Hepatitis B virus infection in HIV-exposed infants in the Western Cape, South Africa

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ContentslistsavailableatScienceDirect

Vaccine

jo u rn al h om ep a g e :w w w . e l s e v i e r . c o m / l o c a t e / v a c c i n e

Hepatitis

B

virus

infection

in

HIV-exposed

infants

in

the

Western

Cape,

South

Africa

Naﬁisah

Chotun

a,∗

_,

_Etienne

_Nel

b

_,

_Mark

_F.

_Cotton

c

_,

_Wolfgang

_Preiser

a

_,

Monique

I. Andersson

a

a_Division_of_Medical_Virology,_National_Health_Laboratory_Service_and_Faculty_of_Medicine_and_Health_Sciences,_Stellenbosch_University,_Tygerberg, SouthAfrica

b_Department_of_Paediatrics_&_Child_Health,_Faculty_of_Medicine_and_Health_Sciences,_Stellenbosch_University,_Tygerberg,_South_Africa c_KID-CRU,_Faculty_of_Medicine_and_Health_Sciences,_Department_of_Paediatrics_&_Child_Health,_Stellenbosch_University,_Tygerberg,_South_Africa

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received25February2015

Receivedinrevisedform31May2015 Accepted19June2015

Availableonlinexxx

Keywords: HBV

Mothertochildtransmission HIV/HBVco-infection Verticaltransmission Vaccination SouthAfrica

a

b

s

t

r

a

c

t

HepatitisBvirusinfection(HBV)isasigniﬁcantpublichealthprobleminsub-SaharanAfrica.Universal infantvaccinationwiththehepatitisB(HB)vaccinehasbeenimplementedwithintheSouthAfrican ExpandedProgrammeofImmunizationsinceApril1995withconcomitantreductioninHBVinfectionin children.However,theﬁrstvaccinedoseisonlyadministeredatsixweeksofage.Thisdelaymayleadto afailuretoreducetheriskofperinatalHBVtransmissiontoinfantsborntoHIV/HBVco-infectedwomen, inwhomHBVinfectionisoftenupregulated.Theaimofthisstudywastodeterminetheprevalenceof HBVinfectioninbabiesborntoHIV-infectedmothersintheWesternCape,SouthAfrica.HBVserological markersweretestedinallinfantserumsamplesandfollowingHBviralloadtesting,sequencingand genotypingwerealsoperformed.Threeof1000samplesscreenedtestedpositiveforHBsAgandHBV DNA.AnadditionalinfanttestedpositiveforHBVDNAalone.AllbabieshadreceivedtheHBvaccineat6, 10and14weeks.TheprevalenceofHBVinfectionwastherefore4/1000(0.4%;95%CI,0.01–0.79%).Three offourinfantsandallfourmotherswerefollowed-up.TwoinfantswerepersistentlypositiveforHBsAg withviralloadsabove108_{International}_Units_per_millilitre._All_four_maternal_samples_were_positive_for

HBsAgandHBeAgandonewasalsopositiveforanti-HBe.Sequencinganalysisoftwomother–childHBV pairsshowed100%sequenceidentity.ThisstudydemonstratesHBVinfectioninHIV-exposedinfants despiteHBvaccinationfrom6weeksofage.Amorestrategicapproachisneededtopreventmotherto childtransmissionofHBV,includingscreeningofpregnantwomen,HBV-targetedantiviraltherapyand HBbirthdosevaccine.

1. Introduction

HepatitisB virusinfection(HBV), a signiﬁcant publichealth problem,isendemicinsub-SaharanAfrica(SSA).Theprevalence hereisamongstthehighestworldwide[1],withsomeregional dif-ferences.Forexample,inSouthAfrica,HBsAgprevalenceinadults rangesfrom3%to25%,withthehighestratesinHIV-infectedadults

[1–6].AntenataldatafromMalawiandZimbabwereport

preva-lencesof13%[7]and25%,respectively[8].

SSAhasmorethan70%oftheworld’sHIVinfections; includ-ing 6.1 million HIV infected people in South Africa alone [9]. ChronicHBVinfectionintheHIV-infectedpatientisanimportant co-morbidity[10,11]withmorerapidprogressionofcirrhosis[12],

∗ Correspondingauthor.Tel.:+27219389360;fax:+27219389361. E-mailaddresses:naﬁisahc@sun.ac.za,naﬁisah.chotun@gmail.com(N.Chotun).

higher hepatitis B(HB) viralloads [13,14]and a higher HBeAg prevalence[13].

