Lesional HPV types of cutaneous warts can be reliably identified by surface swabs
Koning, M.N.C. de; Khoe, L.V.; Eekhof, J.A.H.; Kamp, M.; Gussekloo, J.; Schegget, J. ter; ... ; Quint, W.G.V.
Citation
Koning, M. N. C. de, Khoe, L. V., Eekhof, J. A. H., Kamp, M., Gussekloo, J., Schegget, J. ter,
… Quint, W. G. V. (2011). Lesional HPV types of cutaneous warts can be reliably identified by surface swabs. Journal Of Clinical Virology, 52(2), 84-87. doi:10.1016/j.jcv.2011.06.016
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ContentslistsavailableatScienceDirect
Journal of Clinical Virology
j ourna l h o me p a g e :w w w . e l s e v i e r . c o m / l o c a t e / j c v
Lesional HPV types of cutaneous warts can be reliably identified by surface swabs
M.N.C. de Koning
a, L.V. Khoe
b, J.A.H. Eekhof
c, M. Kamp
a, J. Gussekloo
c, J. ter Schegget
a, J.N. Bouwes Bavinck
b, W.G.V. Quint
a,∗aDDLDiagnosticLaboratory,Voorburg,TheNetherlands
bDepartmentofDermatology,LeidenUniversityMedicalCenter,Leiden,TheNetherlands
cDepartmentofPublicHealthandPrimaryCare,LeidenUniversityMedicalCenter,Leiden,TheNetherlands
a r t i c l e i n f o
Articlehistory:
Received28March2011
Receivedinrevisedform3June2011 Accepted28June2011
Keywords:
Cutaneouswart HPV
Humanpapillomavirus Skin
Genotyping Wart
a b s t r a c t
Background:LargenumbersofHPVtypesinfectthehumanskinandmembersfromtheHPVgeneraalpha, gammaandmuareassociatedwithcutaneouswarts.
Objectives:TheaimofthisstudywastotestiftheHPVgenotypesinswabsoftheoverlyingskinare identicaltothetypespresentwithinthesewarts.
Studydesign:Tothispurpose,25personsbeingtreatedforpersistentcutaneouswartswereenrolled.
Swabsoftheoverlyingskinofthewartwerecollectedfromeachparticipant.Additionally,scabsofthe wartanddeeperportionsofthewartsweresurgicallyremoved.HPVgenotypingwasperformedonall samplesusingthenovelHSL-PCR/MPGassayandtheHPVgenotypingresultswerecompared.
Results:Fromthe25wartbiopsiesonewasHPVnegative.15werepositiveforHPV27,3forHPV57,2for HPV2,2forHPV1,1forHPV3and1wartbiopsywaspositiveforbothHPV41andHPV65.Scabsandswabs ofthewartsbothshowedidenticaltypingresultsasthebiopsiesin24ofthe25cases(sensitivity:96%).
Conclusions:TherewasanexcellentagreementbetweenHPVtypesintheswabsoftheskinthatoverlies thewartsandthebiopsiesofthesewartsvalidatingtheuseofwartswabsforfuturestudiesofwart- associatedHPVtypes.HPV27washighlyprevalent(70%)intheinadultsoftheinvestigatedpopulation ofpatientswithpersistentcutaneouswarts.
© 2011 Elsevier B.V. All rights reserved.
1. Background
Large numbers of HPV types, distributed over five papillo- mavirus genera, infect the human skin.1 HPV types belonging to four of those genera, i.e., Alphapapillomavirus, Gammapa- pillomavirus, Mupapillomavirus and Nupapillomavirus have been associated with cutaneous warts, mainly with foot and hand warts.2–13Reliabledetectionandsamplingtechniquesaremanda- tory to be able to study the epidemiology of these HPV infections.
