www.bjorl.org
Brazilian
Journal
of
OTORHINOLARYNGOLOGY
ORIGINAL
ARTICLE
Herpes
viruses
and
human
papilloma
virus
in
nasal
polyposis
and
controls
夽
Dimitrios
Ioannidis
a,
Vasileios
A.
Lachanas
b,∗,
Zoe
Florou
a,
John
G.
Bizakis
b,c,
Efthymia
Petinaki
a,
Charalampos
E.
Skoulakis
b,caMicrobiologyDepartment,MedicalSchool,UniversityofThessaly,Larissa,Greece bDepartmentofOtorhinolaryngology,UniversityHospitalofLarissa,Larissa,Greece cDepartmentofOtorhinolaryngology,MedicalSchool,UniversityofThessaly,Larissa,Greece
Received17September2014;accepted18January2015 Availableonline8September2015
KEYWORDS
Sinusitis; Nasalpolyps; Viruses; Herpesviridae
Abstract
Introduction:Chronicrhinosinusitiswithnasalpolypsisamultifactorialdiseaseentitywithan unclearpathogenesis.Contradictorydataexistintheliteratureonthepotentialimplicationof viralelementsinadultpatientswithchronicrhinosinusitis.
Objective:Tocompare theprevalenceofhumanherpes viruses(1---6)andHuman Papilloma Virusinadultpatientswithchronicrhinosinusitiswithnasalpolypsandhealthycontrols.
Methods:ViralDNApresencewasevaluatedbyreal-timepolymerasechainreactionapplication tonasalpolypsspecimensfrom91chronicrhinosinusitiswithnasalpolypspatientsandnasal turbinatemucosafrom38healthycontrols.
Results:Epstein---Barrviruspositivitywashigherinnasalpolyps(24/91;26.4%)versuscontrols (4/38;10.5%),butthedifferencedidnotreachsignificance(p=0.06).Humanherpesvirus-6 positivitywaslowerinnasalpolyps(13/91;14.29%)versuscontrols(10/38;26.32%,p=0.13). Inchronicrhinosinusitiswithnasalpolypsgroup,1samplewasherpessimplexvirus-1-positive (1/91;1.1%),andanotherwascytomegalovirus-positive(1/91;1.1%),versusnoneincontrols.No samplewaspositiveforherpessimplexvirus-2,varicella-zostervirus,high-risk-humanpapilloma viruses(16,18,31,33,35,39,45,51,52,56,58,59)andlow-risk-humanpapillomaviruses(6, 11).
Conclusion:Differences in Epstein---Barr virus and human herpes virus-6 positivity among patientswithchronicrhinosinusitiswithnasalpolypsandhealthycontrolsarenotstatistically significant,weakeningthelikelihoodoftheirimplicationinchronicrhinosinusitis withnasal polypspathogenesis.
© 2015Associac¸ãoBrasileira de Otorrinolaringologiae CirurgiaCérvico-Facial. Publishedby ElsevierEditoraLtda.Allrightsreserved.
夽 Pleasecitethisarticleas:IoannidisaD,LachanasVA,FlorouZ,BizakisJG,PetinakiaE,SkoulakisCE.Herpesvirusesandhuman
papillo-mavirusinnasalpolyposisandcontrols.BrazJOtorhinolaryngol.2015;81:658---62. ∗Correspondingauthor.
E-mail:vlachanas@gmail.com(V.A.Lachanas).
http://dx.doi.org/10.1016/j.bjorl.2015.08.010
1808-8694/©2015Associac¸ãoBrasileiradeOtorrinolaringologia eCirurgiaCérvico-Facial. PublishedbyElsevierEditoraLtda.All rights
PALAVRAS-CHAVE
Sinusite; Póliposnasais; Herpesviridae; Papilomavírus humano
Herpesvírusevírusdopapilomahumanonapoliposenasalecontroles
Resumo
Introduc¸ão: Arinossinusitecrônicacompóliposéumadoenc¸amultifatorialdeetiopatogênese aindanãodefinida.Existemdadoscontraditóriosnaliteraturasobreaimplicac¸ãopotencialde elementosviraisnaetiologiadepóliposnasossinusais.
Objetivo: Compararaprevalênciadeherpesvírushumanos(1---6)epapilomavírushumanoem pacientesadultoscomrinossinusitecrônicacompóliposnasais(CRwNP)econtrolessaudáveis.
