Presence of highly oncogenic human papillomavirus in the oral mucosa of asymptomatic men

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

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

Original

article

Presence

of

highly

oncogenic

human

papillomavirus

in

the

oral

mucosa

of

asymptomatic

men

Ana

Paula

Machado

_,

_Flávia

_Gatto

_de

_Almeida

_,

_Camila

_Mareti

_Bonin

_,

Thiago

Theodoro

Martins

Prata

_,

_Leandro

_Sobrinho

_Ávilla

_,

Cacilda

Tezelli

Junqueira

Padovani

_,

_Alda

_Maria

_Teixeira

_Ferreira

_,

Carlos

Eurico

dos

Santos

Fernandes

_,

_Inês

_Aparecida

_Tozetti

a_{PostgraduateProgramofInfectiousandParasitaryDiseasesfromMedicineSchool,UniversidadeFederaldeMatoGrossodoSul/UFMS,}

CidadeUniversitária,CampoGrande,MatoGrossodoSul,Brazil

b_{BiologicalandHealthCenterfromUniversidadeFederaldeMatoGrossodoSulCidadeUniversitária,CampoGrande,}

MatoGrossodoSul,Brazil

c_{MedicineSchoolfromUniversidadeFederaldeMatoGrossodoSul/UFMS,CidadeUniversitária,CampoGrande,MatoGrossodoSul,}

Brazil

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o

Articlehistory:

Received29April2013

Accepted24July2013

Availableonline3January2014

Keywords: Humanpapilomavírus Men Viraltype Oralmucosal

a

b

s

t

r

a

c

t

Objectives:Theaimofthisstudywastoidentifyhighlyoncogenicformsofhuman

papillo-mavirusintheoralmucosaofasymptomaticmen.

Methods:Inthisstudy,weanalyzedsamplesofexfoliatedcellsfromtheoralcavityof559

asymptomaticmen.DNA-humanpapillomaviruswasdetectedusingtheconsensusprimers

PGMY09/11;viralgenotypingwasperformedusingtype-specificPCRandrestriction

frag-mentlengthpolymorphism.

Results:DNA-humanpapillomaviruswasdetectedin1.3%ofthestudyparticipantsandof

those42.8%wereinfectedbymorethanonetypeofvirus.ViraltypesincludedHPV6,11,89

(lowoncogenicrisk),andHPV52,53(highoncogenicrisk).Increasedvulnerabilitytohuman

papillomavirusinfectionwasobservedinindividualsagedover26years,amongthosewho

reportedoralsexpractices,andinthosewhohavehadmorethan16sexualpartnerssince

firstengaginginsexualintercourse.

Conclusions:Therewasalowprevalenceofhumanpapillomavirusdetectionintheoral

mucosaofasymptomaticmen.Highlyoncogenichumanpapillomavirustypesandinfection

bymorethanoneviraltypewasobserved.Oralsexpracticesandalargenumberofsexual

partnersmayincreasetheriskofacquiringhumanpapillomavirusinfection.

©2014PublishedbyElsevierEditoraLtda.

∗ _{Correspondingauthorat:FaculdadedeMedicina,UFMS,CampoGrande,Brazil.}

E-mailaddress:inestozetti1408@hotmail.com(I.A.Tozetti).

1413-8670/$–seefrontmatter©2014PublishedbyElsevierEditoraLtda.

