A high prevalence of human papillomavirus 16 and 18 co-infections in cervical biopsies from southern Brazil

brazilian journal of microbiology49S(2018)220–223

h ttp : / / w w w . b j m i c r o b i o l . c o m . b r /

Short

communication

A

high

prevalence

of

human

papillomavirus

16

and

18

co-infections

in

cervical

biopsies

from

southern

Brazil

Sheile

Pinheiro

de

Jesus

_,

_Ana

_Carla

_Marques

_da

_Costa

_,

_Regina

_Bones

_Barcellos

_,

Rubia

Marília

de

Medeiros

_,

_Cláudia

_Maria

_Dornelles

_da

_Silva

_,

_Maria

_Lucia

_Rossetti

a_{UniversidadeLuteranadoBrasil,CursodeBiomedicina,Canoas,RS,Brazil}

b_{UniversidadeLuteranadoBrasil,ProgramadePós-Graduac¸ãoemBiologiaCelulareMolecularAplicadaàSaúde,Canoas,RS,Brazil} c_{SecretariaEstadualdeSaúde,CentrodeDesenvolvimentoCientíficoeTecnológico,PortoAlegre,RS,Brazil}

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Articlehistory:

Received29August2017 Accepted6April2018 Availableonline24April2018 AssociateEditor:GilianeTrindade

Keywords: HPV16 HPV18 Cervicalbiopsy SouthernBrazil

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HPVtypes16and18werestudiedinparaffin-fixedcervicalbiopsycollectedinsouthern Brazil.HPV16,HPV18andco-infectionHPV16/18wereidentifiedin10/57(17.5%),4/57(7%) andin43/57(75.4%)samples,respectively.SouthernBrazilhasoneofthehighestprevalence ratesofHPV16/18reported.

©2018SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

licenses/by-nc-nd/4.0/).

Introduction

Human papillomavirus (HPV) is a cause of cervical cancer whichisthefourth-mostcommoncanceramongwomenin theworldwide.AccordingtotheWorldHealthOrganization, thereweremorethan528,000newcasesand266,000deaths from cervical cancer in 2012. Furthermore, approximately

∗ _{Correspondingauthorat:CentrodeDesenvolvimentoCientíficoeTecnologico(CDCT),Av.Ipiranga,5400,PortoAlegre,RioGrandedoSul}

90610-000,Brazil.

E-mail:cmdornelles@gmail.com(C.M.Silva).

85% of cervical cancer cases are diagnosed in less devel-opedregions.1 _{InBrazil,cervicalcanceristhesecond-most}

commoncancerinwomenandaparticularlyhighincidence (16.3/100,000) for this disease has been reported in South regionofBrazil.2,3

WhilemostHPVinfections are characterizedby sponta-neous viral clearance, someinfections are persistent. This condition involves oncogenic or high-risk HPV types (e.g.,

https://doi.org/10.1016/j.bjm.2018.04.003

1517-8382/©2018SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

brazilian journal of microbiology49S(2018)220–223

221

16,18, 31,33, 35,39, 45,51, 52,56, 58,and 59) and these havebeenconsistently associatedwithgrades2and3 cer-vical intraepithelial neoplasia (CIN), as well as cases of cervical cancer.4 _{Among these types, HPV 16 and HPV 18}

infectionshavebeenpresentinmorethan70%ofall exam-ined cervical cancer specimens.5 _{In Brazil, approximately}

5.4%ofwomeninthe generalpopulation areestimated to harbor a cervical HPV-16/18 infection at any given time, and 68.2%ofcervical cancers havebeen attributedto HPV types16or18.6_{InBrazil,threeHPVvaccineswerelicensed}

(bivalent/quadrivalent/nonavalent),butonlythequadrivalent (Gardasil®,MerckSharp&Dohme,USA)thatprotectsagainst HPVtypes6,11,16,and18wasincorporatedintothepublic healthsystemanditiscurrentlyofferedtogirls11–13yearsof age.7

DeterminingHPVprevalenceandexaminingthegenotype distributionofHPVinpremalignantandmalignantlesionsare importantparametersforestimatingthe impactof screen-ingprogramsandthe efficacyofHPVvaccines,particularly inrelationtovariationsintheprevalentHPVtypes accord-ingtoregion.Theaimofthepresentstudywastoevaluate theprevalenceofhigh-riskHPVtypes,16and18,inarchival cervicalbiopsysampleswithhigh-gradelesionscollectedin southernBrazil.

