Absence of hepatitis delta infection in a large rural HIV cohort in Tanzania

Short

Communication

Absence

of

hepatitis

delta

infection

in

a

large

rural

HIV

cohort

in

Tanzania

Annja

Winter

,

Emilio

Letang

,

Aneth

Vedastus

Kalinjuma

,

Namvua

Kimera

,

Alex

Ntamatungiro

_,

_Tracy

_Glass

_,

_Darius

_Moradpour

_,

_Roland

_Sahli

_,

_Fre´de´ric

_Le

_Gal

_,

Hansjakob

Furrer

,

Gilles

Wandeler

*

and

the

KIULARCO

Study

Group

a_{DepartmentofInfectiousDiseases,BernUniversityHospital,UniversityofBern,CH-3010Bern,Switzerland} b_{SwissTropicalandPublicHealthInstitute,Basel,Switzerland}

c

IfakaraHealthInstitute,Ifakara,UnitedRepublicofTanzania

d

ISGlobal,BarcelonaCtr.Int.HealthRes.(CRESIB),HospitalClı´nic—UniversitatdeBarcelona,Barcelona,Spain

e

DivisionofGastroenterologyandHepatology,CentreHospitalierUniversitaireVaudois,UniversityofLausanne,Lausanne,Switzerland

f

InstituteofMicrobiology,CentreHospitalierUniversitaireVaudois,UniversityofLausanne,Lausanne,Switzerland

g

HoˆpitauxUniversitairesParis-Seine-Saint-Denis,Paris,France

h_{DepartmentofInfectiousDiseases,UniversityofDakar,Dakar,Senegal} i_{InstituteofSocialandPreventiveMedicine,UniversityofBern,Bern,Switzerland}

1. Introduction

Worldwide,5%ofindividualswithchronichepatitisBvirus (HBV)infectionhaveserologicalevidenceofexposuretohepatitis deltavirus(HDV).1 _{HBV/HDVco-infectedindividualsgenerally}

havemoresevereliverdiseasethanHDV-uninfectedindividuals.

Only limited data on HDV infection are available from Sub-SaharanAfrica (SSA); prevalenceestimates rangefrom zeroin hepatitis B surface antigen (HBsAg)-positive individuals in Mozambiqueto53%intheCentralAfricanRepublic.2,3_Theaim

ofthisstudywastodeterminetheprevalence,aswellasthemain epidemiologicalandclinical determinants,of HDVinfectionin HIV/HBVco-infectedindividualsinrural Tanzania.Toestimate theproportionoffalse-positiveHDVscreeningtests,allpositive sampleswereretestedwithasecondserologicalassayandnucleic acidamplification.

InternationalJournalofInfectiousDiseases46(2016)8–10

ARTICLE INFO Articlehistory:

Received13January2016

Receivedinrevisedform8March2016 Accepted13March2016

CorrespondingEditor:EskildPetersen, Aarhus,Denmark. Keywords: Hepatitisdelta HIV Tanzania False-positiveserology Sub-SaharanAfrica SUMMARY

Objectives:Theepidemiologicalandclinicaldeterminantsofhepatitisdeltavirus(HDV)infectionin

Sub-SaharanAfricaareill-defined.TheprevalenceofHDVinfectionwasdeterminedinHIV/hepatitisBvirus

(HBV)co-infectedindividualsinruralTanzania.

Methods:AllHBV-infectedadultsunderactivefollow-upintheKilomberoandUlangaAntiretroviral

Cohort(KIULARCO)werescreenedforanti-HDVantibodies.Forpositivesamples,asecondserological

test and nucleic acid amplification were performed. Demographic and clinical characteristics at

initiationof antiretroviraltherapy(ART)werecomparedbetweenanti-HDV-negative and-positive

patients.

Results:Among222HIV/HBVco-infectedpatientsonART,219(98.6%)hadastoredserumsample

availableandwereincludedinthestudy.Medianagewas37years,55%werefemale,46%hadWorld

HealthOrganizationstageIII/IVHIVdisease,andthemedianCD4countwas179cells/ml.Theprevalence

ofanti-HDVpositivitywas5.0%(95%confidenceinterval2.8–8.9%).Therewasnosignificantpredictorof

anti-HDVpositivity.HDVcouldnotbeamplifiedinanyoftheanti-HDV-positivepatientsandthesecond

serologicaltestwasnegativeinallofthem.