Método: Apresenc¸adeDNAviralfoiavaliadaporPCRemtemporeal,emamostrasdepólipos nasaisde91 pacientescomCRwNPenamucosadasconchasnasaisde38 controlessaudáveis.
Resultados: A positividade do EBV foi maiornos pólipos nasais (24/91; 26,4%) do que nos controles(4/38;10,5%),masadiferenc¸anão foisignificante(p=0,06).OHHV-6 apresentou positividade menornospólipos nasais(13/91;14,29%)doqueoscontroles(10/38;26,32%), (p=0,13).NogrupoCRwNP,umaamostrafoipositivaparaovírusherpessimples(HSV-1)(1/91; 1,1%),eumaparacitomegalovírus(CMV)(1/91;1,1%);enenhumaamostrafoipositivanogrupo controle.NãohouveamostrapositivaparaHSV-2,VZV,HR-HPV(16,18,31,33,35,39,45,51, 52,56,58,59)eLR-HPV(6,11).
Conclusão:Diferenc¸as de positividade do EBV e HHV-6 entre pacientes comCRwNP e con-trolessaudáveisnãosãoestatisticamentesignificantes,enfraquecendoaprobabilidadedesua implicac¸ãonapatogênesedaCRwNP.
©2015Associac¸ãoBrasileiradeOtorrinolaringologiaeCirurgiaCérvico-Facial.Publicado por ElsevierEditoraLtda.Todososdireitosreservados.
Introduction
Chronicrhinosinusitiswithnasalpolyps(CRSwNP)is a sub-division of idiopathic chronic rhinosinusitis (CRS).1 It is a clinicalsyndromecharacterizedbypersistentsymptomatic inflammation of thenasal andparanasal sinusesmucosa.1 The etiopathogenesisofCRSwNP is mainlyattributed toa dysfunctionalhost---environment interaction.2 Even though theidentificationofexogenousagentsdrivingthesecondary inflammatory mechanisms has been a field of extensive research,thepotentialinvolvementofviralinfectionis rel-ativelyunstudied.1
Herpes simplex virus-1 (HSV-1), herpes simplex virus-2 (HSV-2), varicella-zoster virus (VZV), cytomegalovirus (CMV), Epstein---Barr virus (EBV),and humanherpes virus-6(HHV-6), alongwithhuman papillomaviruses (HPV),are DNAvirusesthathavethecapacitytoincorporateintohost DNA, to establish lifelong latent infections in the upper respiratory mucosa, and to reactivate in immunocompro-misedconditions.3---6Onlyafewstudies7---17haveinvestigated theirpotentialroleinCRSwNP,whiletheirresultsare con-troversial. Furthermore, the highly sensitive quantitative real-time polymerase chain reaction (PCR) technique has beenusedfordetectionofthesevirusesinCRSwNPbyonly twostudiessofar.11,17
The aims of the present study were to evaluate and compare theprevalence of HHV, high-risk HPV types (HR-HPV; subtypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59), and low-risk HPV types (LR-HPV; subtypes 6, 11) in nasal tissue samples of patients with CRSwNP and healthy controls by employing the highly sensitive quantitative PCR technique, and to review the related literature.
Methods
Thiswasacross-sectionalcontemporarycohortstudy,which wasconductedprospectively,fromJanuaryof2009to Jan-uary of 2013, on adult patients with CRSwNP undergoing functional endoscopic sinus surgery (FESS). CRSwNP diag-nosiswasmade according tothe criteriaofthe European PositionPaper onRhinosinusitis and NasalPolyps (EPOS).1 Thecontrolgroupconsistedofhealthyadultpatientswith nasalseptaldeviation undergoingseptoplastywithout CRS accordingtoEPOS criteria.1 Pediatric subjects,aswell as patientswithasthma, cysticfibrosis,primaryciliary dysk-inesia, allergic fungal sinusitis, allergic rhinitis, inverted papilloma,and HIV seropositivitywereexcluded fromthe study.Subjectsinbothgroupswhohadhadanupper respi-ratorytractinfectionwithintwoweeksbeforesurgery,and thosewhohadtakenanynasalorsystemicsteroidswithin the last month prior to surgery were excluded from the study.
In CRSwNP patients, nasal polyp specimens were obtainedfromtheparanasal sinusesduring FESS,while in thecontrolgrouptissuebiopsiesfromtheinferiorturbinate mucosa were taken during septorhinoplasty. Nasal polyps andnasaltissuesextractedduringsurgerywereimmediately transferredinsteriledrycontainersandshippedtothe lab-oratory.Byuseof asurgicalknife,thetissues werecutin half,andseveralpieces(2---4mm)weretakenfromthedeep tissueanddividedintothreeparts:forconventionalculture, formoleculartechniquesandforstorageat−80◦C.
