Rev. Bras. Hematol. Hemoter. vol.37 número5

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w w w . r b h h . o r g

Revista

Brasileira

de

Hematologia

e

Hemoterapia

Brazilian

Journal

of

Hematology

and

Hemotherapy

Original

article

Prevalence

of

Torque

teno

virus

in

healthy

donors

of

Paraná

State,

southern

Brazil

Jocimara

Costa

Mazzola

a

_,

_Patrícia

_Keiko

_Saito

a

_,

_Roger

_Haruki

_Yamakawa

a

_,

Maria

Angélica

Ehara

Watanabe

b

_,

_Waldir

_Veríssimo

_da

_Silva

_Junior

a

_,

Alessandra

Cristina

Gobbi

Matta

a

_,

_Sueli

_Donizete

_Borelli

a,∗

a_Universidade_Estadual_de_Maringá_(UEM),_Maringá,_PR,_Brazil

b_Universidade_Estadual_de_Londrina_(UEL),_Londrina,_PR,_Brazil

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Articlehistory:

Received21October2013 Accepted2July2015 Availableonline29July2015

Keywords:

Torquetenovirus Blooddonorspolymerase Chainreaction

Anellovirus Virology

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Objective:TodeterminetheprevalenceoftheTorquetenovirusinhealthydonorsinthe

northernandnorthwesternregionsofthestateofParaná,southernBrazil.

Methods:TheTorquetenoviruswasdetectedbyanestedpolymerasechainreactionusing

asetofoligoprimersfortheN22region.

Results:Theprevalenceoftheviruswas69%in551healthyblooddonorsinsouthernBrazil.

Therewasnostatisticallysignificantdifferencebetweenthepresenceofthevirusandthe variablesgender,ethnicityandmaritalstatus.Therewassignificantdifferenceinthe preva-lenceofthevirusregardingtheageofthedonors(p-value=0.024)withahigherincidence (74.7%)in18-to24-year-olddonors.

Conclusion:AhighprevalenceofTorquetenoviruswasobservedinthepopulation

stud-ied.Furtherstudiesareneededtoelucidatetheroutesofcontaminationandtheclinical implicationsofthevirusinthehealthypopulation.

Introduction

TheTorquetenovirus(TTV)wasfirstdetectedin1997inthe bloodofJapanesepatientswithpost-transfusionhepatitis.1,2

Theviruswasalsodetectedintheliverandbloodofpeople withhepaticpathologiesofunknownetiology.2 _The

associ-ation betweenTTVand liver diseases isstill controversial,

∗ _{Corresponding}_author_at_:_Laboratório_de_{Imunogenética,}_Universidade_Estadual_de_Maringá_(UEM),_Av._Colombo,_5790,_Zona_07,_87020-900

Maringá,PR,Brazil.

E-mailaddresses:sueliborelli@gmail.com,sdborelli@uem.br(S.D.Borelli).

andseveralstudieshavebeenundertakentoidentifyinfection sources.3–5

Epidemiologicalstudieshaveevidencedtheprevalenceof TTVinotherpathologicalconditions,suchasinautoimmune diseases,4_respiratory_conditions6_and_cancer.7_However,

infor-mationisstilllackingonTTVinfectionandthedevelopmentof pathologies,aswellasthechangeinthecourseofaparticular disease.3,5

http://dx.doi.org/10.1016/j.bjhh.2015.07.005

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Table1–PrevalenceofTorquetenovirusinhealthypopulationsofdifferentBrazilianstates.

Reference Yearofpublication State Samplestested TTVDNA

n (%)

Nieletal.17 ₁₉₉₉ _Rio_de_Janeiro ₇₂ _62.0

Bassitetal.18 ₂₀₀₂ _São_Paulo ₁₉₇ _85.3

deCastroAmaranteetal.19 ₂₀₀₇ _São_Paulo ₂₇₀ _50.5

Pintoetal.20 ₂₀₀₇ _Pará ₁₈₆ _60.0

Nasseretal.4 ₂₀₀₉ _Paraná ₁₀₀ _6.0

Costaetal.5 ₂₀₁₂ _Mato_Grosso_do_Sul ₄₆ _15.2

Massaúetal.21 ₂₀₁₂ _Rio_Grande_do_Sul ₁₅₀ _73.3

TTV:Torquetenovirus.

