High prevalence of hepatitis E virus antibodies in Sao Paulo, Southeastern Brazil: analysis of a group of blood donors representative of the general population

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

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

Original

article

High

prevalence

of

hepatitis

E

virus

antibodies

in

Sao

Paulo,

Southeastern

Brazil:

analysis

of

a

group

of

blood

donors

representative

of

the

general

population

Ana

Maria

Passos-Castilho

a,∗,♦

_,

_Mônica

_Renata

_Reinaldo

a,♦

_,

_Anne

_de

_Sena

a,b

_,

Celso

F.H.

Granato

a,b

a_Division_of_Infectious_Diseases,_Federal_University_of_Sao_Paulo,_Sao_Paulo,_SP,_Brazil b_Fleury_SA_Group,_Sao_Paulo,_SP,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received24February2017

Accepted11May2017

Availableonline9June2017

Keywords: HepatitisEvirus Blooddonors Seroprevalence Brazil

a

b

s

t

r

a

c

t

Brazilisanon-endemiccountryforhepatitisEvirus(HEV)infectionwithseroprevalence

from1%to4%inblooddonorsandthegeneralpopulation.However,dataonseroprevalence

ofHEVinthecountryarestilllimited.Thisstudyevaluatedtheprevalenceofpastorpresent

HEVinfectioninagroupofblooddonorsrepresentativeofthegeneralpopulationofthe

cityofSaoPaulo,SoutheasternBrazil.Serumsamplesfrom500blooddonorsweretested

fromJulytoSeptember2014byserologicalandmolecularmethods.Anti-HEVIgGantibodies

weredetectedin49(9.8%)subjectsandcategorizedagegroupsrevealedanage-dependent

increaseofHEVseroprevalence.Amongtheanti-HEVIgGpositivesubjects,only1had

anti-HEVIgMwhilenonetestedpositiveforHEV-RNA.Thepresentdatademonstrateahigher

seroprevalenceofanti-HEVIgGthanpreviouslyreportedintheregion.

openaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/

by-nc-nd/4.0/).

Introduction

HepatitisEvirus(HEV)infectionisamongthemostfrequent

causesofacutehepatitisworldwide.Itisestimatedthat2.3

billionpeoplehavealreadybeeninfectedwithHEVwith70,000

deathsattributedtothisviruseveryyear.1,2

∗ _{Corresponding}_author.

E-mailaddress:anampassos@gmail.com(A.M.Passos-Castilho).

♦ _These_authors_contributed_equally_to_this_work.

Hepatitis E presents as large epidemics and sporadic

cases in endemicareas, including genotype 1in Asia and

Africa,genotype2inMexicoandAfrica,andgenotype 4in

Asia. Sporadic cases of genotype 3 occur worldwide. HEV

detectedfromArgentinaandBrazilaremorerelatedtoviruses

from industrialized countries (North America and Europe),

whereastheHEVintheCaribbeanandMexicoincludeviral

http://dx.doi.org/10.1016/j.bjid.2017.05.004

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genotypes more closely related tooutbreaks in Africa and

Asia.3,4

ThetotalprevalenceofantibodiesagainstHEVinendemic

countriesisvariable (3%–27%).5 _In_non-endemic_areas _with

propersanitaryconditionsandawell-controlledwater

sup-ply,theprevalenceofantibodiesagainstHEVinthegeneral

populationisrelativelyhigh(upto7%–10%).5

DataonHEVseroprevalenceinBrazilarestillscarce.HEV

isnotroutinelyinvestigatedinthecountry,evenincasesof

unexplained liverenzymeelevationoracute hepatitis,and

onlya few laboratories perform anti-HEV tests. Brazil is a

non-endemiccountryforHEV,withrarecasesconﬁrmedby

molecularmethods.HEVseroprevalenceinthecountryranges

from1%to4%inblooddonorsorthegeneralpopulation,13%

inindividualsfromanagriculturalsettlementintheAmazon

Basinand15%inrenaltransplantrecipients.6–9_Nonetheless,

mostavailablestudiesare limited,outdatedand cannotbe

comparedproperlybecauseoftheirsmallsamplesizesand/or

diversemethodology.Therefore,theoccurrenceand

charac-teristicsofhepatitisEinBrazilarepoorlyunderstood.

Theaimofthisstudywastoassesstheprevalenceof

hep-atitisEinSaoPaulo,SoutheasternBrazil,throughtheanalysis

ofanti-HEVantibodiesinagroupofblooddonors

representa-tiveofthegeneralpopulation.