Thehyperkeratoticskinlesion(HSL)PCR/MPGassayhasbeen describedrecently14anditdetectsandidentifiesDNAofallknown wart-associatedHPVtypesfromthealpha-(HPV2,3,7,10,27,28, 29,40,43,57,77,91and94),gamma-(4,65,95,48,50,60and88), mu-(HPV1and63)andnu-genus(HPV41).Biopsiesofthelesions arethegoldstandardfordeterminingwhichHPVtypeispresent withinthewarts.However,takingabiopsyandextractingtheDNA
∗ Correspondingauthorat:DelftDiagnosticLaboratory,Fonteijnenburghlaan7, 2275CXVoorburg,TheNetherlands.Tel.:+310703401670;fax:+310703401671.
E-mailaddress:w.g.v.quint@ddl.nl(W.G.V.Quint).
isnotonlylabourintensiveandpainfulforthepatient,but will alsobesubjectofethicalrestraintsbecauseofthebenignnatureof thesewarts.Large-scaleepidemiologicalstudies,therefore,should notrelyonbiopsiesonly.Ifnon-invasivesamples,likewartswabs wereagoodtargetfordetectionofHPVtypescausingthespecific wart,largerstudiescouldbemoreeasilydonetoinvestigatethe epidemiologyoftheseHPVtypes.Inpreviouswork,15–17focused onhealthyskin,actinickeratoses,basalcellandsquamouscellcar- cinomasitwasshownthatskinswabsareanefficienttargetfor thedetectionofcutaneousHPVtypesfromthebetaandgamma genera,but,sofarcutaneouswartsandtheirassociatedHPVtypes werenotevaluated.
2. Objectives
Theaimofthisstudywastovalidatetheuseofswabsofthe skinthatoverliesthewartforreliabledetectionoftheHPVtype(s) present in thedeeperportions of thesamewart. Our expecta- tionwasthattheseswabswouldcontainthesameHPVtype(s) as the underlyingwarts. Swabs from theperilesional skin and from theforehead were taken toinvestigate whetherthe HPV 1386-6532/$–seefrontmatter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.jcv.2011.06.016
DNA found in the wart was also detectable elsewhere on the body.
3. Studydesign 3.1. Clinicalmaterials
Twenty-five consecutiveimmunocompetent personsseeking treatment for persistent cutaneous warts in general prac- tice (8) or outpatient dermatology department (17) were enrolled.
Swabsoftheoverlayingskinofthewartweretakenfromeach participantbyfirmlyrubbingapre-wettedcotton-tippedstickfor 5timesoverthesurfaceofthelesion(Swabofwart,Table1).Sim- ilarly,swabsoftheskinareaaroundthewart(Swabperilesional, Table1)andtheforehead(Swabforehead,Table1)werecollected bysamplingaskinareaof5by5cm.Next,thecotton-tippedsticks wereputin1mlofsalinesolution,movedaroundinthesolution andwerepressedagainsttheinnerpartofthetubetoremovemost oftheliquidinthecotton-tips.Tenlofthesalinesolutionwas useddirectlyinthenovelHSL-PCR/MPGassay.Inordertoreduce thelikelihoodofcrosscontaminationduringsampletakingfirstthe swabsoftheforehead,thentheperilesionalswabsandfinallythe swabsofthewartsweretaken.Additionally,scabsofthewartand deeperportionsofthewartsweresurgicallyremovedandincu- batedin100–250lofa1mg/mlproteinaseKsolutionat70◦C for16htoreleasetheDNA,afterwhich,proteinaseKwasinac- tivatedat95◦Cfor10min.Allsampleswereanalysedinrandom order.
3.2. HSL-PCR/MPGassay
The HSL-PCR/MPG assay was performed as described by the manufacturer (Labo Bio-medical Products BV, Rijswijk, the Netherlands). Briefly, it comprises a primer set with 27 non- degenerate primers (13 forward and 14 reverse) generating a biotinylatedamplimerof76–84bpfromtheL1regionin35ampli-
ficationcycles.Genotypingisperformedwithbead-basedxMAP suspensionarraytechnologywhichisabletosimultaneouslyiden- tify23wart-associatedHPVtypesfromthealpha-(HPV2,3,7,10, 27,28,29,40,43,57,77,91 and94),gamma-(4, 65,95,48,50, 60and88),mu-(HPV1and63)andnu-genus(HPV41).14Theassay designresultsinverylowaspecificbackgroundsignalsandthere- forerequiresnobackgroundsubtraction.Cut-off’swereappliedas describedbefore.14Negativeextraction,PCRandgenotypingcon- trolswereincorporatedandremainednegativeuponanalysiswith theHSL-PCR/MPGassay.