Bloodtransfusionwasinitiallyindicatedastheprincipal viaofviraltransmissionduetodirectcontactwith contam-inatedblood.Despite theprogressinpretransfusion safety, bloodrecipientsarenotfreefromriskofcontamination.8

TheserologicaltestsperformedonblooddonorsinBrazil are established bythe nationalhealth surveillance agency (ANVISA),andincludeserologyforHIV1andHIV2,HTLVIand HTLVII,hepatitisB(HBV),hepatitisC(HCV),Trypanosomacruzi

(Chagasdisease),Treponemapallidum(syphilis)andPlasmodium

inendemicareasofmalaria.9

Besidesthe serologicaltestsconductedaccordingtothe MinistryofHealthprotocol,thereisconcernaboutthe emerg-ing and re-emerging diseases that can affect transfusion safety.10

However,new routes oftransmission have been identi-fied,duetothepresenceofthevirus indifferentbiological excretionssuchasinfeces,11_saliva12_and_also_in_river_water

contaminatedbysewage.13

Currently,wide variabilityin theprevalenceofTTV has beenobservedinhealthypopulationsindifferentcountries, suchasinAlexandriainEgypt(48.4%),14_United_Arab_Emirates

(75.0%)15 _and_Iran_(13.4%).16 _In_Brazil_the_prevalence_of_TTV

variesfrom6to85%indifferentstates(Table1).

Several factors may contribute to the variability of the resultsofTTVprevalencestudies,suchasthegeographical distributionofthepopulationunderanalysis,thediagnostic methodused,thesizeofthestudygroupandthedifficultyof makingasinglesetofprimersabletoidentifythemajorityof viralgenotypes.10,15

TTVinfectioniscommoninhealthydonorsworldwide.15,19

KnowledgeoftheprevalenceoftheTTvirusinspecificregions, servesasaresourcetoelucidatethetransmissionroutesand thepossiblecause ofdisease,and mayassistindeveloping guidelinesforactionstocontrolvirustransmissionin popula-tions.Theaimofthisstudywastodeterminetheprevalence ofTTVinhealthydonorsinthenorthernandnorthwestern regionsofParanástate,asthereisalackofstudiesshowing theprevalenceoftheTTVvirusinhealthydonorsinsouthern Brazil.

Methods

Thistransverse quantitativeanalysis involves humanDNA samplesobtainedfromhealthydonorsin2010.Thepopulation comprised551volunteers,agedbetween18and55years.The samplesweregroupedaccordingtothegeographiclocationof

thesource,inoneofthesevenmesoregionsofParanáState, accordingtotheBrazilianInstituteofGeographyandStatistics (IBGE)(Northwest,Central-West,North-Central,theregionof NortePioneiro,Central-Eastern,Mid-SouthandMetropolitan regionofCuritiba)andotherstates.

Anestedpolymerasechainreaction(nestedPCR)with spe-cificprimersfortheN22codifyingregion(ORF1OpenReading Frame1)wasemployedtodetectTTVDNA.Asenseprimer (RD037)followedbyoligonucleotideprimers5′_GCA_GCAGCA

TATGGATATGT3′_and_RD038₍₅′_TGA_CTG_TGC_TAA_GGC_CTC

TA3′₎_were_employed_in_the_first_{amplification.}_The_product

ofthefirstamplificationandtheantisenseprimersRD0515′

CATACACATGAATGCCAGGC3′_and_RD052₅′_GTA_CTT_CTT

GCTGGTGAAAT3′_were_used_in_the_second_{amplification.}_All

reagentswereidenticalinbothreactionswithafinalvolume of25.0␮Lcomprisedof2.5␮LPCRbuffer,0.75␮L50mM

mag-nesiumchloride(MgCl2),2.0␮L1.25mMdeoxyribonucleotide

phosphate(dNTP),1.0␮Lofeachsense(2.5␮M)andantisense

(2.5␮M)primer,2.5␮LTaqDNApolymerase(InvitrogenLife

TechnologiesBrazil),and12.75␮LsterileMilliQwater.A

fur-ther2.5␮LgenomicDNAwasusedforthefirstamplification,

and2.5␮Loftheproductfromthefirstamplificationwasused

forthesecondamplification.Bothreactionsoccurredina ther-mocycler(AppliedBiosystems)devicewithdenaturingat94◦_C

