Comparison among the BED capture enzyme immunoassay test and AxSYM avidity index assay for determining recent HIV infection and incidence in two Voluntary Counselling and Testing Centres in Northeast Brazil

(1)

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

Comparison

among

the

BED

capture

enzyme

immunoassay

test

and

AxSYM

avidity

index

assay

for

determining

recent

HIV

infection

and

incidence

in

two

Voluntary

Counselling

and

Testing

Centres

in

Northeast

Brazil

Daniela

Medeiros

Salustiano

a,b,∗

,

Kledoaldo

Oliveira

de

Lima

b,c

,

Ana

Maria

Salustiano

Cavalcanti

d

_,

_Ricardo

_Sobhie

_Diaz

e

_,

_Heloisa

_Ramos

_Lacerda

b

a_The_Herbert_de_Souza_Counselling_and_Testing_Centre,_Cabo_de_Santo_Agostinho,_PE,_Brazil

b_{Post-graduation}_in_Tropical_Medicine,_Universidade_Federal_de_Pernambuco,_PE,_Brazil

c_The_Paulista_Counselling_and_Testing_Centre,_Paulista,_PE,_Brazil

d_Department_of_Virology,_Laboratório_Central_de_Pernambuco_(LACEN-PE),_Recife,_PE,_Brazil

e_{Retrovirology}_Laboratory,_Universidade_Federal_de_São_Paulo,_SP,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received24October2013

Accepted17March2014

Availableonline26April2014

Keywords: HIV

Recentinfection

Avidityindexassay

BED-CEIA

a

b

s

t

r

a

c

t

TheaimsofthisstudyweretocomparetheautomatedAxSYMavidityassayindexwith

theBEDcaptureenzymeimmunoassaytestandtocalculatetheHIV-1incidenceusingthe

BEDcaptureenzymeimmunoassayandAxSYMavidityassayindexalgorithmswithina

populationseekingtheVoluntaryCounsellingandTestingCentresintwomunicipalities

intheMetropolitanRegionofRecife,NortheastofBrazil.Ananalysiswasconductedin365

samplesthattestedpositiveforHIVinfectionfromfrozenserumcollectedduringtheperiod

2006–2009.Therewasasimilarproportionofmalesandfemales;mostpatientswere

hetero-sexual(86%)withamedianageof29years.Ofthe365samples,102(28%)and66(18.1%)were

identifiedasrecentinfectionsbyBEDcaptureenzymeimmunoassayandAxSYMavidity

assayindex,respectively.TheHIV-1totalincidenceintheBEDcaptureenzyme

immunoas-sayandAxSYMavidityassayindexalgorithmswere:0.79(95%CI:0.60–0.98)and0.34(95%

CI:−0.04to0.72),respectively.Incidencewashigheramongmen.Therewasgood

agree-mentbetweenthetests,withakappaof0.654andaspecificityof95.8%.AxSYMavidity

assayindexmaybehelpfulinimprovingthequalityoftheestimatesofrecentHIVinfection

andincidence,particularlywhenusedinacombinedalgorithmwithBEDcaptureenzyme

immunoassay.

∗ _{Corresponding}_author_at:_Av._Prof._Moraes_Rêgo,_s/n,_Bloco_A,_Térreo_do_Hospital_das_Clínicas_da_Universidade_Federal_de_Pernambuco,

50670-420,CidadeUniversitária,Recife,PE,Brazil.

E-mailaddress:danisalustiano@yahoo.com.br(D.M.Salustiano).

http://dx.doi.org/10.1016/j.bjid.2014.03.001

(2)

Brazilhasreported656,701 casesofAIDS, andinrecent

yearstherehasbeenanincreaseintherateofcasesinthe

NorthandNortheastregionsofthecountry,whencompared

totheSouthandSoutheastregions.Furthermore,the

North-easternstateofPernambucoanditscapitalRecifepresentthe

highestratesofnewcasesofAIDSofanystateorstatecapital

inNortheastBrazil.1

ThegrowinginterestinestimatingHIV-1incidencehasled

tothe developmentof a varietyoflaboratory assays. This

methodologyis usedonlyforepidemiologicalinvestigation

purposes,andisreferredtobytherecentlyintroducedterm

RITA (RecentInfectionTestingAlgorithm),used todescribe

laboratoryassaysthatareabletoidentifyatransitionalperiod

ofrecentseroconversionofHIVinfection.2

In 1998, an adaptation of a diagnostic test for HIV-1

that would revolutionize epidemiological methodsthrough

itsabilitytoidentifyrecentHIV-1infectionwasdescribedin

theliterature.3_The_test_{differentiated}_recent_infections_(less

thansixmonths)fromestablishedinfections(morethansix

months),duetothekineticsofantibodiesaftertheonsetof

HIV-1infection.ThistestallowedtoestimateHIV-1incidence

fromcross-sectionalstudies,usingasingleserumsampleper

patient.