In a retrospective cross-sectional study, conducted between 2008 and 2009, a total of 60 paraffin-embedded samplesgradedascervicalintraepithelialneoplasiaI–III(CIN I, CIN II, CIN III)and cervical cancer (stained with hema-toxylinandeosin)werecollectedattheHospitalofLutheran UniversityofBrazil(ULBRA),Canoas,RioGrandedoSul(RS) (thesouthernmost statein Brazil). Thecity ofCanoas is a medium-sizedurbancenterlocatedinthemetropolitanarea ofthecapital(PortoAlegre,RS).Allsampleswereclassifiedby acertifiedpathologist.Thestudyobtainedapprovalfromthe EthicsCommitteeoftheLutheranUniversityofBrazil (proto-colnumber 2008-601H)beforereleasing archivalspecimens thatwerekeptcodedandconfidential.Informedconsentwas notneededbecauseoftheretrospectivenatureofthestudy.

Ten microtome serial sections of 10␮m (approximately 25mgpersample)weresenttotheCentrodeDesenvolvimento CientíficoeTecnológico(CDCT),PortoAlegre,RioGrandedo Sul,Brazil.Excessparaffinwasremovedwithsterileblade,and theremainingmaterialplacedinthemicrotube.

DNAextractionwasperformedinbiopsyspecimensusing theDNAkitQIAmp® FFPETissue(QIAampDNAFFPETissue Handbook,QIAGEN-Hilden,Germany) withminor modifica-tionsaccordingtotheprotocolsuggestedbySimonatoetal.8

andVilanova-Costa9_{.Inbrief,1000␮Lofxylenewasaddedto}

microtubescontainingthesamples.Thetubeswerevortexed vigorouslyfor30s,andcentrifugedfor5minat10,000rpm.To thepelletwasaddedagain1000␮Lofxylene,vortexed vig-orously and incubated at 65◦C for 30min. Thetubes were centrifugedat10,000rpmfor5min,andthenthepelletwas washedtwicewith1000␮Lof96%ethanol,vortexedfor30s andcentrifugedat10,000rpmfor5min.Thetubeswereopen andincubated at42◦Cfor20minforcomplete evaporation ofresidualethanol.Afterthesestepsofdeparaffinization,the kitprotocolwasfollowedasindicatedinthemanufacturer’s manual.

ThegeneralprimersGP5+andGP6+,whichspanaregion of140–150-bpfromtheL1openreadingframeofabroad spec-trumofHPVgenotypes,wereusedinthePCR,asdescribedby DeRodaHusmanetal.,10 _{withtheexception thatthe GP6+}

primer was biotinylated.A negative control (no DNA) was includedineachPCRruntoensurethatnocross contamina-tionhadoccurred.Theamplificationreactionwasperformed under the following conditions: 95◦C for 5min for initial denaturation,followedby40cyclesof95◦Cfor1min,52◦Cfor 1min,72◦Cfor1min,and72◦Cfor10minforfinalextension. Tubes were keptat4◦C forstorage ofthe amplified mate-rial.PCRproductswereelectrophoresedon1.5%agarosegel containing0.5mg/mLethidiumbromideandvisualizedunder ultravioletlight.AsacontrolofDNAquality,allsampleswere pre-screenedwith␤-globin primersPCO3 (5

ACACAACTGT-GTTCACTAGC3)andPCO4(5CAACTTCATCCACGTTCACC3) (110bpfragment).11

BiotinylatedPCRproductsgeneratedbytheGP5+/GP6+PCR were identified using microplate colorimetric hybridization assay(ImmobilizerAminoSurface,Nunc,Roskilde,Denmark) developedinourlab.12_{Eachampliconwashybridized}

sepa-ratelyusingprobesforthetwomostprevalentHPVtypesin cervicalcancer(HPV16and18).

