcantcauseofmorbidity

andmortality,particularlyintheyoungandelderly.3

Accord-ing to the virus gene homology, there are three types of

inﬂuenza virus, A, B and C. Theinﬂuenza A’s variation is

thelargest,andthemostpopular.Therefore,differentiation

ofinﬂuenzavirusfromotherrespiratoryvirusesisofprime

importancebecauseinﬂuenzaisassociatedwithhigherrates

ofmorbidityandmortality,ispotentiallypreventableby

vacci-nation,andcannowbetreatedwithspeciﬁcantiviraldrugs.4

Sincethediagnosisofinﬂuenzacanbedifﬁcultwhenbased

solelyonclinicalsymptoms,rapiddiagnosisofinﬂuenza

per-mits the initiation of antiviral therapy within a beneﬁcial

time frame, can result in discontinuation ofinappropriate

antibiotics,andpromptsinfectioncontrolmeasurestoreduce

spreadinhealthcaresettings.5–7

Thereareseveraldifferentmethodstodiagnoseinﬂuenza.

The“goldstandard”fordiagnosisofinﬂuenzaistissue

cul-tureandvirusisolation,whichmaytake2–14days.Detection

ofvirus-infectedcellsinnasopharyngealsecretionsbydirect

orindirectimmunoﬂuorescentstainingiswidelyusedbutis

quitetechniqueandtechniciandependent.Inaddition,itstill

requiresatleast2htoﬁnish.8_RT-PCR_remains_the_method_of

choiceforclinicaldiagnosisofS-OIVH1N1virusinrespiratory

specimensandfordifferentiatingitfromseasonalinﬂuenza

viruses.9_However,_such_tests_are_of_{high-complexity}_and

can-notbereadilyperformedinprimaryhealthcaresettings.10_The

rapidinﬂuenzadiagnostictest(RIDT),basedon

immunochro-matographiclateralﬂowtests,iscurrentlythebestchoicefor

screeningsamplesforthediagnosisofinﬂuenzavirusdueto

itsrapiddetectionability,simpleoperationandlowcost.RIDT

isunabletofurtherclassifyinﬂuenzaAvirussubtypesandis

notassensitiveasRT-PCR.However,itcanprovidearapid

diag-nosisofinﬂuenzaAorBinfectiontoaidclinicalmanagement

inhalfanhour.

Inthepresentstudywehaveinvestigatedtheclinical

per-formanceoftheWondfoinﬂuenzaA&Btestfortheirabilityto

detectanddifferentiateinﬂuenzaAandBantigens.

Further-more,theiranalytical sensitivity, speciﬁcityand interfering

factorsweredetermined.Thesensitivity,speciﬁcityand

pos-itivepredictivevalue(PPV)andthenegativepredictivevalue

(NPV)werecomparedwithcommerciallyrapiddiagnostickit,

RT-PCR, and virus isolationin cell culture for detection of

inﬂuenza H1N1 viralantigen inrespiratory specimens

col-lectedduringthe2009pandemicperiod.

Materials

and

methods

Specimencollection

The virus strains used to evaluate Wondfo inﬂuenza A&B

test included inﬂuenza A viruses A/14160 (H1N1), A/30

(H1N1),A/44045(H3N2),A/924(H3N2),avaininﬂuenzavirus

A/Beijing/302/54(H5N1)andA/swine/Guangdong/2/01(H1N1),

inﬂuenzaB virusesB/1715,B/1704, B/179, B/668,as well as

otherviruses,suchasadenovirus,respiratorysyncytialvirus,

herpessimplexvirustype1,herpessimplexvirustype2,

rhi-novirustype2,parainﬂuenzavirustype2,parainﬂuenzavirus

type 3,mumpsvirus. Theywere obtainedfrom the Center

forDiseaseControland PreventionofGuangdongProvince.