TheBED capture enzymeimmunoassay(BED-CEIA), the

assayforHIV-1 incidence,haspeptidesasantigensderived

from the immunodominant region of gp41, containing

sequencesofsubtypesB,EandD,whicharerepresentativeof

multiplesubtypesofHIV-1.ThetimeatwhichHIVinfection

occurredcanbeestimated,giventhattheratiobetweenthe

totalIgGandanti-HIVIgGvariesaccordingtotheprogression

ofthedisease.4_This_technique_has_been_widely_used_in

con-junctionwithmathematicaltoolsforestimatingtheincidence

ofHIVinfection.5

Another method for distinguishing between recent and

establishedHIVinfectionsbasedonantibodyavidity(affinity)

wasdescribedin2002.6_This_methodology_is_based_on_the

prin-ciplethattheantibodyavidityproducedintheinitialphaseof

theinfection(withinsixmonthsofseroconversion)islow,in

contrasttothatobservedinestablishedinfections(morethan

sixmonths).6_Besides_the_simplicity_and_automated_nature_of

thetechnique,intheory,itwouldalsobeabletodetectahigh

proportionofsubtypesofHIV-1andwouldnotbeaffectedby

thestageoftheinfection.Theassociationofthetwotestshas

beenusedtoconfirmthepositiveresultsofBED,resembling

resultsofincidencedataofcohorts thathavedocumented

seroconversion.7

Inviewoftheimportanceofcomprehendingthedynamics

oftheHIVepidemic,theaimofthisstudyweretocomparethe

magnitudeofagreementbetweentheautomatedtestAxSYM

avidity assay index (Ax-AI) with the BED-CEIA test and to

calculatetheHIV-1incidenceusingBEDandAx-AIwithina

populationseekingVoluntaryCounsellingandTesting

Cen-tres(VCTs)intwomunicipalitiesoftheMetropolitanRegion

ofRecife,inNortheastBrazil.

FrozensamplesfrompatientsdiagnosedwithHIV

infec-tion during the period 2006–2009 were obtained from two

VCTsinthemunicipalitiesofCabodeSantoAgostinhoand

Paulista, in the Metropolitan Region of Recife – Northeast

Brazil.Threehundredandeighteenpatientswerediagnosed

asHIV-infectedattheVCTinCabodeSantoAgostinho,and

aliquotsfrom206(64.8%)ofthemwereavailableforfurther

testing.AtthePaulistaVCT,179casesofHIVinfectionwere

detected,butonly159(94%)hadserumavailablefortesting.

Thusatotalof365serumsampleswereanalyzed.

Samples from patientswith previoususe of

antiretrovi-raltherapy(ART),infectedthroughverticaltransmission,and

thosewithpreviousdiagnosisofHIVinfectionoccurringmore

than sixmonthspriortothecollectionofthesamplewere

excluded.

Datarelatingtotheindividuals’gender,age,and

sociode-mographicdatawereretrievedfromthedatabankoftheHIV

testresultinformationsystem(SIREX-HIV)attheVCTs.This

studywasapprovedbytheResearchEthicsCommitteeofthe

HospitalAgamenonMagalhães(protocolnumber518/2009).

For theAx-AIthesamplewas defrosted,twoaliquotsof

20␮Lwereobtainedanddiluted1:10.Onealiquot(PBSaliquot)

wasdilutedwith180␮Lofphosphate-bufferedsaline(PBS)and

theother(Galiquot)wasdilutedwith180␮Lof1Mguanidine

solution(G)(Sigma–Aldrich).Afterdilution,allsampleswere

vortex-stirredandincubatedfor10minatroomtemperature.

Thetwoaliquotsofeachsampleweretestedusingthe

auto-matedAxSYMHIV-1/2gOassay(AbbottDiagnosticsDivision,

Wiesbaden-Delkenheiem,Germany),inaccordancewiththe

manufacturer’sinstructions.6

Additionofadenaturingagent(guanidine)breaks

hydro-genbridges,whichhelpsdeterminingthesecondarystructure

of the antibodies and leads to a residual effect from the

antigen–antibody interaction. This procedure has greater

effectonrecentantibodies,whicharelow-avidityantibodies.8

ThisprocedureresultsinanS/COratiothatislowerfortheG

aliquotthanforthePBSaliquot.Theavidityindex(AI)is

calcu-latedafterobtainingtheS/COratiosforthePBSandGaliquots

asfollows:6,9

AI=GuanidinealiquotS/CO

PBSaliquotS/CO .