3.3. Statisticalanalysis
ThesensitivityofHPVgenotypingonthedifferentswabsand thewartscabswasdeterminedbycomparisonwiththeHPVgeno- typingresultsgeneratedfromthewartbiopsies.Wedidnottest the specificity of the swabs, because we did not intentionally includeskinlesionsthatwerenotclinicallydiagnosedascutaneous warts.
4. Results
Thestudypopulationwascomposedof25participants,includ- ing11womenand14men.Theirmeanagewas31.7years,ranging from6to53years(Table1).Sevenofthemhadhandwartsandthe other18hadfootwarts.
Twenty-fourofthe25wartbiopsies(96%)wereHPVpositive (Table1).15werepositiveforHPV27,3forHPV57,2forHPV2,2for HPV1and1forHPV3.OnewartbiopsywaspositiveforbothHPV41 andHPV65andonewasHPVnegative.Remarkably,intheadult participants70%(14of20)oftheHPVpositivewartscontained HPV27(Table1).
Genotypingresultsobtainedfromscabsandswabsofthewarts wereidenticaltotheresultsfromthedeeperwartportionsin24 ofthe25(96%)cases(Table1).Intwocasestheresultbetweenthe wartbiopsyandscabofthewartorwartswabwerenotidentical (Table1).Thescabandwartswabwerenegativeinthesetwocases
Table1
HPVgenotypingresultsobtainedwiththeHSL-PCR/MPGassay.Resultsindicatedinboldfontarenotidenticaltotheresultfromthewartbiopsy.
Participant Ageinyears Locationwart Wartbiopsy Scab Swabofwart Swabperilesional Swabforehead
1 25 Hand 27 27 27 27 27
2 19 Foot 27 27 Negativea 2,27 2,27,57
3 27 Foot 27 27 27 27 3
4 19 Hand 3 3 3 3 Negative
5 62 Hand Negative Negative Negative 48 48
6 23 Foot 27 27 27 27 2,27
7 41 Foot 2 2 2 2 2
8 42 Foot 27 27 27 27 27
9 25 Hand 27 27 27 27 27
10 21 Hand 27 27 27 27 27
11 46 Foot 2 2 2 2 2
12 44 Foot 57 Negativea 57 57 Negative
13 53 Foot 27 27 27 27 27
14 48 Foot 27 27 27 27 Negative
15 42 Foot 27 27 27 27 Negative
16 34 Foot 27 27 27 27 Negative
17 30 Hand 57 57 57 57 Negative
18 49 Foot 57 57 57 57 Negative
19 19 Foot 27 27 27 27 27
20 53 Foot 27 27 27 27 Negative
21 35 Hand 27 27 27 Negative Negative
22 11 Foot 41,65 41,65 41,65 1,41,65 10
23 6 Foot 1 1 1 1 1
24 7 Foot 1 1 1 1,2 Negative
25 10 Foot 27 27 27 Negative Negative
aThesetwosamplesshowanelevatedsignalforHPV27(119MFI)andHPV57(134MFI),respectivelythatiswellabovethebackgroundsignaloftheprobebutstillbelow thecut-offoftheassayforpositivity(200MFI).
butclearlyshowedanelevatedsignalforthesameHPVtypepresent inthewartbiopsies.Thisresultsinanequalsensitivityof96%for thescabsandswabsofthewarts.
Swabstakenfromaroundthewartandfromtheforeheadpro- vided identical HPV genotyping results as the wart biopsy for 19 (19/25,76%) and 9 (9/25, 36%) participants,respectively. In theperilesionalandforeheadswabs3and2multipleinfections weredetectedand 2/25 and 11/25were HPVnegative,respec- tively(Table1).Theresultingsensitivityofperilesionalswabswas higher than the sensitivity of foreheadswabs at 92% and 46%, respectively.