Mycoplasmapneumoniae,Chlamydiapneumoniae,and Mycobac-terium tuberculosis were isolated at the Microbial Research

CenterwithinSunYat-SenUniversity.156inﬂuenzaA(H1N1)

specimensconﬁrmedbyRT-PCRwereisolatedintheCenter

forDiseaseControlandpreventionofGuangdongProvince.

The1928suspected specimensofinﬂuenzaA (H1N1)virus

infection including766specimensdetectedbyRT-PCRwere

collectedduringthe2009pandemicperiodintheCenterfor

Disease Control and preventionof Guangzhou, Guangzhou

WomenandChildrenMedicalCenter,GuangzhouNo.8

Peo-ple’sHospital,People’sLiberationArmyNo.302Hospitaland

GuangdongEntry-ExitInspectionandQuarantineTechnology

Center.

Analyticalsensitivity,speciﬁcityandcrossreactivity

Ten virus isolates of A/14160 (H1N1), A/30 (H1N1),

A/44045 (H3N2), A/924 (H3N2), A/Beijing/302/54 (H5N1),

A/swine/Guangdong/2/01(H1N1),B/1715,B/1704,B/179,B/668

wereculturedinMardinDarbyCanineKidney(MDCK)cells.

Theculturesupernatantswerealiquotedandfrozenat−80◦C.

Theywereevaluatedtodeterminethelimitofdetectionofthe

Wondfoﬂu A&Btest(WondfoBiotechCo.,Ltd., Guangzhou,

China). An aliquot of each virus was thawed and a series

of 10-fold dilution prepared. The testing was carried out

according to the manufacturer’s instructions. Furthermore,

a series of twofold dilutions were done in duplicate from

the end point obtained above. The diluted virus isolates

were usedtoinfectMDCKcellstodetermineTCID50ofthe

virus. To determine the speciﬁcity and cross reactivity of

the Wondfo inﬂuenza A&Bfast test strip, commensal and

pathogenic micro-organisms(inﬂuenzaBvirus,inﬂuenzaA

virus,respiratorysyncytialvirus,adenovirus,herpessimplex

virus type 1, herpes simplex virus type 2, rhinovirus type

2, parainﬂuenza virus type 2, parainﬂuenza virus type 3,

mumpsvirus,Mycoplasmapneumoniae,Chlamydiapneumoniae,

Streptococcus pneumoniae, Mycobacterium tuberculosis) that

may be present in the nasal cavity or nasopharynx were

tested. Bacteria were cultured and suspended in sterile

Dulbecco’s phosphate buffered saline at concentrations of

108_CFU/ml. _Viral _isolates_were _tested_at_titers_between₁₀3

and108_TCID50/ml.

ComparisonofcharacteristicofWondfotestandreference kit

One thousand nine hundred twenty-eightsuspected cases

ofinﬂuenzaA(H1N1)virusinfectionweretestedbyWondfo

inﬂuenzaA&Bkitandothercommerciallykit(Hangzhou

Gen-esis Biodetection & Biocontrol Ltd., called as GENESIS) as

referencekit.Thespecimenswithinconsistentresultsinthe

abovetestingwerefurtherconﬁrmedbyvirusisolationincell

culture.Theresultispositivedetectedbythebothkitsorthe

resultispositivedetectedbyonekitandisconﬁrmedpositive

byvirusisolationincellculture.Otherresults,notpositive,

aretruenegative.Thesensitivity,speciﬁcity,PPVandNPVof

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brazj infect dis.2013;17(2):247–250

249

ComparisonofcharacteristicofRDTsandRT-PCR

Sevenhundredsixty-sixsuspectedcasesofinﬂuenzaA(H1N1)

virusinfectionweretestedbytworapiddiagnostictests(RDTs,

Wondfo inﬂuenza A&B Kit and GENESIS kit) with RT-PCR

(UT-BIOMEDInternational,Canada)asthereferencemethod.

Sensitivityandpredictivevaluesweredeterminedandthe

dif-ferencesofsensitivityandpredictivevalueswerecompared

betweenRDTsandRT-PCR.