Acutoffvalueof0.8distinguishesaninfectionoflessthan

sixmonths6,10_from_a_long-term_infection_(a_duration_of_more

thansixmonths).

TheBED-CEIAtest(CalypteBiomedicalCorporation,

Ore-gon, USA) is an IgG-capture ELISA technique based on

detectingincreasingproportionsofspecificanti-HIV-1IgG

fol-lowingseroconversion.Anti-HIVIgGandnon-specificIgGsare

capturedinthesolidphaseofthemicroplate.After

incuba-tionandwashing,peptidesfromtheimmunodominantregion

ofgp41 thatcontainsequences ofthe subtypesB,Eand D

wereadded.Afterfurtherincubationandwashing,a

strepta-vidinconjugatewasadded,followedbyanincubationperiod

of90min.Theresponseisascertainedaftertheadditionof

tetramethylbenzidine(TMB)withaspectrophotometer

(wave-length450nm).4,11

Following the immunoassays and reading of the

spec-trophotometer,thenormalizedopticaldensities(ODns)were

obtained,asfollows:

ODnofthecontrols= medianODofthecontrols

medianODofthecalibrator

ODnofthesample= ODofthesample

(3)

Thetests were interpreted as follows: all samples with

ODn≤1.2 were tested in triplicate to confirm whether it

was a recent or long-term infection. Confirmation

inter-pretation: samples with OD>0.8 were characterized as

long-termseroconversionandthosewithOD≤0.8asrecent

seroconversion.12

HIV-1 incidence and 95% confidence interval (95% CI)

for the BED-CEIA and Ax-AI algorithms were calculated

using the spreadsheet providedbythe South African

Cen-tre for Epidemiological Modelling and Analysis (SACEMA)

(http://www.incidence-estimation.com/page/tools-for-incidence-from-biomarkers-for-recent-infection).13

Thefollowingparameterswerereportedonthespreadsheet:

the Estimated Mean Duration of Recent Infection (MDRI),

whichwas180daysfortheAx-AIand155daysforthe

BED-CEIA;4 _the _estimated_False _Recent _Rate _(FRR%),_which _was

5.6%,14_and_10.6%_for_the_BED-CEIA_and_Ax-AI,_{respectively;}7,14

numberofHIV-positiveindividuals;numberofHIV-negative

individuals and number of recent HIV-1 infections.

Inci-denceestimateswereexpressedasthenumberofnewHIV

infectionsper100person-years.

Sensitivity,specificity,positiveandnegativepredictive

val-ues,accuracy,likelihoodratiosforpositiveandnegativetests,

andkappawereobtainedbycomparingtheresultsofAx-AI

tothoseofBED-CEIA,usingtheMSOfficeExcelversion2010

(Microsoft Corporation)and the SPSS forWindows version

12.0.Significantassociationsbetweenthevariableswere

con-sideredwhenp-valueswerelessthan0.5.

Thestudy population consistedof177 men(48.5%)and

188women(51.5%),ofwhom53(28.2%)werepregnant.The

medianagewas29years.Otherepidemiologicandbehaviour

dataarelistedinTable1.Ax-AIandBED-CEIAresultswere

comparedusing 365samples. One hundred and two

sam-pleswereidentifiedasrecentinfectionbyBED-CEIA,and77

samplesbyAx-AI.Forthestatusoflong-terminfection,the

BED-CEIAandAx-AIidentified263and288samples,

respec-tively.Bothtestswereconcordantin66samplesclassifiedas

recentinfectionand252asalong-terminfection.

WhencomparedtotheBED-CEIA,withacutoffof0.8,the

Ax-AIshowed64.7%sensitivityofand95.8%specificity,with

ratesofpositivepredictivevalues(PPV)and negative(NVP)

greaterthan85%.Theagreementbeyondchancebetweenthe

twotechniqueswasgood(kappa=0.654,p<0.001).

BED-CEIAandAx-AIresultsforsixpatientswhose

sero-conversiondatewasequaltoorlessthanayear,confirmedby

anegativeassayforHIVfollowedbyapositiveassaywithin

12months,demonstrateddisagreementinonlytwopatients

theclassificationstatusofrecentinfection.Inoneofthese

casesseroconvertedaftersevenmonthsandBED-CEIAODn

wasborderline(atthecutoffthreshold)(datanotshown).