HBVinfectionispreventable.Asafeandeffectivevaccinehas beenavailableforoverthreedecades.Remarkablesuccessin reduc-inglong-termcomplicationsinhighprevalenceareas,likeTaiwan, hasbeenreported[15].WorldHealthOrganisationguidelines advo-catethatthefirstdoseofinfantHBvaccinebeadministeredin thefirst fewdays of life[16].However,in mostSSA countries, thefirstdoseisadministeredatsixweeksofage.Thisschedule isbasedonepidemiologicalstudiesshowingthatAfricanmothers arepredominantlyHBeAg-negative,[17]thereforeatlowriskof transmittingHBVvertically[18].Studiesconfirmingthislowrisk showedlittletonoearlyHBVinfectioninchildren,butincreasing acquisitionbetweentheagesofoneandfiveyears,suggestiveof horizontaltransmissionamongstsiblingsandplaymates[18–22]. WithHIV/HBVco-infection,HBviralloadmaybehighand HBeA-gaemiamorecommon.Therefore,delayingfirstvaccinationtosix http://dx.doi.org/10.1016/j.vaccine.2015.06.076

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weeksmayleadtoafailuretoreducetheriskofperinatalHBV trans-missiontoinfantsborntoHIV/HBVco-infectedwomen,inwhom HBVinfectionisoftenupregulated[23].Theaimofthisstudywas todeterminetheprevalenceofHBVinfectionininfantsbornto HIV-infectedmothersintheWesternCape,SouthAfrica.

2. Materialsandmethods

2.1. Studypopulation

Thiswasacross-sectional study.EDTAplasmasamplesfrom infantsbetweentheagesof0and18months,whosemotherswere HIV-infected,andwhosesamplesweresubmittedforroutine HIV-1PCRtestingtotheDivisionofMedicalVirology,NationalHealth LaboratoryService(NHLS),StellenboschUniversity,betweenJune 2011andFebruary2012,wereselectedforthisstudy.Sampleswith lessthan100␮lresidualvolumewereexcluded.Age,sexandHIV statusofallinfantsweredocumented.Ethicsapprovalwasgranted by theHealth Research Ethics Committee of Stellenbosch Uni-versity(EthicsReferenceNumber:N11/05/151).Allsampleswere testedforHBsAg.HBVDNAtestingwasperformedinallsamples bypoolingfoursamplesperrunasdescribedpreviously[24].The limitofdetectionofthisassaywas50InternationalUnitsper millil-itre(IU/ml).AllHBsAgorHBVDNApositiveinfantsweretraced andmaternalandinfantfollowupsamplesunderwentserological andmolecularHBVtesting.Additionaldemographicinformation includingtheagesofthemothersandchildrenatfollow-up,the vaccinationstatusofthechildrenatfollow-upandtheCD4counts ofthemothersatoraroundthetimeofdeliverywerecollected. 2.2. HBVserologicaltesting

AllsamplesweretestedforHBsAgusingtheMurexHBsAg Ver-sion3enzymeimmunoassay(EIA)(Murex,BiotechLtd.,Dartfort, UK)accordingtomanufacturer’sinstructions.Positiveresultswere conﬁrmedusinganin-houseneutralisationassayperformedwith theMurexHBsAgVersion3EIAand10IU/mlanti-HBs(suppliedby theBloodBorneVirusesUnit,PublicHealthEngland,London,UK). AllconﬁrmedHBsAgpositivesamplesweretestedforHBeAgand anti-HBebyEIAusingtheETI-EBKPLUSandETI-EBKABPLUSkits fromDiasorin(Saluggia,Italy)respectivelyaccordingto manufac-turer’sinstructions.

2.3. HBVmoleculartesting 2.3.1. HBviralload

DNAwasextractedusingtheQIAamp®MinEluteVirusSpinKit andthepresenceofHBVDNA wasdetectedbyareal-time PCR assay[25].Theﬁnalreactionvolumewas25␮landthereal-time PCRwasperformedontheRotorGeneTM₆₀₀₀_(Corbett_Life_Science,

Australia)usingthefollowingcyclingparameters:aninitial dena-turationfor15minat95◦Cfollowedby45cyclesof95◦Cfor15s and60◦Cfor60s.

2.3.2. Sequencing

DNApositive samplesweresequencedusing primers target-ingthepol/surfaceregions[26].TodeterminetheHBVgenotype and identifyany drug-resistance mutationsin the HBV strains, thesesequencesweresubmittedonlinetoHBVSeq(http://hivdb.

stanford.edu/HBV/HBVseq/development/HBVseq.html).