Introduction

Thehumanpapillomavirus(HPV)isaprimaryriskfactorfor

cervicalneoplasiaandisassociatedwith25%ofcancers

affect-ingtheheadandneckregion,1_{andmayinducedevelopment}

oforopharynxcarcinoma.2

TherearetwoformsofHPV,includinghighoncogenicrisk

(HR)and low oncogenicrisk (LR)types. Low-risk typesare

associatedwithbenignlesionsinthehostcharacterizedas

ordinaryor condylomatouswartsandincludeHPV6,11,40,

42,43,44,54,61,70,72,and81.High-risktypeshave

carcino-genicpotentialandincludeHPV16,18,26,31,33,35,39,45,

51,52,53,56,59,66,68,72,and81.3

Themoreanindividualengagesinriskybehaviorssuchas

oralsexandhashighnumberofsexualpartners,thegreater

theriskofacquiringthevirus,withsubsequentdevelopment

of cellular differentiation and progression to neoplasia.4,5

However, some individuals develop oropharynx carcinoma

withoutpriorexposuretotheseriskfactors,suggestingthat

potentiallyoncogenicvirusesmayleadtochangesincontrol

andcellproliferationmechanisms.6

Previousstudies have examined the prevalence of HPV

in the oral mucosa of asymptomatic men and suggested

its association with neoplastic development; however, the

assessmentmethods have shown disparate frequencies of

infection,rangingfrom 0%to100%.7 _{Thus, theaimofthis}

studywastoassessthefrequencyofhighlyoncogenicforms

ofHPVintheoralmucosaofasymptomaticmen.

Methods

and

subjects

Exfoliated cells from the oral cavity of 559 asymptomatic

men were analyzed. Ethical approval was granted by the

Ethics Committee in Research of UFMS, protocol CAAE

0251.0.049.000-11.Allparticipantscompletedaquestionnaire

containinginformationregardingriskbehaviorsthatmay

pre-disposethemtoHPVinfection.

CollectionofspecimensanddetectionofHPV-DNA

Samples were collected through 5–10brushings inregions

of pre-establishedoral mucosa, including the right buccal

mucosa (positionfrom top to bottom); left buccal mucosa

(positionfromtop tobottom);right, leftand dorsal sideof

thetongue;andinnerregionsofupperandlowerlips.After

collection, DNA wasextracted from the samplesusing the

WizardGenomicDNAPurificationkit(Promega,Fitchburg,WI,

USA)and quantified using a NanoDrop (ThermoScientific,

Waltham,MA,USA)(180–260nm).

HPV-DNA detection was performed using PCR with a

pool ofconsensus primers that amplify PGMY09/11 450-bp

DNA sequences within the L1 region ofHPV, as described

previously.8_{AnendogenouscontrolwasusedtoverifyDNA}

integrityusingprimersfortheˇ-globingene,PC04andGH20,

whichamplifya286-bpregionofhumanDNA.Negative

con-trolsforbackgroundcontaminationwereaddedtotheDNA

template.PCRproductswereanalyzedusing1.5%agarosegel

electrophoresiswithethidiumbromidestainingtovisualize

theDNAunderultraviolet(UV)light.Molecularweightswere

determinedbycomparisonwitha100-bpDNAladder.

Genotypingusingtype-specificPCR(TS-PCR)and restrictionfragmentlengthpolymorphism(RFLP)

HPV-DNApositivesamplesweregenotypedbyPCRusing

TS-PCRfortheE6andE7geneDNAsequencesofHPV6,11,16,18,

31,33,and45.9_{PCRproductswereanalyzedona2.5%agarose}

gelwithethidiumbromidestainingtovisualizeDNAunderUV

light.Molecularweightsweredeterminedbycomparisonwith

100-bpand50-bpDNAladders.Thesamesampleswere

ana-lyzedusingRFLP.Next,thePGMY09/11PCRproductofthese

sampleswaspurifiedfromtheagarosegelusingtheQIAEX

IIGelPurificationKitQiagen(Hilden,Germany)accordingto

themanufacturer’sprotocol.Theconcentrationofextracted

materialswas determined using a NanoDrop(180–260nm);

samplescontainingthePCRproductweresubjectedto

enzy-maticdigestionforonehourat37◦C.Theenzymesusedfor

reaction included BamHI, DdeI, HaeIII, HinfI, RsaI, PstI, and

Sau3A.Thedigestionpatternwasanalyzedona3%agarosegel

withethidiumbromideunderUVlightandinterpretedusing

analgorithmdescribedpreviously.10

Statisticalanalysis

ThedistributionofpositivityforHPV-DNAwasinvestigated

according to age range (≤25 years and ≥26 years), marital

status, report oforal sexpractices, and estimated number

ofsexualpartners.Statisticalanalysiswasperformedusing

SPSS,version10.011_ofPearson2_{testforcontingencytables,}

adjustedforPhiCramer’sV.Tocomparefrequenciesbetween

agesubgroups,whensignificant,theGtestcorrectedbyYates

indexwasused.Theproportionofpositivefindingswithinthe

groupwasanalyzedusingthebinomialtestfortwo

indepen-dentsamples.