GlobalHIV-1incidencesaccordingtoBED-CEIAandAx-AI

were0.79(95%CI:0.60–0.98)and0.34(95%CI:0–0.72),

respec-tively. Thus,the incidencerate estimatedbythe BED-CEIA

was significantly higher (p>0.0001).The number ofrecent

infectionsamongHIVpositiveindividuals,fortheBED-CEIA,

Ax-AIandBED+Ax-AIwere 105,77 and66 (p=0.004).Only

theAx-AIdemonstratedasignificantincreasingtendencyof

HIVincidencewithtime (p=0.02). Theincidenceestimates

variedbetween0.66and0.97%/yearand0.19and0.45%/year

for the BED-CEIA and Ax-AI, respectively. All methods for

estimatingHIV-1incidenceshowedsignificantlyhigherrates

Table1–Demographicsandbehaviouraldataofpatients newlydiagnosedasHIV-positiveattwoVCTsin

Pernambuco–Brazil,2006–2009.

Variables HIV-positivepatients

Gender

Male 177(48.5)

Female 188(51.5)

Pregnant

Yes 53(28.2)

No 135(71.8)

Medianage(years) 29

Sexualorientation

Heterosexual 304(86)

MSMa ₅₀₍₁₄₎

Risksituationb

Bloodtransfusion 14

Sexworker 08

HIV+partner 26

Schooling

≤8years 231(63.3)

>8years 123(33.7)

Notinformed 11(3.0)

Employmentsituation

Employed 69(18.9)

Self-employed 42(11.5)

Unemployed 61(16.7)

Student 25(6.8)

Housewife 117(32.1)

Retired 09(2.5)

Notinformed 42(11.5)

a _MSM,_men_who_have_sex_with_men.

b _Not_expressed_in_percentage_since_most_patients_did_not_present

thisrisk.

for men than for women (p<0.0001), with a difference of

approximatelythreefold(Table2).

The present study demonstrates that estimates of HIV

incidenceinapopulation seekingVCTsintwo

municipali-tieswithintheMetropolitanRegionofRecife,capitalcityof

Pernambuco,NortheastBrazil,mayvaryaccordingtothe

diag-nosticalgorithmused,witharateof0.79%/yearwhenusing

theBED and0.34%/yearwiththeAx-AI,excludingthoseon

HAART. TheBED-CEIA methodology has been widely used

to measurethe incidence ofHIV-1 incross-sectional

stud-ies,mainlyduetoitscommercialavailability.However,many

questions remain regardingits accuracy,witha suggestion

ofoverestimation,despitetheconsensualuse ofcorrection

factors.2_The_WHO_Working_Group_on_HIV_Incidence_Assays

systematicallyreviewedtheaccuracyofserologicaltestsfor

recent infectionswithHIV.Across 13different assays,

sen-sitivity todetect recent infections ranged from 42 to100%

(median89%).Specificityfordetectingestablishedinfections

wasbetween49.5and100%(median86.8%)andwashigher

forinfectionsofdurationslongerthanoneyear(median98%,

range 31.5–100.0).Serologicalassayshavereasonable

sensi-tivity fordetectingrecentHIVinfection,but are vulnerable

tomisclassifyingestablishedinfectionsasrecent,potentially

leadingtobiasesinincidenceestimates.15_The_use_of_a

(4)

Table2–AnnualHIV-1incidenceestimatedbyusingtheBED-CEIA,Ax-AI,andBED-CEIA+Ax-AIalgorithms.

Year/gender No.ofindividuals No.ofindividuals IncidenceBED

(%)–(95%CI)d

IncidenceAx-AI

(%)–(95%CI)d

HIVpositivea

(BEDsamples)b

(Ax-AIsamples)c

HIVnegative Recent

infection (BED)

Recent infection

(Ax-AI)

Recent infection

(BED+Ax-AI)

2006 111a₍₆₆₎b₍₆₄₎c ₈₉₃₁ ₁₈ ₁₁ ₀₉ _0.70_{(0.38–1.02)} _0.19_(0–0.63)

2007 141(98)(90) 9242 29 20 16 0.97(0.60–1.35) 0.45(0–0.94)

2008 122(118)(118) 9208 34 26 24 0.81(0.47–1.14) 0.38(0–0.81)

2009 123(93)(93) 9998 24 20 17 0.66(0.36–0.95) 0.34(0–0.74)

Male 243(180)(177) 8449 44 37 32 1.46(0.94–1.97) 0.77(0–1.65)

Female 254(195)(188) 28,930 61 40 34 0.59(0.42–0.76) 0.24(0–0.51)