Patients

and

methods

Studyarea

SaoPaulo,themostpopulouscityinBrazil,isalsoconsidered

themostmulticulturalcityinthecountry,withapproximately

12millioninhabitantsandimmigrantsfromallpartsofthe

world.10_The_city_is_divided_in_eight_zones,_with_distinct

socio-economiccharacteristics.

Studydesign

Asamplingplanaccountingfordemographiczone,genderand

agegroupwascreatedtogatherasamplerepresentativeofthe

generaladultpopulationinthecity.

Aprospective,cross-sectionalstudywascarriedout

involv-ing 500 blood donors who consecutively underwent blood

donationfromJulytoSeptember2014intheBeneﬁcent

Associ-ationforBloodCollection(COLSAN)inSaoPaulo,Southeastern

Brazil,strictlyfollowingthesamplingplan.

Thissamplewascalculatedtobesufﬁcienttosuccessfully

estimateHEVseroprevalenceofapproximately10%inthecity

populationata95%conﬁdenceinterval(CI).

Sampleanddatacollection

Fivehundredmilliliteraliquotsoftheserumsamplesroutinely

collectedforserologicaltestingwereseparatedandstoredat

–80◦Cuntillaboratoryanalysis.Dataregardingdateofbirth

andgenderwerecollectedduringenrollment.

Anti-HEVIgGandIgMantibodiesdetection

The presence of anti-HEV IgG antibodies was investigated

through enzymeimmunoassayusing the WANTAIHEV-IgG

ELISA kit (Beijing Wantai Biological Pharmacy Enterprise,

Beijing,China),strictlyaccordingtothemanufacturer

recom-mendations.Specimenswithpositiveresultsweretestedfor

anti-HEVIgMantibodiesusing aspeciﬁckit fromthe same

manufacturer.

RNAextractionandquantitativeRT-PCR

HEV RNA was extracted from the serum samples using

QIAampviralRNAminikit(QIAGEN,Hilden,Germany),strictly

accordingtothemanufacturer’sinstructions.

QuantitativeRT-PCRwasperformedaccordingtoa

mod-iﬁed 1-steptriplex realtimeprotocolpreviouslydescribed11

with a set of primers and probe targeting a highly

con-served70ntlongsequencewithinoverlappingpartsofORF2

and ORF312 _and _a _set _speciﬁc _for _a ₁₁₃_nt _sequence _of

ORF2.13 _A _third _set _of _primers _and _probe _targeting _the

human RNAseP gene was used as endogenous internal

ampliﬁcation controlto certify specimen quality and RNA

extraction.14

AplasmidclonefromaBrazilianhumanHEVstrain

previ-ouslycharacterized(GenBankaccessionnumberKF152884)15

was constructed with TOPO® TA cloning® kit (Invitrogen,

Carlsbad, CA, USA) and the primers described. Plasmid

DNAwaspuriﬁedusingQIAprepspinminiprepkit (QIAgen,

Hilden, Germany), linearized and quantiﬁed withthe

Nan-odropND-1000instrument(Wilmington,DE,USA)following

transcriptiontoRNAwithT7RNApolymerase(Promega,

Madi-son,WI,USA).Standardcurvesweregeneratedusing100_to

1010_copies_of_plasmid_RNA._HEV_viral_loads_were_determined

basedonthestandardcurves.Thelimitofdetectionofthe

real-timeRT-PCRwas5copiesofRNAperreaction,whilethe

limitofquantiﬁcationwassetat50copiesofRNAper

reac-tion.Allscreeningreactionswereruninduplicatesusingwith

propercontrols.

Statisticalanalysis

AlldatawereenteredandanalyzedusingSPSSversion11.0

(SPSSInc.,Chicago,IL,USA).Descriptivestatisticsconsisted

ofthe characterizationofthe studiedpopulationand

anti-HEVIgGseroprevalence throughtherespectivepercentages

and95%conﬁdenceintervals(CI)ormean/medianvaluesand

standarddeviation(SD)forcontinuousvariables.Thebivariate

analysisconsistedofPearson’sChi-squaretesttocompare

cat-egoricalvalues.Non-conditionallogisticregressionwasused

toidentifyassociationsbetweendependentandindependent

variablereportedasoddsratio(OR).Mann–WhitneyUtestwas

usedtocomparemeansofnon-normallydistributedvariables.

Linearregressionanalysiswasusedtoevaluatethetrendsof

anti-HEVpositivitywithrespecttoagegroup.Statistical

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20 15 10 5 0 18–29 n = 138 n = 183 Age group

Anti-HEV IgG positiv

e (%)

n = 131 n = 48

30–44 45–59 60–69

Fig.1–Estimatedprevalenceofanti-HEVIgGbyagegroup inSaoPaulo,Brazil.