Conclusions: NoconfirmedcaseofHDVinfectionwas foundamong over200HIV/HBV co-infected

patients in Tanzania. As false-positive serology results are common, screening results should be

confirmedwithasecondtest.

ß2016TheAuthors.PublishedbyElsevierLtdonbehalfofInternationalSocietyforInfectiousDiseases.

ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(

http://creativecommons.org/licenses/by-nc-nd/4.0/).

* Correspondingauthor.Tel.:+41316322525.

E-mailaddress:gilles.wandeler@ispm.unibe.ch(G.Wandeler).

ContentslistsavailableatScienceDirect

International

Journal

of

Infectious

Diseases

j o urn a l hom e pa ge : ww w. e l s e v i e r. c om/ l o ca t e / i j i d

http://dx.doi.org/10.1016/j.ijid.2016.03.011

1201-9712/ß2016TheAuthors.PublishedbyElsevierLtdonbehalfofInternationalSocietyforInfectiousDiseases.ThisisanopenaccessarticleundertheCCBY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).

2. Methods

Thestudy was conductedwithin theKilomberoand Ulanga AntiretroviralCohort(KIULARCO) inIfakara, Tanzania.4 _AllHIV/

HBV-co-infectedadults(15yearsofage)withatleastabaseline andonefollow-upvisitbetweenJanuary2013andDecember2014, and who started antiretroviral therapy (ART) before December 2014, were included. All participants gave written informed consent.Ethicalapproval wasobtained fromtheIfakaraHealth InstituteinstitutionalreviewboardandfromtheNationalInstitute forMedicalResearchofTanzania.

SerumHBsAgwastestedusingarapidtest(AbonBiopharm, Hangzou,China). The ETI-AB-DELTA-2test (DiaSorin,Brussels, Belgium)was used toscreen foranti-HDV antibodies; results were considered positive when the optical density (OD) was <0.9.Asecondserologicaltestwasusedasconfirmation (HDV-Ab kit; DiaPro, Milan, Italy, used on an ETI-Max (DiaSorin) platform).The resultwas considered negative whenthe ratio (threshold/OD) was <0.9. For the measurement of HDV viral load,totalnucleicacidswerepurifiedfromplasma(QiagenEZ1 DSPkit),andcDNA(HighCapacitycDNAReverseTranscription Kit; Applied Biosystems) was subjected to real-time PCR according to Ferns et al.,5 _{with minor modifications. The}

detectionlimit was 1000 genome equivalentsper milliliter of plasma.

Anti-HDV prevalence was described with a 95% confidence interval(CI).Fisher’sexacttestandtheWilcoxonranksumtest were used to compare individual characteristics at ART start between anti-HDV-positive and negative individuals. Analyses

weredoneusingSASversion9.3software(SASInstitute,Cary,NC, USA).

3. Results

Of 222 HBsAg-positive patients on ART, 219 (98.6%) had a storedsampleavailableforHDVserology.Themedianageofthe patients was37 years,55%werefemale,46%had WorldHealth OrganizationstageIII/IVHIVdisease,andthemedianbaselineCD4 countwas179cells/

m

Theprevalenceofanti-HDVantibody-positivitywas5.0%(11/ 219, 95% CI 2.8–8.9%) with the screening test. In all positive samples, the OD ranged between 0.60 and 0.91(median 0.81, interquartile range(IQR)0.65–0.87),whilethepositivecontrols had OD values between 0.05 and 0.09. Among 11 anti-HDV-positivesamples,nonewaspositivewiththesecondanti-HDVtest (negative controls all had a DiaPro ratio (threshold/OD) <0.9;

Table1).HDVRNAcouldnotbeamplifiedinanyofthepatients witha positive screeningtest. Positive real-timePCR resultsof spiked internalRNAcontrolsconfirmedthatHDVRNA-negative resultsweretruenegatives(datanotshown).