Total 497(375)(365) 37,379 105 77 66 0.79(0.60–0.98) 0.34(0–0.72)

a _A_total_of₁₀_HIV-positive_patients_were_excluded_from_the_incidence_calculation:_eight_had_presented_a_previous_positive_serological_test

forHIV,onewasverticaltransmission,andanotherwasundergoingantiretroviraltreatment.Threeoftheseindividualswerefromthe2006

cohort,twowerefromthe2007cohort,threewerefromthe2008cohort,andtwofromthe2009cohort.

b _Total_number_of_available_aliquots_for_BED-CEIA_testing.

c _Total_number_of_available_aliquots_for_Ax-AI_testing.

d_Results_are_expressed_as_the_{incidence/100}_people/year_(95%_CI).

combinationwithanavidityassaywascitedasthemost

com-monlyusedalgorithm.2

Thepresent study tested this strategy inindividuals in

themetropolitanareaofRecife,NortheastBrazil,wherethe

mostprevalentsubtypesBare(around57%),F(around37.7%)

andC(around3.1%).16_A_significant_difference_was_observed

betweenincidenceratesestimatedbyBED-CEIA(0.79%/year)

andAx-AI(0.34%/year)(p<0.00001).

One major difficulty when discussing estimates of HIV

incidenceisthedifferenceamongpopulationsstudied.Most

studieshaveatargetpopulationofgroupsatrisk,especially

sexworkers,menwhohavesexwithmen(MSM)andinjected

drugusers.Thisresearchevaluatedamixedpopulationfrom

twoVCTsintheBrazilianstateofPernambuco,whichwere

linkedtotheBrazilianMinistryofHealth’sNationalSTDand

AIDSProgramme.Inthisregion,thoseattendingtheVCTs

con-sistofthreemaingroups:peoplefromthegeneralcommunity,

usuallyoflowriskinfection,pregnantwomenfromwithinthe

community,andindividualswithbehaviourpatterns

associ-atedwithanincreasedriskofinfection(e.g.sexworkersand

MSM).Thus,onelimitationofthisstudyamongotherstudies

inBrazilistheheterogeneityofthepopulationstudiedplusthe

factthattheaccuracyoftheestimatesofincidenceby

sero-logicalmethodsdependsontheaccuracyofthemeanRITA

durationandtheRITAFRR,whicharethemeasuresevaluated

forpopulationswithcharacteristicsdifferentfromthoseofthe

presentstudy.17

Therearedataillustratingthattheaviditytestpresentsa

betterperformanceincorrectly classifyingindividuals with

along-termviralload,whiletheBED-CEIAhasgreater

ten-dency to classify them erroneously as recent infections.18

Furthermore,ithasbeenshownthatAI-Axsuffersless

inter-ference from the HIV subtype and antibody titres intheir

performance.6

The incidence estimates presented herein were about

threefoldhigherformenthanforwomen.TheBrazilian

epi-demicisconcentratedwithincertaingroups,especiallyyoung

MSM.1 _However _in_the_present _study,_this_group _accounted

for only14%, thus, underrepresented inthe population of

VCTsstudied.InBrazil,therehavebeenseveralstudies,which

applied serological methods to estimate HIV-1 incidence,

especially inthe South and Southeast. Althougha

consid-erableportion ofthese studiesincluded individuals witha

higherriskforHIVinfection,suchasprisoners,cocaineand

injected drugsusers,MSMand high-riskheterosexualmen

andwomen,incidencedataforbothsexesshowahigherrate

formen,asobservedbySchechteretal.19_and_Alves_et_al.,20

wheretheestimatedincidenceratewasbetween1.9and2%

forwomenand2.8and2.7%formenand,respectively.

Giventhelargenumberofdiagnostictestsperformedfor

HIVinfection(132million/year)andbloodbankscreening(101

million/year),aswellasrecognizingthehugemarketinvolved,

theimportanceofapplyingtheRITAmethodologyshouldbe

emphasized inorder toprovide abettermap ofthe spread

oftheepidemicandallowadequatestructuringofpreventive

strategies.Toourknowledge,thisisthefirststudyinBrazil

toapplytheconceptofthedualtestingalgorithmto

deter-mineestimatesofHIVincidence,basedonthemostrecent

recommendationsoftheWHO.2,14

Funding

ProgramaNacionaldeCooperac¸ãoAcadêmica–Ac¸ãoNovas

Fronteiras(Procad–NF)–Coordenac¸ãodeAperfeic¸oamento

dePessoaldeNívelSuperior(CAPES),MinistériodaEducac¸ão,

Brasil.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

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