ComparisonofdetectionrateofH1N1ofWondfokitand referencekit

Onehundredﬁfty-sixcasesofH1N1virusinfectioncollected

during the inﬂuenza epidemic in 2009were conﬁrmed by

RT-PCRbyCenterforDiseaseControlandPreventionof

Guang-dongProvince.ThetestingresultsofWondfoinﬂuenzaA&B

kitwerecomparedwithothercommerciallykit(GENESIS)as

referencekit.

Results

Analyticalsensitivity,speciﬁcityandcrossreactivity

Theestimatedlimitofdetection forthe 10inﬂuenza virus

isolateswassummarizedinTable1.Itcanbeseenthatthe

Wondfo test could detect as low as 1.7×103_TCID50/ml _of

inﬂuenzaAvirusand6.3×102_TCID50/ml_of_inﬂuenza_B_virus.

Theresults indicated that the Wondfo inﬂuenza A&Btest

coulddistinguishinfectionofinﬂuenzaAandBvirus,butdid

notshowcrossreactivitywithnon-inﬂuenzavirusesthatwe

tested.

ComparisonofcharacteristicofWondfoandreferencekit Table1showstheresultsof1928specimensdetectedbytwo

rapiddiagnostictestkitsandcompareresultswithvirus

iso-lation incell culture as the gold standard.Thesensitivity,

speciﬁcity,PPVandNPVofWondfoinﬂuenzaA&Bwere100%,

98.23%,92.45%,and 100%forﬂuA,respectivelyand96.39%,

99.95%,98.77%,and99.84%forﬂuB,respectively.The

sensi-tivity,speciﬁcity,PPVandNPVofGENESISwere71.72%,99.30%,

95.72%,and94.20%forﬂuA,and98.80%,99.84%,95.35%,and

99.95%forﬂuBrespectively.ForﬂuA,thesensitivityofthe

Table1–Resultsof1928specimensdetectedbytwo rapiddiagnostickits.

Evaluation kits(Wondfo)

Referencekits(GENESIS) FluA(n=1928) FluB(n=1928) Positive Negative Positive Negative

Positive 246 125a ₇₉ ₂c

Negative 11b ₁₅₄₆ ₇d ₁₈₄₀

a ₉₇_cases_were_confirmed_positive. b _All₁₁_cases_were_confirmed_negative. c ₁_case_was_confirmed_positive. d ₄_cases_were_confirmed_negative.

Table2–ResultsoftheWondfoinﬂuenzaA&Btest, GENESISandRT-PCRindetectionofclinicalspecimens suspectedinﬂuenzaA(H1N1)virusinfection(n=766).

RT-PCR

FluA(n=766) FluB(n=766) Positive Negative Positive Negative

Evaluation reagents (Wondfo) Positive 209 1 14 1 Negative 161 395 41 710 Referencereagents (GENESIS) Positive 117 5 13 2 Negative 253 391 42 709

WondfokitwasgreaterthanthatofGENESIS,butforﬂuB,the twomethodshadnosigniﬁcantdifference.

ComparisonofcharacteristicofRDTsandRT-PCR

Table2showstheresultsof766specimensdetectedbytwo

rapiddiagnostic testkits and compareresultswithRT-PCR

method.Itcanbeconcludedthatthesensitivity,speciﬁcity,

PPVandNPVofWondfoinﬂuenzaA&Bwere56.5%,99.75%,

99.52%,and71.04%forﬂuA,respectivelyand25.45%,99.86%,

93.33%, and 94.54% for ﬂu B, respectively. The sensitivity,

speciﬁcity, PPV and NPV of GENESIS were 31.62%, 98.74%,

95.90%, and 60.71% for ﬂu A, and 23.64%, 99.72%, 86.67%,

and 94.41% for ﬂu Brespectively. For ﬂu A, the sensitivity

oftheWondfokitwasgreaterthanthatofGENESIS,butfor

ﬂu B, the two methods had no signiﬁcant difference. The

sensitivitywasgreaterfortheWondfokit(56.50%)than

GEN-ESIS(31.62%) indetectingﬂuA. Thesensitivitywassimilar

fortheWondfokit(25.45%)thanGENESIS(23.64%)in

detec-ting ﬂu B. The speciﬁcity, PPV and NPV of both Wondfo

inﬂuenzaA&Btestandthereferenceinﬂuenzatestswere

con-sistent.