The ageand sexdistribution, aswell as medianbody mass index,CD4cellcount,and alanineaminotransferaselevelswere similar between anti-HDV-positive and -negative individuals (Table 2). The estimated glomerular filtrationrate wasslightly lower in HDV-positive individuals compared to HDV-negative individuals(p=0.05).

4. Discussion

Inthiscohortofover200HIV/HBVco-infectedindividualsin rural Tanzania, no confirmedcase of activeHDV infectionwas found. Five percent of the patients had a positive anti-HDV antibodyscreeningtest,butthesecondserologyandnucleicacid amplificationwerenegativeinallofthem.Asnodemographicor clinicalpredictorofanti-HDVfalse-positivitywasidentified,these resultssuggestthatweaklypositive(ODvalues0.1–0.9)anti-HDV screeningresultsshouldbeconfirmedwithanadditionaltest.

There appeartobenopublisheddataon HDVprevalence in Tanzaniaatthecurrenttime.Inlinewiththeresultsofthisstudy, reports from neighboring countries Mozambique and Burundi haveshowntheabsenceofHDVinfection,whereasananti-HDV antibody prevalence of 53% has been described in theCentral AfricanRepublic.2,3,6_{AsthereseemtobelargedifferencesinHDV}

prevalenceacrossandevenwithincountries,furtherresearchis neededtoimproveourknowledgeonHDVinfectioninSSA. Table1

Serologyresultsfor11patientswithapositiveanti-HDVscreeningtesta

Patient DiaSorinOD DiaProratio

1 0.87 0.49 2 0.62 0.18 3 0.65 0.19 4 0.81 0.21 5 0.87 0.18 6 0.71 0.20 7 0.60 0.19 8 0.83 0.21 9 0.89 0.18 10 0.91 0.27 11 0.73 0.19

HDV,hepatitisdeltavirus;OD,opticaldensity.

a

Positiveresult:DiaSorinOD<0.9,DiaProratio(threshold/OD)>0.9.

Table2

Baselinecharacteristicsbyanti-HDVscreeningresult

Baselinecharacteristics HDVscreeningtestresult p-Value

Anti-HDVnon-reactive(n=208) Anti-HDVreactive(n=11)a Sex,n(%)

Female 114(54.8) 5(45.5) 0.55

Male 94(45.2) 6(54.6)

Age,years,median(IQR) 37(31–44) 37(30–44) 0.77

BMI,kg/m2

,median(IQR) 20.3(18–23) 20.8(17–23.5) 0.98

CD4cellcount,cells/ml,median(IQR) 174(66–275) 214(84–337.5) 0.69

ALT,IU/ml,median(IQR) 23.0(11.0–39.6) 26.6(13.7–54.0) 0.36

eGFR,ml/min,median(IQR) 132.3(110.6–146.1) 115.4(105.1–124.4) 0.05

InitialARTregimen(%) 0.18

3TC/D4T/NVP 39(18.8) 0

3TC/AZT/EFV 63(30.3) 5(45.4)

XTC/TDF/EFV 77(37.0) 6(54.6)

Other 29(13.9) 0

HDV,hepatitisdeltavirus;IQR,interquartilerange;BMI,bodymassindex;ALT,alanineaminotransferase;eGFR,estimatedglomerularfiltrationratebasedontheCKD-EPI formula;ART,antiretroviraltherapy;3TC,lamivudine;D4T,stavudine;NVP,nevirapine;AZT,zidovudine;EFV,efavirenz;XTC,lamivudineoremtricitabine;TDF,tenofovir.

a

UsingETI-AB-DELTA-2(DiaSorin).

Allpatientswithafalse-positivescreeningtesthadaweakly positiveresult.Thus,thecut-offrecommendedbythe manufac-turerappearsinappropriateinthepresentstudysetting. Unfortu-natelyitwasnotpossibletocalculatethespecificityoftheHDV antibodyscreeningtestaspatientswithanegativeresultwerenot testedwith thesecondserology. However, ifthepatients with a negative screening test were all truly HDV-uninfected, the specificitywouldbe95%(95%CI91–97%).Althoughnopredictorof anti-HDVfalse-positivitywasidentifiedinthiscohort,thepower todetectsuchadifferencewasverylimited.Govindarajanetal. suggestedthatlipemicserumorthepresenceofrheumatoidfactor were associated with false-positive anti-HDV immunoassay results.7_{Unfortunatelyitwasnotpossibletotestthesehypotheses}

asdataonlipidswereunavailable.