Results

Samplesofasymptomaticoralmucosafrommenaged18–68

years(mean,23years)wasanalyzed.Ofthe559samples

col-lected, 514(91.9%)were positiveforthe ˇ-globin gene and

thusincludedinthestudy.HPV-DNAwasdetectedinseven

samples,whichaccountedfor1.3%ofthestudyparticipants.

DetectedviraltypesincludedHPV6,11,and89intheLR

groupandHPV52and53intheHRgroup(Figs.1and2).Highly

oncogenicinfectionwasdetectedintwooutoftheseven

sam-ples(28.5%),andinfectionbymorethanoneviraltypewas

foundinthreeofthesevensamples(42.8%).

Amongtheparticipants,71.7%reportedbeingsingleand

27.2%married,withahigherHPV-DNApositivityamong

mar-ried men.Thebinomial test showedthat individuals older

than 26 years of age were more vulnerable to infection

(p<0.01). Oral sexpractices were reported by 71.9% of the

respondents(Table1),particularlyamongthoseover26years

ofage(p<0.01),whereHPV-DNApositivitywashigher.

Amongindividualswhoreportedhavinghadmorethan16

sexualpartnerssincetheirfirstsexualintercourse,HPV-DNA

Table1–EpidemiologicalcharacteristicsofthepopulationassociatedwithpositivityforHPV-DNA.

Variable Total HPV(+) HPV(−) p-Valuea

N◦ _{% N}◦ _{% N}◦ _% Age 0.08 ≤25 397 77.2 3 0.75b _{394 99.25} ≥26 108 21.0 3 2.7b _{105 97.3} Missing 9 1.8 1 11.0 8 89.0 Maritalstatus 0.73 Nevermarried 367 71.4 4 1.0 362 99.9 Married 140 27.2 3 2.1 137 97.9 Missing 5 1.4 0 0.0 5 100.0 Oralsex 0.5 No 131 25.4 1 0.7 130 99.3 Yes 370 71.9 5 1.3 365 98.7 Missing 13 2.7 1 7.6 12 92.4

Sexualpartnerssincetheirfirstsexualintercourse 0.09

1–5 119 23.1 1 0.8 118 99.2 6–15 164 31.9 1 0.6* _{163 99.4} 16–25 56 10.9 1 1.8 55 98.2 >26 90 17.5 3 3.3* _{87 96.7} Missing 85 16.6 1 1.1 84 98.9 HPV,humanpapillomavirus.

a _Pearson2_{test,adjustedforPhiCramer’sV.} b _{Binomialtestfor2independentsamples(p<0.05).}

Discussion

InthisstudyHPVwasdetectedin1.3%oftheparticipants.A

cohortstudyconductedintheUSfrom2000to2006assessing

thepresenceofHPVinoralmucosafoundanHPVfrequency

of6.0%inoralbrushingandoropharyngealscrapings.12_Other

multicenterstudiesconductedtoexaminemalesubjectsin

Brazil,theUS,andMexicofoundapositivityof2.1%,3.6%,and

5.9%,respectively,inmaterialfromgargling.13

Previousstudies haveshownthattherateofHPV

infec-tionishighinmalesubjects,14_{rangingfrom3.5%to45%for}

*100pb Pst I Hae III Dde I Rsa I Controla *50pb

500p

250p

Fig.1–Electrophoresisinagarose3%showingpatternsof polymorphismoftherestrictionfragmentsobtainedwith theproductofPGMY09/11usedforgenotypingofthe HPV52(HR).*100pband50pb=ladderDNA;a_control: PGMY09/11PCRproductof450pbwithoutenzymatic digestion.Restrictionenzyme:PstI=notdigestion;Hae III ∼_{= 200}pb;DdeI ∼_{= 350}pb;RsaI=notdigestion.

alldetectabletypes.15_{However,otherstudiesshowdifferent}

values,withhigherandmoredivergentprevalence.16_These

results may reflect methodological discrepancies used for

HPV-DNAdetection,thereproducibilityofgenotypingassays,

andthesensitivityandspecificityofprimersusedfor

diagno-sis.