Foreach patient,thetissuepieceswere inoculatedon Sabouraudagarat 30◦C for 10daysandthen on5%sheep blood Columbia agar incubated in a 5% CO2 atmosphere
Table1 Epidemiological data on sex, age, and type of surgery(primaryorrevision)forchronicrhinosinusitiswith nasalpolyps(CRSwNP)andcontrolgroups.
CRSwNPgroup Controlgroup Total 91 38
Male(%) 63(69.2%) 22(57.9%) Female(%) 28(30.8%) 16(42.1%) Meanage(range) 53 (19---77) 43(18---54) Primarysurgery(%) 65(71.4%) N/A Revisionsurgery(%) 26(28.6%) N/A
wasperformed on all specimens in ordertoevaluate the presenceofleukocytesandmicrobialflora.
Samples of tissue from each patient were chosen for DNAextraction usingcommercial kits (Invitrogen) accord-ingtothemanufacturer’sinstructions.TheefficiencyofDNA extractionandthepossiblepresenceofinhibitorsinthe sam-plewereconfirmedbythedetectionofthe2-globingene usingtheprimers RS42 (5′-GCTCACTCAGTGTGGCAAAG-3′)
andKm(5′-GGTTGGCCAATCTACTCCCAGG-3′).
Theextracted DNAspecimensweresubmittedto quan-titative real-time PCR (Applied Biosystems 7500 Fast Real-Time PCR System) by using commercially available assaysaccordingtothemanufacturer’sinstructions:HSV1 Q-PCRAlertAmpliMIX,HSV2Q-PCRAlertAmpliMIX,VZVQ-PCR AlertAmpliMIX,EBVQ-PCRAlertAmpliMIX,Q-CMVRealTime Complete,HHV-6Q-PCRAlertAmpliMIX(NanogenAdvanced DiagnosticsS.r.L),theHPVHighRiskScreenRealTimePCR (SacaceBiotechnologies)andHPV 6/11Real-TMRealTime PCRkit(SacaceBiotechnologies).
All data, including patients’ demographic information (age,gender, history)wereplacedin adatabase. For sta-tisticalanalysis,Fisher’sexacttestwasused.Dataanalysis wasperformed withSPSSv.20software(IBM,Chicago,IL, UnitedStates).p-values<0.05wereconsideredas statisti-callysignificant.
ThesampleswereanalyzedattheDepartmentof Microbi-ology,MedicalSchool,UniversityofThessaly.Thestudywas approvedby theinstitutional review board (approval pro-tocolNo.10/28-11-2007). Awritten informedconsentwas obtainedfromallpatientsandcontrolsubjects.
Results
There were129 whitesubjects enrolledin thestudy.The nasalpolypsgroupconsistedof91patients(63males;mean age 53 years;range19---77 years)while thecontrol group consistedof38subjects(22males;meanage43years;range 18---54years).Primarysurgerywasperformedin65patients andrevisionsurgeryin26patients(epidemiologicaldataare summarizedinTable1).
Conventionalcultures showed that all specimens were negative for fungi, while they were positive for Gram-positive cocci ofmicrobial flora, includingStaphylococcus aureus, coagulase-negative staphylococci, and viridans streptococci.
DNAextraction,indicatedby 2-globingenedetection, wassuccessfulinallthesamples.
EBVpositivitywashigher innasalpolyps thanthe con-trolgroup(polypsgroup:24/91;26.4%versuscontrolgroup: 4/38; 10.5%). This difference did not reach significance (p=0.06).
HHV-6 positivity was lower in nasal polyps than the control group(polyps group:13/91;14.29% versus control group:10/38;26.32%).Thisdifferencewasalsonot signifi-cant(p=0.13).
Onenasalpolypsample(1/91;1.1%)wasfoundtobe HSV-1positiveandonesample(1/91;1.1%)wasfoundtobeCMV positive. All control group sampleswere negative for the HSV-1andCMV.Thesedifferenceswerenotsignificant.
In nasal polyps group, the specimens of four patients werepositiveforbothEBVandHHV-6,whileinonepatient theywerepositiveforbothEBVandCMV.Simultaneous pos-itivityinEBVandHHV-6wasalsonoticedintwosubjectsof thecontrolgroup.
All specimens of study and control groups were nega-tive for HSV2, VZV, HR-HPV,and LR-HPV DNA(results are summarizedinTable2).