Ethicalaspects

ThestudyprotocolwasapprovedbytheInstitutionalEthics

Committee(162/09and86730/12).

Results

Atotalof49samplesof500subjectstestedpositivefor

anti-HEVIgGantibodies,whichrelatestoaseroprevalenceof9.8%

(95%CI7.5–12.7).Fromthese,only1presentedwithanti-HEV

IgM.NonetestedpositiveforHEV-RNA.

Themeanageoftheenrolledsubjectswas38.8yearswitha

SDof13,withinarangeof18–67andamedianageof36years

old.Meanageofsubjectstestedpositiveforanti-HEVIgGwas

43.7yearswithaSDof11.7,range24–67years,signiﬁcantly

higherthanthatofthosewhotestednegativeforanti-HEV

IgG(38.3±13.0;p=0.006).Categorizedagegroupsrevealedan

age-dependentincreaseofHEVseroprevalencewithR2_of_0.99

forupto59yearsandR2_of_0.70_for_up_to₆₉_years_(p₌_0.023)

asshowninFig.1.Only4.3%(95%CI2.0–9.2)ofthesubjects

showedpositivityforanti-HEVIgGintheagegroupof18–29,

whereas15.3%(95%CI10.1–22.4)werepositiveforanti-HEV

IgGintheagegroupof45–59years.Theseresultsshowa4-fold

increaseoftherisktoundergoaHEVinfectionorpresentwith

positiveanti-HEVIgGantibodiesatanadvancedage(45–59)

thanatanearlyage.

15.9% 3.7% 6.7% 21.1% 5.6% 19.4% 5.8% 12.5% Center West South center South Southeast East Northeast Northwest

Fig.2–CitymapofthecityofSaoPaulo,Southeastern Brazil,andanti-HEVIgGseroprevalenceaccordingtocity zone.

Inall,245(49%)patientswere male(asobservedforthe

general population inSao Paulo). There was no difference

indistributionofanti-HEVpositivecasesbetweenmaleand

female subjects. Table1presents the characteristics ofthe

studiedindividuals.

Anti-HEV IgG prevalencevaried signiﬁcantlyamong the

demographiczonesinthe cityofSaoPaulo(Fig.2).Highest

rateswereobservedintheCentral(21.1%)andSouth-Central

Table1–Characteristicsofthestudiedindividualsaccordingtoanti-HEVIgGstatus,SaoPaulo,SoutheasternBrazil.

Characteristic Anti-HEVIgGpositive p OR(95%CI) Total

Gender Male 24(9.8) 0.998 – 245(49.0) Female 25(9.8) – 255(51.0) Agegroup 18–29 6(4.3) 0.023a ₁_(Ref) ₁₃₈_(27.6) 30–44 17(9.3) 2.25(0.86–5.88) 183(36.6) 45–59 20(15.3) 3.96(1.54–10.22) 131(26.2) 60–69 6(12.5) 3.14(0.96–10.23) 48(9.6)

Resultsarepresentedasnumberandpercentage. –:notapplicable.

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Table2–Seroprevalenceofanti-HEVIgGaccordingtodemographiczone,SaoPaulo,SoutheasternBrazil.

Cityzone Anti-HEVIgGpositive p OR(95%CI) Total

Central 4(21.1) 0.031a _6.93_{(1.15–41.61)} _19(3.8) South-Central 7(19.4) 6.28(1.22–32.22) 36(7.2) Northwest 7(15.9) 4.92(0.97–25.03) 44(8.8) East 16(12.5) 3.71(0.82–16.75) 128(25.6) West 3(6.7) 1.86(0.30–11.63) 45(9.0) South 6(5.8) 1.61(0.31–8.25) 103(20.6) Southeast 4(5.6) 1.55(0.27–8.81) 71(14.2) Northeast 2(3.7) 1(Ref) 54(10.8)

Resultsarepresentedasnumberandpercentage.

a _Signiﬁcant_at_p_<_0.05.

(19.4%)zones,whiletheNortheast(3.7%),Southeast(5.6%)and

South(5.8%)presentedwiththelowestprevalences(p=0.031).

TheCentralandSouth-Centralzonespresentedwitha7-and

6-foldincreasedriskofHEVpastinfection,respectively,when

comparedtotheNortheast(Table2).