TheglobaldistributionofdifferentHPVtypesexhibitslarge

variation,evenfortheoralcavityofsubjectsindifferent

geo-graphicalregions.StudiesexaminingHPVprevalenceintheUS

conductedin2009–2010showedthatamongHRtypesinthe

oralmucosa,theHPVgenotypes16,66,and51wereprevalent,

*100pb Pst I Hae III Dde I Rsa I Controla _*50pb

250p 500p

Fig.2–Electrophoresisinagarose3%showingpatternsof polymorphismoftherestrictionfragmentsobtainedwith theproductofPGMY09/11usedforgenotypingofthe HPV89(LR).*100pband50pb=ladderDNA;acontrol: PGMY09/11PCRproductof450pbwithoutenzymatic digestion.Restrictionenzyme:PstI=notdigestion;Hae III ∼_{= 350pb;}DdeI ∼_{= 250}pb;RsaI ∼_{= 380}pb.

and among LR types, 62, 55, and 89 were predominant,5

in contrast to the findings of this study. Other authors

demonstrated thatHPV types6, 11,and 16 were the most

prevalent.16

Theoralmucosaofhealthyindividualscanhost

approx-imately 4.4% of carcinogenic types and 7.5% of

non-carcinogenic types of HPV.7 _{The difference between HPV}

typesdetectedintheoralcavityandcervical–vaginalregion

showsabroaderviewofdistributionfordifferenttissuetypes.

Thus,theseregionscanbecolonizedbytypesnotpreviously

detected,whichmayexplaineventssuchasthelackofcell

cyclecontrolinthesetissues.

Inmen,theprevalenceofinfectionisnotrelatedtoage,nor

istheprevalencehigherinyoungerindividuals,incontrastto

trendsobservedinwomen.Thesefindingssuggestthatmen

mayexperiencepersistentlong-terminfectionwithahighrate

ofre-infection.OtherstudieshaverevealedthatHPVpositivity

increaseswithage.5,13,17

Thepresentstudyobservedhigherpositivityamong

indi-viduals aged more than 26 years; however, this may be

associatedwiththeadoptionofriskysexualbehaviorbythis

agegroup.

Oralsexpracticeshavebeenwidelyassociatedwiththerisk

ofHPVinfection,althoughitisimpossibletoidentifyan

inde-pendentfactorresponsibleforinfection.1,18_{Ourstudyshowed}

that oral sex practices were prevalent in 71.9% of

partici-pants.AstudyconductedinMexicoexaminedtheincidence

ofHPVintheoralmucosaofwomenwithcervicallesionswho

reportedoralsexpracticesandalsoshowed72%positivityfor

HPV.19

Despite the hypothesis that HPV is transmitted to a

particular region through oral sex practices, there is no

consensusforcollectingparticipants’secondarydata.Thus,

studies minimizing sample failures, such as the effect of

mouth kissing, which is associated with infection among

those who reported no oral sex practices, should be

examined.12

The number of sexual partners may be a marker of

other risk factors.20 _{Previous studies have analyzed the}

correlation between HPV infection in couples and in men

with a large number of sexual partners.18,21 _{The present}

study showed a higher positivity of HPV among

individ-ualswithalargernumberofsexualpartners;however,this

observation should not be considered in isolation since a

combinationofpracticespredisposing toinfectionmust be

considered.

In conclusion, the three primary findings of this study

includealowprevalenceofHPVinfectionintheoralmucosa

ofasymptomaticmen,theabilityofthemouthmucosatohost

high-riskHPVtypes,andthepresenceofmultipleviraltypes

withoutalwayspresentingwithclinicallesions.

Riskysexualbehaviorssuchasoralsexandhavingalarge

numberofsexualpartnersmayberelatedtoindividual

pre-dispositiontoHPVinfection,makingthemmorevulnerableto

HPVinfection.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

Thisstudy wassupportedbyFoundationtoSupport

Devel-opmentofEducation,ScienceandTechnologyoftheStateof

MatoGrossodoSul,Brazil(Fundect-MS), NationalResearch

Council(CNPq),andCoordinationofImprovementofHigher

Level(CAPES).

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