ComparisonofdetectionrateofH1N1ofWondfotestand referencekit

The156inﬂuenzaA(H1N1)specimenswereconﬁrmedby

RT-PCR.Theresultsareshown inTable3. Itcanbeconcluded

that66.67%(104/156)werepositivebyWondfoinﬂuenzaA&B

testwhileonly18.59%(29/156)werepositivebytheGENESIS

kit.

Table3–ResultsoftheWondfoinfluenzaA&Btestand similarproductindetectingRT-PCR-confirmedinfluenza A(H1N1)virusinfectioninclinicalspecimens.

WondfoinﬂuenzaA&Btest Referencekit(GENESIS) Positive Negative

Positive 29 75

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Discussion

Althoughthegoldstandardfordetectinginﬂuenzavirus

infec-tionisvirusisolationincellculture,ittakestoolongtoget

results,whichinturnwoulddelaytheactiontoinitiate

antivi-raltreatmentandtotakemeasurestopreventﬂu epidemic

andtransmission.Therefore,rapidtestsinatimeofminutes

aredeﬁnitelynecessary.

The Wondfo inﬂuenza A&B test used in our study has

beenpreviouslyshowntobeeffectiveindetectingseasonal

inﬂuenzaviruses.10_The_test_was_able_to_{speciﬁcally}_detect_all

inﬂuenzaviruses(typeAandB)includingH1N1,H3N2,H5N1

virus(Table1).Theanalyticalsensitivity(detectionlimitrange:

6.3×102_–3.2_×₁₀7_TCID50/ml)_of_the_ﬂu _A&B_test_was

estab-lishedforsevenﬂu Aand fourﬂu Bisolates.For thecross

reactivitystudy,noneofthe14microorganismstested

(bacte-riaandviruses)turnedoutpositivebytheWondfoinﬂuenza

A&Bfasttest,indicatingverygoodspeciﬁcity.

Ourresultsshowthatthesensitivitywasgreater forthe

Wondfokitthanothercommerciallyavailablekits in

detec-tingﬂuA.Comparedwithothercommerciallyavailablekits,

thesensitivityoftheWondfoinﬂuenzaA&Bfasttestwas100%

forinfluenzavirusAand96.36%forinfluenzavirusBforflu

A.Theresultsofthetwomethodshadsigniﬁcantdifference

(p<0.05)andthetwoRIDTreagentswerecommonconsistent

results(Kappa=0.743,p=0.021,p<0.05).Comparedwith

RT-PCRassay,thesensitivityoftheWondfoinﬂuenzaA&Bfast

testfor inﬂuenzavirus Awas alsomuchhigher than that

ofothercommerciallyavailablekits.Thehighsensitivityof

Wondfo test forinﬂuenza A was conﬁrmed bytesting 156

inﬂuenzaA(H1N1)specimensthatwereconﬁrmedbyRT-PCR.

ThepositivedetectionrateoftheWondfoinﬂuenzaA&Btest

forH1N1was66.67%.However,withtheothercommercially

availablekititwas18.59%.Theseresultssuggestedabetter

performanceoftheWondfoinﬂuenzaA&Bfasttestthanthat

oftheothercommerciallyavailablekitsusedinourstudy.

Conﬂict

of

interest

Allauthorsdeclaretohavenoconﬂictofinterest.

Acknowledgements

WeexpressourthankstoProf.ShujuanYuandDr.Shixing

Tangfortheirexcellenttechnicalhelpandcriticalcomments.

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