for30s,followedby35cyclesat53◦_C_for₃₀_s_for_primer

anneal-ing,72◦_C_for₄₅_s_for_primer_extension,_with_a_final_extension

at72◦_C_for₁₀_min._The_amplified_DNA _products_were

ana-lyzedby2%agarosegelelectrophoresis,stainedwithSYBR®

Safe(1␮L/10mLgel),andphotographedunderUVlight.The

DNALadder(Invitrogen)consistedof50basepairs(bp)andthe amplifiedDNAproductsof197bp.Alltestsincluded a pos-itive control(TTV genomic DNA).Data were analyzedwith theStatistic7.0computerprogramusingthechi-squaredtest, Yates’scontinuitycorrectionandFisher’sexacttestwith sig-nificance set atalevel of5%.The assaycomplied with all ethicalguidelinesandwasapprovedbytheResearchEthics CommitteeoftheUniversidade EstadualdeMaringá(UEM), Paraná,Brazil(Process271/2011).

Results

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Table2–DemographiccharacteristicsandprevalenceofTorquetenovirusamonghealthyblooddonors.

TTV TTV Total p-Value

Positive n(%)

Negative n(%)

Gender 0.785

Male 144(70.0) 62(30.0) 206

Female 236(68.4) 109(31.6) 315

Age(years) 0.024

18–24 124(74.7) 42(25.3) 166

25–38 154(70.3) 65(29.7) 219

39–53 97(63.0) 57(37.0) 154

54–55 5(41.7) 7(58.3) 12

Ethnicbackground 0.999

Caucasian 316(69.0) 142(31.0) 458

Africandescent 64(68.8) 29(31.2) 93

Maritalstatus 0.916

Married 183(68.0) 86(32.0) 269

Single 170(69.7) 74(30.3) 244

Othersa ₂₇_(71.1) ₁₁_(28.9) ₃₈

a _Widowed,_separated,_divorced_or_cohabiting.

sociodemographic variables,the proportionofTTV-positive individualsdifferedinrespecttoage(Fisher’sexact test:p -value=0.024),withtheratebeinghigherinthe18–24yearolds. Therewasnostatisticallysignificantdifferenceinthe preva-lence of the virus between gender and ethnic background (p-value>0.05).

The prevalence of TTV in healthy blood donors was assessedbymesoregionofthestateofParaná,southernBrazil (Table3).Thestatisticstest(ChisquaredwithYatescorrection) indicatednosignificantdifferencesinthepresenceofthevirus andthedifferentmesoregions(p-value=0.576).

Discussion

TheprevalenceofTTVinblooddonorsinthemesoregionsof thestateofParanáinsouthernBrazilwas69%.Other stud-iesinBrazilianpopulationsshowed60%prevalenceinBelém, Pará20_and_50.5%_in_the_southeastern_region_of_the_state_of_São

Paulo.18_In_southern_Brazil,_studies_showed_a_high_prevalence

ofthevirusinhealthydonors(73.3%)inthemunicipalityof Pelotas,RS21_and_also_the_presence_of_the_virus_in_samples_of

drinkingwaterandsewagewater.13,22

TTVtransmissionbybloodtransfusionhasbeena recur-ringconcernsincethe1990s.1_In_Brazil,_a_study_conducted_at

auniversityhospitalshowedconcernabouttheriskofviral transmissionbybloodtransfusion,23_the_serological_screening

ofdonorscannotprovidecompleteprotectionfromthe trans-missionofinfectiousagents.

SimilartoTTV, othervirusesrelatedtoliverdamageare overlooked inblood donors,including thehepatitisGvirus (HGV).Somestudies haveshownthe prevalenceofHGVin healthypopulationsofJapan(0.9%)andSouthAfrica(18.9%).24

InBrazilaprevalenceof7.1%wasalsoshowninthestateof Goias25_and_9.7%_in_São_Paulo.26

AlthoughTTVcontamination can occur from both con-taminated blood and blood products,2 _there _is _no _specific

legislationthatrequirestestingofblooddonorsforthevirus. Therefore, little isknownabout the routesoftransmission anddiseasesoriginatingfromthepresenceofthevirusinthe humanpopulation.