Discussion
Kozaketal.7werethefirsttoinvestigatethepotentialrole of EBVin theetiology ofnasal polyps.In apilot studyon nine patients with CRSwNP and six controls using in situ
Table2 RatesofHSV-1,HSV-2,VZV,CMV,EBV,HHV-6,andHPV(HR-HPVandLR-HPVtypes)inchronicrhinosinusitiswithnasal polyps(CRSwNP)andcontrolgroups.
CRSwNP(n=91) Control(n=38) Significancea
Positive---n(%) Positive---n(%)
HSV1 1(1.1%) 0(0%) NS
HSV2 0(0%) 0(0%) NS
VZV 0(0%) 0(0%) NS
EBV 24(26.4%) 4(10.5%) NS
CMV 1(1.1%) 0(0%) NS
HHV-6 13(14.29%) 10(26.32%) NS
HR-HPV 0(0%) 0(0%) NS
LR-HPV 0(0%) 0(0%) NS
NS,non-significant;HSV-1,herpessimplexvirus-1;HSV-2,herpessimplexvirus-2;VZV,varicellazostervirus;CMV,cytomegalovirus;EBV,
Epstein---Barrvirus;HHV-6,humanherpesvirus-6;HPV,humanpapillomaviruses;HR-HPV,high-riskHPV;LR-HPV,lowriskHPV.
hybridization(ISH),theyreportedthatEBVDNAwasabsent inbothgroups.SimilarfindingswithISHwerepublishedby Sham etal.8 in 2012, in 30 CRSwNP patients and 32 con-trols.Incontrast,Taoetal.9werethefirsttoreportnasal polypmucosaasoneofthesitesofEBVpersistence.They studied 13 CRSwNP patients and, by using Southern blot hybridazation (SBH), qualitative PCR, andISH, found EBV positivityin15%, 69%,and85%, respectively.However,no controls were usedin theirstudy. Zaravinos etal.10 com-parednasalpolypsamplesof23patientstoacontrolgroup of 13inferiorturbinate specimensfrompatients undergo-ingnasalcorrectivesurgery,byusingqualitativePCR.They foundEBVpositivityin35%ofCRSwNPpatientsversus0%in theircontrolgroup,demonstratingasignificantcorrelation betweenEBVpresenceandnasalpolypformation.Recently, Costaetal.11 werethefirsttousequantitativePCR in 35 patientswithCRSwNP,inordertocomparetheoccurrence ofEBVinnasalpolypsandadjacentinferiorturbinates tis-sue samples.Theyfound that EBVpositivitytended tobe higherinCRSwNP,suggesting apotentialcausativeroleor persistence in the inflammatory lymphoid tissue, but this difference did not reach significance. However, the main limitationoftheirstudywasthelackofacontrolgroupof healthysubjects.Thepresentresultsaresupportivetothose publishedbyCostaetal.,11sinceEBVpositivitywashigher inCRSwNPthanhealthycontrols,butthisdifferencedidnot reachsignificance(p=0.06).
Regarding HPV, controversial findings have also been reported.Bradnsma etal.12 and Gaffey et al.,13 by using SBH and ISH respectively, reportedzero HPV positivityin CRSwNPpatients.Beckeretal.14andShametal.,8byusing qualitativePCR,alsodidnotfindanyHPVpositivityinboth CRSwNP patients and controls. In 2000, Hoffman et al.15 reportedasinglesuspiciousHPVpositivesampleinagroup of33nasalpolyppatientsbyusingSBHandqualitativePCR, whileZaravinosetal.,10utilizingqualitativePCR(GP5+/6+ non-type-specificprimers),foundanonsignificantpresence ofHPV inCRSwNP patients(3/23, 13%)comparedtotheir controls(0/13inferiorturbinates).