2.3.3. Phylogeneticanalyses

AMaximumLikelihoodphylogenetictreebasedontheKimura 2-parametermodel[27]withabootstrapof1000replicateswas constructedinMEGA6[28]usingmother–childpairedsequences,

Table1

Demographicsoflaboratoryandstudypopulations.

Characteristic Laboratory population* (n=2582) Study population (n=1000) Age

Median,days(range) 45(0–730) 46(0–540) Sex

Male,% 41.2 48.7

HIVstatus

HIV-infected,%(95%CI) 4.6(3.3–5.9) 6.1(4.62–7.58) *_Total_number_of_samples_received_for_HIV-1_PCR_testing_by_the_NHLS_during_the samplecollectionperiod;CI:ConﬁdenceInterval.

antenatalHBVsequencesfromtheWesternCape[6]andadditional HBVsequencesobtainedfromGenBank.

3. Results

3.1. Studypopulation

From2582infantplasmaspecimensreceivedfrom203Western CapeclinicsbetweenJune2011andFebruary2012forHIV-1PCR testing(laboratorypopulation),1000individualsamples(39%of thelaboratorypopulation)from106clinicscomprisedthestudy population.Table1showsnosigniﬁcantdifferencesbetweenthe demographicsofthestudyandlaboratorypopulations.

3.2. Screeningtestsresults

Threeof1000sampleswerereactiveforHBsAgandconﬁrmed positivebyHBsAgneutralisation.Allthreesampleswerealso pos-itive forHBV DNA.One additionalinfant’s sample waspositive

onlyforHBVDNA(Table2).SinceaverylowlevelofHBVDNA

(<200IU/ml) was detected in the plasma of this infant in the absenceofHBsAg,hewasconsideredtohaveanocculthepatitis Bvirusinfection(OBI).OBIischaracterisedbythepersistenceof detectableHBVgenomesinHBsAgnegativeindividuals[29].The prevalenceof HBVinfection in this studycohort wastherefore 4/1000(0.4%;95%conﬁdenceinterval(CI),0.01–0.79%).Noneof thefourinfantswasHIV-infected.Theywereallmale.

3.3. Followup

Oneofthefourinfectedinfants(theonewiththeoccult infec-tion)couldnot betraced (thisinfantwasnolongerinthecare ofhismother);theremainingthreeHBsAgpositiveinfantswere reviewedatasinglefollowupvisit.Themedianinfantageat follow-upwas195days(range174to232days).Themeanageofthefour motherswas29.5years(range26to35years).

3.4. Followupserologicalandmoleculartestresults

AllthreeinfantshadreceivedthreedosesoftheHBvaccineat 6weeks,10weeksand14weeks,conﬁrmedbychildvaccination records.TwowerepersistentlypositiveforHBsAgandHBeAg.All foursamplesfromthemotherswereconﬁrmedpositiveforHBsAg andHBeAg.Onemother’ssamplewasalsopositiveforanti-HBe.

TwoHBsAgpositiveinfantsampleswerepositiveforHBVDNA, withviralloadsabove108_IU/ml._HBV_DNA_was_not_detected_in_the

thirdinfant’ssample.AllfourmothershaddetectableHBVDNA, three ofwhomwithHBviral loadsgreaterthan 108_IU/ml._The

fourthmother,whowasalsoanti-HBepositive,hadanHBviral loadof103_IU/ml₍_Table₂_).

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Table2

Serologicalandmolecularresultsoffourinfectedmother–childpairsatscreeningandfollow-up.

Patient Screening Follow-up

HBsAg HBVDNAIU/ml HBsAg HBeAg HBVDNAIU/ml AntiHBe

MH1 ND ND + + 1×108 ₋ BH1 + 1×102 ₋ _ND _UD ₋ MH2 ND ND + + 1×103 ₊ BH2 + 2×108 ₊ ₊ ₆_×₁₀8 ₋ MH3 ND ND + + 4×108 ₋ BH3 + 5×104 ₊ ₊ ₂_×₁₀9 ₋ MH4 ND ND + + 4×107 ₋ BH4* ₋ ₅_×₁₀1 _ND _ND _ND _ND

*_Patient_was_lost_to_follow-up;_IU/ml:_{International}_Units_per_millilitre;_HBsAg:_hepatitis_B_surface_antigen;_HBeAg:_hepatitis_B_e_antigen;_AntiHBe:_antibody_to_hepatitis Beantigen;BH:Infectedinfant;MH:Motherofinfectedinfant;−Negative;+Positive;ND:Notdone;UD:Undetectable.