Data wereevaluated usingthesoftwareStatistical Pack-ageforSocialSciences(SPSS)v.16.0.Fisher’sexacttestswere usedtocomparetheageandthedifferentdegreesof cervi-callesions.Fisher’sexacttestswerealsousedtocomparethe prevalenceofsingleandcombinedHPVinfectionacross histo-logical(CINIvsCIN2orworse)strata.p<0.05wasconsidered significant.

Ofthe60paraffin-embeddedcervicalbiopsieswithlesions thatwereexamined,39wereconfirmedtobeCINI(65.0%),7 wereCINII(11.7%),11wereCINIII(18.3%),and3cervical can-cer(5.0%).Thedistributionoftheavailablebiopsyspecimens accordingtopatientagewere:15/60samples(25.0%)werefrom women16–25yearsofage,38/60(63.3%)fromwomen26–35 yearsofage,4/60(6.7%)fromwomen36–45yearsofage,and 3/60(5.0%)fromwomen46–55yearsofage.Themeanageof thepatientswas29.6years.Therewasnosignificant differ-encebetweenmeanpatientageanddegreesofcervicallesions (p>0.05).

DNAwasextractedfromeachbiopsysamplesandwas sub-jectedtoPCRtoamplifya␤-globinfragmenttoassesssample integrity.Positiveamplificationwasdetectedfor57/60ofthe cervical biopsysamples.Theremainingthreesamples,two CINI samplesand oneCINII sample,were excludedfrom furtheranalysis.These57samplesweresubjectedto ampli-ficationoftheHPVL1regionandtheampliconsanalyzedin agarosegelandusingamicroplatecolorimetrichybridization assay.Inthefirstcase,amplificationproductswereobservedin 33/57(57.9%)samples.However,usingamicroplate colorimet-richybridizationassaythatemployedspecificprobestodetect HPV16 and HPV18,theampliconswere detectedinall 57 (100%)samples.Amongthesesamples,HPV16wasdetected in53/57(93%)ofthesamplesandHPV18wasdetectedin47/57 (82.5%)ofthesamples.Regardingsingleinfectionsversus co-infections,HPV16waspresentin10/57samples(17.5%),HPV 18waspresentin4/57samples(7%),andco-infectionofHPV 16/18wasdetectedin43/57samples(75.4%).

222

CIN I CIN II CIN III CC

F req u en cy (% ) HPV types HPV 16 HPV 18 HPV 16/18

Fig.1–HPVtypesdistributionaccordingto

histopathologicaldiagnosis.CIN,cervicalintraepithelial neoplasiaI–III;CC,cervicalcancer.

Correlations between the type of HPV present and the histopathologicaltypeofthelesionswerealsoexamined.Of thesamplesdiagnosedasCINI,HPV16wasdetectedin5/37 (18.5%)ofthesamples,HPV18wasdetectedin3/37(8.1%),and HPV16/18wasdetectedin29/37(78.4%).Amongthesamples diagnosedasCINII,3/6(50.0%)presentedwithHPV16and 3/6(50.0%)wereco-infectedwithHPV16/18.AmongtheCIN IIIsamples,HPV16waspresentin1/11(9.1%)samples,HPV 18waspresentin1/11(9.1%)samples,whileco-infectionof HPV16/18wasdetectedin9/11(81.8%)samples.Amongthe cervicalcancersamples,HPV16wasdetectedin1/3(33.3%) samplesand2/3(66.7%)sampleswereco-infectedwithHPV 16/18(Fig.1).Therewerenostatisticallysignificantdifferences betweentheprevalenceofsingleversuscombinedHPV infec-tionsacrossthehistologicalstrata(p>0.05).