Onthecontrary,FeiPeietal.,16inalarge-scaleChinese study,usedqualitativePCRandflow-throughhybridization aswellasgenechip technologyfor detectionof low risk-HPV(LR-HPV)andHR-HPVin 204CRSwNP patientsand 36 healthycontrols(middleturbinatemucosa).Theyreported 40.2%HPVpositivityinCRSwNPversus0%incontrols.Intheir study,13HPVgenotypeswerefoundinCRSwNPsamples (LR-HPVsubtypes:11,6,34,70,44;andHR-HPVssubtypes:58, 52,18,16,68,53,31,33),withLR-HPV-11themost preva-lent(45.28%).Thepresentstudydidnotconfirmtheresults publishedbyFeiPeietal.16Thedifferentfindingscouldbe attributedtodifferencesinstudypopulations(Asianversus white)or themethodusedtodetect HPV infection (qual-itive versus quantitativePCR). Recently, Rizzoet al.,17 in a white population of 20 CRSwNP patients and ten con-trols, used quantitative real time PCR for the first time with HR-HPV (subtypes: 16, 18, 31, 33, 35, 39, 45, 52, 53,56, 58,59,66, and70)and LR-HPV(subtypes:6, 11). No HR-HPV positivity wasfound in their study. Regarding LR-HPV,they reporteda 50%HPV-11 presence in the sub-groupoftenCRSwNPpatientswithoutallergicdisease,while no positive samples were found in the subgroup of ten CRSwNP patients withallergicdisease, aswell asin their
controlgroup.Theauthorssuggestedthatclinical parame-ters,suchallergy,couldbeaconfounderfortheHPVresults observed.Furthermore, theysuggested thatthe presence of HPV-11 might be a prognostic marker in the follow-up ofCRSwNPwithoutallergicdisease,sincetheynoticedthat thefiveHPV-11 positivepatients presentedwitha relaps-ingnasalpolyposis.17 InthepresentstudyquantitativePCR wasusedinalargepopulationof 91CRSwNPpatientsand 38controls.TheseresultsconfirmthosepublishedbyRizzo etal.17regardingtheabsenceofHR-HPVinbothpatientsand controls.However,thisstudydidnotconfirmthehigh preva-lenceofLR-HPV-11reportedintheirstudy.Thedifferences betweenthepresentfindingsmaybeattributedtothe dif-ferencesinthesizeofthesamples.Itshouldbenotedthat thisstudydidnotfindanyLR-HPVpositivity,whileallergic patientswereexcluded,and26patientsunderwentrevision surgery.
Zaravinosetal.10werethefirsttoinvestigateHHV-6 pres-encein nasalpolyptissue. By usingqualitative PCR, they foundthatHHV-6DNAwasabsentinbothnasalpolypsand inferiorturbinate samples of thecontrol group. However, Costa et al.11 used quantitative PCR in nasal polyps and inferiorturbinatesamplesof35patientswithCRSwNP,and foundanHHV-6 positivityof 8%innasalpolypsand35%in adjacentturbinatemucosa.Thisdifferencewasnot signifi-cant,andtherewasnocontrolgroupofhealthysubjectsin theirstudy.The presentdatasupportthefindingsofCosta etal.,11sinceHHV-6positivitywaslowerinnasalpolypsthan inferiorturbinatesamplesofthecontrolgroup(14.29% ver-sus26.32%respectively),whilethisdifferencewasalsonot significant.Itis probablethatthedifferencebetweenthe presentresultsandthosepublishedbyZaravinosetal.10may beduetothehighersensitivityandspecificityof quantita-tiveversusqualitativePCR.11
ThepresenceofHSV-1,HSV-2,VZV,andCMVwas investi-gatedbyZaravinosetal.10withqualitativePCRandbyCosta etal.11withquantitativePCR.Bothreportedsimilarfindings (twopositiveHSV-1samplesandoneCMVsampleeach,while noHSV-2orVZVpresencewasfound)inlinewiththepresent data.
Themainweaknessofthisstudyisthattherewasa dif-ferenceinthesamplesizeoftheCRSwNP(n=91)andcontrol (n=38)groups.Thesesamplesizesaresodifferentbecause, withinthestudyperiod,only38oftheseptoplastypatients acceptedtoparticipate.Thisdifferenceinsamplesizemight have influencedthe results and may explainsome differ-enceswith the resultsalready reportedin the literature. Furthermore,asstatedabove,theresultsmaybedifferent withthosealreadypublishedduetothedifferentmethods usedtodetect theviruses (ISHversus SBHversus qualita-tivePCRversusquantitativePCR).Itshouldbenoticedthat apartfromthisstudy,onlyCostaetal.11haveused quantita-tivePCR.Thisisalsooneofthestrengthsofthisstudy,since quantitativePCRisthemethodwiththehighestsensitivity andspecificity.
Conclusion
thatHR-HPV andLR-HPVwere absentin nasal polypsand controls.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgements
The authorswould like toexpressour appreciationto Dr. TimoleonTerzisandMrs.ParaskeviTsirevelouforthe assis-tance they provided in sample collection, their advice, andtheir continuous moralsupportthroughout the entire project.
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