Fig.1.Phylogenetictreeofmother–childpairswithHBVstrainsbelonging to subgenotypeA1basedonpol/surfaceregion.BH:Infectedinfant;MH:Motherof infectedinfant;SequencesD001801,D002438,D002911D002784andD003920 weresequencedfrompatientsfromtheWesternCape[6].Sequenceswith acces-sionnumbersAY233290.1,AY233279.1,AY233287.1,AY903452.1,JN182327.1, JN182333.1andGQ184326.1weredownloadedfromGenBank.Theevolutionary historywasinferredusingtheMaximumLikelihoodmethodbasedontheKimura 2-parametermodel[27].Therewereatotalof917positionsintheﬁnaldataset.

TheHBVpol/surfaceregionsfromthetwoHBsAgpositiveinfant samplesandfourmaternalsamplesweresequenced.Allbelonged tosubgenotypeA1.Nomutationsassociatedwithdrug-resistance orvaccine-escapewereidentiﬁedinthepol/surfaceregions.The HBVsequencesfromtwo mother–childpairswereusedto con-structa phylogenetictree(Fig.1).TheHBVsequencesfromthe twomother–childpairs(MH2/BH2andMH3/BH3)showed100% sequenceidentity.

4. Discussion

Thisstudyshowsa prevalenceofHBV infectionof0.4%in a cohortof1000HIV-exposedinfants.Allfourmotherswho trans-mittedHBVtotheirinfantswereHBeAgpositiveandwerenoton antiretroviraltherapy(ART).Twoofthethreefollowed-upinfants remainedpersistentlyinfected,withhighHBviralloadsataround sevenmonthsofage.Thesedatasuggestverticaltransmissionof HBVintheseHIV-exposedinfants,despiteroutineHBvaccination. Severalobservationssupportaverticalrouteoftransmissionin thefourHBV-infectedinfants.First,allwerebeloweightmonths of agewhen HBsAg positivity wasconﬁrmed (Table 3).At this age,exposuretoothersourcesofinfectionislimited.Second,all fourmotherswereHBeAgpositive,aknownriskfactorforvertical

transmission. Although the HB viral load in one mother was 103_IU/ml_at_review,_we_do_not_know_what_it_had_been_at_delivery.

Itispossiblethatshewasseroconvertingintheearlypost-natal periodand mayhavehada higherviralloadduringpregnancy. Lastly,inthetwosequencedmother–childpairs,phylogenetic anal-ysisshowedthatthestrainswereidentical,supportingamaternal source.

TheHBvaccinewasintroducedintoSouthAfricaalmosttwo decadesago,whentheHBsAgprevalenceinchildrenwas approx-imately10%[30].Subsequently,therehasbeenadecreaseinHBV infectionsinchildren.InLimpopo, anorthernprovinceofSouth Africa,HBsAgwasfoundin3of303(0.9%)childrenbetween5and 24monthsofage[31].Inanearlierstudycarriedoutinthesame province,noneof598childrenhadanHBVinfection(HIVstatus notreported).OnlyonemotherinthatcohortwasHBeAgpositive

[32].Anothercross-sectionalstudyfromSouthAfricacomparing pre-andpost-vaccinationcohortsoveranalmost20yearperiod showedadecreaseinHBsAgprevalencefrom4.2%to1.4%[33].

HBeAgstatusofmothersisanimportantmarkerofHBV trans-missionrisk. HBeAg positivitywas consideredrare inpregnant womeninSSA[17].However,wehavepreviouslyshownHBeAg positivityin 18.9% ofHIV-infected and17.1% ofHIV-uninfected HBsAgpositivewomen[6].InHIV/HBVco-infectedwomenwith lowCD4counts,42%(5/12)wereHBeAgpositive[23].Inamore recentSouthAfricanstudy,HBeAgprevalencewas43%andHBV mothertochildtransmissionratewas28%inHIV/HBVco-infected women[34].InaMalawianstudyinHIV-infectedwomenandtheir HIV-exposeduninfectedinfants,of5%ofHBsAgpositivewomen, 38%werealsopositiveforHBeAg.Inthatstudy,theHBV transmis-sionratewas10%,withallinfectedinfantsborntoHBeAgpositive women[35].