Oneof thewarts(participant5)wasHPVnegative withthe HSL-PCR/MPGassay.AdditionalPCRanddirectsequencinganaly- sesrevealedthepresenceofHPV107,aBetapapillomavirus(BetaPV) type,intheswaboftheforehead,perilesional,intheswaband thescabof thewart,butnotinthedeeperportionofthewart.
BetaPVareubiquitousviruses16,18–20andtheclinicalrelevanceof findingHPV107inthisparticipantwithrespecttothewartwasfur- therinvestigated.Inretrospect,thewart-likelesionconsistedofan accumulationofcallusasaresultofbagpipeplaying,anddidnot haveaviralgenesis.
Limiteddataareavailableaboutmethodologies ofpreparing skinwartsamplesforPCRanalyses.Therefore,weusedsamples from thefirst ten participants for limited optimization experi- ments.Pre-treatmentofwartswabsbyproteinaseKdigestionor manualcolumnbasedDNApurificationdidnotresultsinadditional positivityascomparedtodirectuseof10lofswabsampleinthe PCR(datanotshown).Forthescabsanddeeperwartportionsthe proteinaseKtreatmentasdescribedabovewasoptimalascom- paredtowashingthematerialin200lofwaterandusing10lof watereitherdirectlyinthePCRorafteranincubationof5minat 100◦C(datanotshown).
5. Conclusions
In 24 of the 25 cases (96%), swabs of the overlying skin showedthesameHPVgenotypingresultastheunderlyingwart.
Theuseoftheseswabsis thereforea highlysensitivesampling technique for the determination of the HPVtype in cutaneous wartsandthismethodcouldbereliablyandeasilyusedinlarge scaleepidemiological studies.Scabscouldalsobeusedbecause this method is equally reliable, but this methodis more inva- sive and more prone to contamination and more difficult to standardize.
Inthetwocasesthatthegenotypingresultofthewartbiopsy wasnotconfirmedbythegenotypingresultofthescabofthewart orofthewartswab(participant2and 12,Table1)therewasa clearlyelevatedsignalvisible.Thesetworesultsillustratethatcut- offsaredifficulttoestablishforassaysbasedonxMAPsuspension arraytechnology.Apparently,thereisa‘grey-zone’foreachprobe signalandtheuseofacut-offwillleadtoanunderestimationof positivity.
Genotyping performed on swabs from perilesional skin is less sensitiveand using swabs from theforehead is much less sensitive, so these locationsshould not be used for the deter- mination ofHPV type in thewart.In addition, theagreements ingenotypingresultsbetweenperilesionalskinswabsandfore- head swabs and wart biopsies were lower at 76% and 36%, respectively.
Eventhough theagreementbetweenthegenotyping results of thewartbiopsies and swabs of theoverlyingskin wasvery high in this study, the swabs need to be taken very carefully if warts exist in close proximity of each other as thesamples mightgetcross-contaminatedifthesewartsharbordifferentHPV types.
Remarkably, in the adultparticipants 70% (14 of 20) of the HPVpositivewartscontainedHPV27.Itshouldbenotedthatthe participantsofthisstudy,enrolledinanoutpatientdermatology department and in generalpracticewere treated for persisting wartswhereasskinwartsusuallyresolvespontaneously.Itcould thereforebespeculatedthatHPV27isespeciallyassociatedwith persistingskinwartsinimmunocompetentadults.
Inconclusion,thedatapresentedinthisstudyshowthatHPV genotypinginswabstakenfromthesurfaceofpersistentcutaneous wartsaccuratelyidentifiestheHPVtypethatispresentinthebiopsy fromthedeeperwartportionsandthusprovideausefulresearch toolforepidemiologicalstudies.
Funding None.
Conflictsofinterest
Theauthorsdeclarednoconflictsofinterest.
Ethicalapproval
Nonerequiredbecauseprocedurewaspartofclinicalpatient care.
Acknowledgments
We thank J. Lindeman and Labo Bio-medical Products B.V.
(Rijswijk,Netherlands)forprovidingtheHSL-PCR/MPGassay.
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