Discussion

Thepresentdatademonstrateahigherseroprevalenceof

anti-HEVIgGinSaoPaulo,SoutheasternBrazil,thaneverreported

forthe generalpopulation (or blood donors)inthe region,

witha cumulativeincrease withage. In studies from 1997

to2006,anti-HEVIgGprevalencevariedfrom2.0%to4.3%in

blooddonorsfromSoutheasternandNortheasternBraziland

from0%to4.3%inindividualsfromruralandurbanareasand

thoselivinginlowsocioeconomiccommunities.6_A_study_on

699patientsfromalowsocioeconomicgrouplivinginRiode

Janeiroin2002reporteda2.4%HEVseroprevalence.16

Thehigherprevalenceobservedinthepresentstudymay

beduetoanincreaseinHEVseroprevalenceinrecentyears.A

recentstudyfrom 15years oflaboratoryresultsofpatients

clinically suspected of HEV infection in the same region,

describedanoverallanti-HEVpositivityrateof2.1%(47/2271),

rangingfrom0%(0/183)to8.6%(12/139)withyear.Thehighest

frequencieswereobservedfrom2011to2013:5.9%(2/34),8.6%

(12/139),and6.1%(9/148),respectively.9

Theperformanceofanti-HEVIgG assays mayalso have

playedaroleinthehigherseroprevalenceofanti-HEV

anti-bodiesfoundinthepresentstudy.Manyoftheearlierstudies

inBrazilusedanti-HEVassaysofpoorsensitivity,whichcould

resultinsigniﬁcantunderestimatedratescomparedtothis

studythatistheﬁrsttouseavalidated,highlysensitiveassay

intheregion.ThesamewasobservedinEnglandwhereHEV

seroprevalencerosefrom3.6%to16%whenamoresensitive

IgGassay,i.e.,theWantaitest,wasused.17_A_recent_study,_also

usingtheWantaitest,demonstratedasimilarseroprevalence

ofanti-HEVIgG(10%)amongblooddonorsinthemetropolitan

areaofItajaiValley,SouthernBrazil.18

Theeatinghabitsofthepopulationcouldalsoinﬂuence

thehigherseroprevalenceofHEVantibodies,sincethe

inges-tionofraworundercookedporcinemeathasbeenassociated

withHEVinfection19,20 _and_probable_zoonotic _transmission

ofHEVhasbeendemonstratedinBrazil,asveryhigh

homol-ogywasfoundbetweenhumanandswineHEVstrains.15,21,22

The consumption of pork varies according to multiple

factors,includingculturalandsocioeconomicalaspects.23_In

thepresentstudy,theanti-HEVIgGprevalencevaried

signif-icantlyamongdemographiczones,withthehighestratesin

theCentral(21.1%)andSouth-Central(19.4%)zones.

Interest-ingly,thesezonesarebothclassiﬁedashavinghighguarantee

ofeconomicandsocialrightsofallinhabitantsintheregion,

whichaccountsforemployment,income,housing,healthand

education.Moreover,thesearetheregionswiththehighest

incomepercapitainthecity,withlessthanﬁvepercent

low-incomeinhabitants.24_Thus,_the_higher_prevalence_of_anti-HEV

IgGantibodiesintheseregionscouldbearesultofhigherpork

consumption,whichisknowntoberelatedtobettereconomic

conditions.25 _Accordingly,_the_South_zone,_which_presented

withoneofthelowestHEVseroprevalence(5.8%),is

classi-ﬁedashavingpoorguaranteeofeconomicandsocialrights

and isthezonewiththehighestpercentualoflow-income

inhabitants(20%ormoreearninglessthanhalftheminimum

wage).24

Only1ofthe49anti-HEVIgGpositivesubjectsstudied

pre-sentedwithanti-HEVIgM,amarkerofacuteinfection.Noneof

thesubjectspresentedwithHEV-RNA.Nonetheless,thisstudy

demonstratesahigherrateofpastHEVinfectionthanitwas

knowntooccurintheregion,showingthatthisinfectionis

probablyunderdiagnosedinthecountry.

Theauthorsacknowledgesomelimitationstothisstudy.

It wasnotpossible toenquiry the subjectson clinical

his-tory,possibleriskfactorsand eatinghabits thatcouldhelp

understandtheoccurrenceofhepatitisEinthispopulation.

Thus, furtherand largerepidemiological studies shouldbe

performed.

In conclusion, a high prevalence of anti-VHE IgG was

observedinSaoPaulo,whichisthehighestprevalenceever

reportedintheregionandthesecondhighestinthecountry.

Financial

support

FAPESP2012/22925-3,2013/03701-0.

Conﬂicts

of

interest

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Acknowledgments

TheauthorswithtothanktheCOLSANblooddonorcenterand

allemployeesfortheirsupport.Thisworkwassupportedby

theFundac¸ãodeAmparoàPesquisadoEstadodeSãoPaulo

(FAPESP).

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