Inthepresentstudy,with0.4%ofHIV-exposedinfantsbeing HBV infected and a background HBsAg prevalence of 3.5% in HIV-infectedwomenintheWesternCape[6,23],theHBV trans-missionrate is similarto that reportedelsewhere [35]. During thisstudy,HIV-infectedpregnantwomenwithCD4countsbelow 350cells/mm3_received_ART,_including_tenofovir_and_lamivudine.

Tenofovirandlamivudinebothhaveanti-HBVactivityandwould therefore reduce HB viral loads and risks of vertical

transmis-sion[36,37].ThewomenwhotransmittedHBVverticallyhadCD4

counts above 350cells/mm3_, _so _received_neither_tenofovir _nor

Table3

AgeofHBV-infectedinfantsatdiagnosis.

Infant Ageatdiagnosis,days

BH1 53

BH2 113

BH3 41

BH4 234

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Table4

Numberofinfantstestedperagegroup.

Age,days Numberofinfants

0–1 7 2–4 8 5–10 7 11–20 35 21–30 14 31–42 199 43–70 451 71–98 115 99–180 64 181–365 72 >365 28

lamivudine.Morerecently,withtheadoptionof“OptionB+”by

SouthAfricain2013,allHIV-infectedpregnantwomenshouldnow

receiveARTirrespectiveoftheirCD4count.However,HIV

infec-tionisstillbeingmissedinpregnancy,withmothersthereforenot

receivingART[38]andincreasingtheriskforbothHIVandHBV

transmission.

AbirthdoseoftheHBvaccinereducesHBVvertical transmis-sionfromHBeAg-positivemothersbyapproximately75%[39].Data fromSSAhasshownthecosteffectivenessofaddingHBbirthdose vaccine[40].HepatitisBimmunoglobulin(HBIG)hasadded ben-eﬁttopreventtransmission[41]; howeveritscostandlogistics areprohibitiveformostAfricansettings.MotherswithhighHB viralloads(>105_IU/ml)_are_at_risk_of_infecting_their_infants_[42]

despiteabirthdoseofHBvaccinetoneonates[43].Itisnowwell establishedthat treatingthesewomenduringthelatestages of pregnancywithnucleos(t)idesanalogues willreducetherisk of transmission[36,37,44].AlthoughHBV-activeARTisnowavailable toallHIV-infectedpregnantwomeninSouthAfrica,theinfantsof HBVmonoinfectedmothersremainvulnerable.

Theresultspresentedhereshouldbeinterpretedinthecontext ofthestudy’slimitations.Thesmallproportionofinfantstested forHBsAgbeforetheageofonemonthinthiscohort(7.1%)were allnegative(Table4).However,becauseoftheiryoungage,these couldhavebeenfalse-negativeresultsandwemaythereforehave underestimatedthenumberofverticaltransmissionsinthiscohort. Furthermore,the HBviral loadof themothers at deliverywas unknown.However,giventhattheywerenotonARTduring preg-nancyandwereHBeAgpositive,itisplausiblethattheirviralloads atdeliverywerehigh. In addition,this studyonlyincludedthe infantswhoattendedclinicforHIVtesting.Thereforeourresults mayhavebeenbiasedagainstthoseinfantswhosemothersdonot regularlyusehealthfacilitiessuchasantenatalclinics.Asonly HIV-exposedinfantswererecruited,theresultscannotbeextrapolated toHIV-unexposedchildren.Furtherworkisrequiredtoinvestigate theprevalenceofHBVinfectioninHIV-unexposedinfants.

5. Conclusion

TheadditionoftheHBvaccinetotheSouthAfricanExpanded ProgrammeofImmunizationhassuccessfullydecreasedHBV infec-tionprevalencefromaround10%toaround1%inonetoﬁveyear oldsina periodoftwodecades.However,HBVmothertochild transmissionisstilloccurringinHIV-exposedchildren,and reduc-ingthistozeroispossible.ScreeningforHBVduringpregnancy, accesstoHBV-targetedantiviraltherapyandHBbirthdosevaccine arestrategicandfeasibleapproachestoachievingthis.

Conﬂictofintereststatement

Theauthorsdeclarethattheyhavenoconﬂictofinterest.

Acknowledgements

We thankthe PoliomyelitisResearch Foundation (PRFGrant Number:11/01)and theHarryCrossleyFoundationfor funding thisstudyandProfRichardTedderandDrSamreenIjazfortheir adviceandsupport.Weexpressoursinceregratitudetothe ded-icatednursesfromtheclinicswhowentoutoftheirwaytotrace theinfectedinfantsandtheirmothers.

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