Inthepresent study,HPVDNAwasdetectedin100%of thepremalignantandmalignantlesionsthatwerepresentin thecervicalbiopsyspecimensanalyzed.Thislevelof preva-lenceissimilartothelevelsreportedbystudiesconductedin othercountriesthatalsoexaminedparaffin-embedded sam-ples graded asCIN I–III and cervical cancer: United States (95%),13_Zambia(94%),14_{Ethiopia(93%),}15_{andPakistan(88%).}16

These findings reinforce the causal role of HPV infections in developing cervical malignancy5 _{and the importance of}

archivalbiopsysamplesasasourceofmaterialforstudies relatedtotheidentificationofHPVtypes.

TheprevalenceratesofHPVtypes16and18inthepresent setofsampleswerehigh(93%and82.5%,respectively),with HPV16beingthemostfrequentgenotypeidentifiedinallof thestagesofdiseasethatwereanalyzed.Thispredominance ofHPV16 isinagreementwithpreviousstudies ofvarious geographicalregionsofBrazil17_{andpopulationsworldwide.}18

Concurrent infections with multiple HPV genotypes in high-grade lesions of the cervix are a common finding, and some studies have shown that women with multiple infections, particularly those with co-infections involving oncogenic HPV types, have a significantly higher risks of progressingto cervicalcancer comparedwithwomen with singleinfections.19_{Thefollowingprevalenceratesofmultiple}

HPVgenotypesinhigh-gradesquamousintraepitheliallesions (HSIL)havebeenreported:36.9%inPortugal,20_43.2%inCosta

Rica,21_52%inBrazil,22_{62.5%intheUnitedStates}23_and64.9%

inPakistan.24 _{Toourknowledge,thepresent studyshowed}

oneofthehighestprevalenceratesofHPVco-infection(75.4%) reported,andcorroboratewithstudiesthatfoundHPV16/18as afrequentcombinationofHPVinfectionfoundinhigh-grade lesions worldwide.18 _{Incontrast,somestudies haveshown}

thatotherHPVtypescombinationscanbemorefrequentthan HPV16/18.InPortugal,therateofco-infectionsofHPV16/51 (12.3%)washigherthanHPV16/18(4.6%).InCampinas(Brazil), co-infectionswithHPV16/58(12.7%)andHPV16/52(8%)were morefrequentthanco-infectionwithHPV16/18(6.7%)in high-gradelesions.22

It is noteworthyto mention that the samplesanalyzed in the present study isfrom a region (Canoas city) which the mortality rate(age-standardized) for cervical cancerin 2008 was higher (8.72/100,000) than the Brazilian popula-tion(5.12/100,000)inthesameyear,25_{andtheglobalaverage}

(6.8/100,000)in2012.26 _{Furthermore,from1979to1998,the}

metropolitanareaofPortoAlegre,whichincludesCanoas,had the highestmean annualmortalityrate(9.72/100,000) com-paredtoothermeso-regionsoftheStateofRS.27_{So,theserates}

observedinthetargetpopulationsuggestacontinuumfailure ofthecervicalcancerscreeningprograms.

Inthepopulationscreenedinthepresentstudy,themean agewas29.6years.Similarly,anotherstudyreportedthemean age ofwomen infectedwithtwo or moreHPVtypesas 31 years.19_{Youngerwomenofthispopulationcouldbebenefited}

withtheprophylacticHPVvaccines,thusreducingtheburden ofcervicalcancer,probablyassociatedtoHPV16and18.Future studieslikethisinCINI-III/cervicalcancerlesions,usinglarger sampleand,afterHPVvaccination,couldbehelpfulto char-acterizetheoncogenicHPVtypesandanalyzeifHPV16and 18arestillthemostprevalentinthisparticularregion.

Inconclusion,thepresentstudyshowedoneofthehighest prevalenceofHPV16and18co-infectioninCINI–III/cervical cancerlesionsreported.Inthissetting,measurestoensure betterhealthforthe populationareurgentlyneededwhich includetheadvocacyofadministrationofprophylactic vac-cines currently available by these two oncogenic types in Brazil.

Conflicts

of

interest

Theauthorshavenoconflictsofinterest.

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