Insummary,HDVinfectionwasfoundtoberareinHIV/HBV co-infected individuals in rural Tanzania. To avoid the over-estimation of HDV prevalence, future studies should confirm serologicalresultswithasecondtest,especiallyinpatientswitha weaklypositiveETI-AB-DELTA-2testresult.

Acknowledgements

We thank all patients and staff for their participation. The Kilombero and Ulanga Antiretroviral Cohort and the Chronic Diseases Clinic of Ifakara receive financial support from the GovernmentoftheCantonofBasel,Switzerland,theSwissTropical and Public Health Institute, the Ifakara Health Institute, the GovernmentofTanzania,andUSAIDthroughTUNAJALI-Deloitte. GWwassupportedbyanAmbizione-PROSPERfellowshipfromthe SwissNationalScienceFoundation(PZ00P3_154730).

The members of the KIULARCO Study Group are: Aschola Asantiel,Manuel Battegay, AdolphinaChale, DianaFaini, Ingrid

Felger,GideonFrancis,HansjakobFurrer,AnnaGamell,TracyGlass, Christoph Hatz, Speciosa Hwaya, Aneth Vedastus Kalinjuma, BrysonKasuga,NamvuaKimera,YassinKisunga,ThomasKlimkait, Emilio Letang, Antonia Luhombero, Lameck B. Luwanda, Herry Mapesi,LeticiaMbwile,MengiMkulila,JuliusMkumbo,Margareth Mkusa,DorcusKMnzava,GermanaMossad,DoloresMpundunga, AthumaniMtandanguo,KimD.Mwamelo,SelerineMyeya,Sanula Nahota,ReginaNdaki,AgathaNgulukila,AlexJohnNtamatungiro, Leila Samson, George Sikalengo, Marcel Tanner, and Fiona Vanobberghen.

Conflictofinterest:Theauthorsdeclarenoconflictsofinterest.

References

1.RizzettoM.HepatitisD:thirtyyearsafter.JHepatol2009;50:1043–50.

2.CunhaL,PlouzeauC,IngrandP,GudoJP,IngrandI,MondlaneJ,etal.Useof replacementblooddonorstostudytheepidemiologyofmajorblood-borne viruses in the general population of Maputo, Mozambique. J Med Virol 2007;79:1832–40.

3.BekondiC,MobimaT,OuaveneJO,KoffiB,KonamnaX,BereA,etal. [Etiopatho-logicalfactorsofhepatocellularcarcinomainBangui,CentralAfricanRepublic: clinical,biologicalcharacteristicsandvirologicalaspectsofpatients].PatholBiol (Paris)2010;58:152–5.

4.HarakaF,GlassTR,SikalengoG,GamellA,NtamatungiroA,HatzC,etal.Abundle ofservicesincreasedascertainmentoftuberculosisamongHIV-infected indi-vidualsenrolledinaHIVcohortinruralSub-SaharanAfrica.PLoSOne2015;10: e0123275.

5.FernsRB,NastouliE,GarsonJA.Quantitationofhepatitisdeltavirususinga single-stepinternallycontrolledreal-timeRT-qPCRandafull-lengthgenomic RNAcalibrationstandard.JVirolMethods2012;179:189–94.

6.deLallaF,RizzardiniG,RinaldiE,SantoroD,ZeliPL,VergaG.HIV,HBV, delta-agentandTreponemapalliduminfectionsintworuralAfricanareas.TransRSoc TropMedHyg1990;84:144–7.

7.GovindarajanS,ValinluckB,Lake-BakkarG.Evaluationofacommercial anti-deltaEIAkitfordetectionofantibodiestohepatitisdeltavirus.AmJClinPathol 1991;95:240–1.

A.Winteretal./InternationalJournalofInfectiousDiseases46(2016)8–10 10