Based on the results presented, the association of the virus withthestudy variables canbedetermined(Table2). The prevalence of TTVinfection was (380/69%) in healthy donorsfromthenorthernandnorthwesternregionsofParaná, slightlybelowthatfoundinRioGrandedoSul(73.3%)21_and

the regionofSãoPaulo(85.3%).18_However,_one_should_also

notethattheprevalenceofthevirusinothercountriesranged from2.7to79.5%.10_The_variables_of_gender,_race_(Caucasian

orAfricandescent),andmaritalstatusshowednostatistical associationwiththepresenceofthevirus.Theresultsofthis studyareinagreementwithotherstudiesthathavesuggested thatTTVinfectionisrelativelycommonindifferent popula-tionsandindifferentregionsoftheworld.1,2

Withrespecttoagegroups,thestudyincludedindividuals between18and55 yearsandrevealedahighprevalenceof infectedyoungpeoplebetween18and24(74.7%).However, it was found thatthe prevalencedeclined inover 24-year-old individuals, especiallythose of 54 and 55 (41.7%).This contrasts with previous studies that showed a cumulative prevalencewithincreasingage,orthepresenceofthevirus independentlyofage.27

Several studies conducted in different countries and individuals in different age groups demonstrated varying prevalencesforTTV.18,28_The_discussion_of_other_age_groups

islimitedbytheparticularpopulationselectedforthisstudy. The results of this study indicated that the presence of TTVwassignificantlyassociatedwithage(p-value=0.024),in agreementwithastudyinPelotas,southernBrazil.21

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Table3–DistributionoftheprevalenceofTorquetenovirusinhealthyblooddonors.

TTV TTV Total p-Value

Positive n(%)

Negative n(%)

n(%)

Mesoregions 0.576

North-West 169(68.7) 77(31.3) 246(45.0)

Central-West 4(80.0) 1(20.0) 5(0.9)

North-Central 200(68.5) 92(31.5) 292(53.0)

NortePioneiro 1(100.0) 0 1(0.2)

Central-East 0 1(100.0) 1(0.2)

Central-South 2(100.0) 0 2(0.4)

MetropolitanCuritiba 1(100.0) 0 1(0.2)

OtherStates 3(100.0) 0 3(0.5)

Total 380(69.0) 171(31.0) 551(100.0)

Mostofthedonorswere fromthenorth-centraland

north-westParanámesoregions(98%)withprevalencesof68.5%and 68.7%,respectively.Theseregionsbelongtothe15thRegional HealthDistrictofParanáandreferthecityofMaringa,Parana, Brazil.Abeetal.demonstratedthattheTTVvirusiswidely distributedindifferentregionsoftheplanet,andwithhigh prevalencerates.29

Asisapparentfromtheliterature,severalfactorsmay influ-encethevariabilityofresultsforTTVprevalence,amongthem thegeographicaldistributionofthepopulationsstudied,the diagnosticmethodsofdetection,thesizeofstudygroupsand thesetofprimersusedinthestudy.3,15,28

Thehighratesofviralprevalencemaybedirectlyrelated totheformsofcontamination.Astudyofsamplesofblood andsalivafrom thesame individualsshowedthe presence ofthevirusinthesameproportionsregardlessofthe biolog-icalsampleused.30 _The_presence_of_the_virus _in_water_has

beeninvestigatedovertime,andalthoughthepurposeofthe presentstudywasnottodemonstratethepresenceofvirus inenvironmentalsamples,theimportanceofthisanalysisfor studyingtheviralprevalenceinagivenregionisimportant.

StudieshavedetectedTTVin97%ofwatersamples col-lectedinJapan31_and _in_Brazil,_in_92%_of_samples_collected

fromriversandstreamsinManaus.32_The_viral_genome_was

alsoreportedinsamplesofdrinkingwaterinRioGrandedo Sul.13_The_presence_of_TTV_in_the_water_of_rivers,_lakes,_and

treatmentplantsandespeciallyindrinkingwaterhashada majorimpactinspreadingthevirus.Thismayberelatedto thehighprevalenceofthevirusinhealthyindividuals.

Conclusion

ThisstudyfoundahighprevalenceofTTVinhealthyblood donors,inagreementwithotherstudiesintheBrazilian pop-ulation.Theclinicalsignificanceofthepresenceofthevirus inthesedonorscannotbeevaluatedbasedonthisstudy,but canserveasabasisforfuturestudies.Inviewofthe differ-enttransmissionroutesandthelackofcompleteinformation about the pathogenesis ofTTV, it is important to develop measurestominimizethe riskoftransmissionofthis and othervirusesamonghealthcareproviders.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

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