www.bjorl.org
Brazilian
Journal
of
OTORHINOLARYNGOLOGY
ORIGINAL
ARTICLE
Prevalence
of
the
Helicobacter
pylori
in
the
tonsils
and
adenoids
夽
Tuba
Bayindir
a,
Yuksel
Toplu
a,
Baris
Otlu
a,
Yusuf
Yakupogullari
a,
Ozge
Yildirim
a,
Mahmut
Tayyar
Kalcioglu
b,∗aInonuUniversity,Malatya,Turkey
bIstanbulMedeniyetUniversity,Istanbul,Turkey
Received31May2014;accepted24August2014 Availableonline28March2015
KEYWORDS Helicobacterpylori; Reservoirs;
Adenoids; Palatinetonsil
Abstract
Introduction:ThereisanongoingdebateabouttheexistenceandeffectsofHelicobacterpylori
(Hp)inadenotonsillartissue.
Objective: AclinicalstudywasconductedtoassesstheexistenceofHpintheadenoidand/or adenotonsillartissues,whichweresurgicallyexcisedduetochronicadenotonsillitis.
Methods:PhosphoglucosaminemutasegeneforthedetectionofHpandcytotoxin-associated geneasvirulencegenewereexaminedin84adenotonsillartissuesobtainedfrom64patients andpatients’serumbyusingpolymerasechainreaction.
Results:Hp IgG was detected in57 (89%) patients’ serum.A total ofseven tissuesamples from64patients(10.9%)werefoundpositiveforHpDNA,ofwhichfivewereadenoidsandtwo weretonsiltissues.Allpolymerasechainreactionpositivesampleswerealsopositiveforthe cytotoxin-associatedgene,whichisavirulencedeterminantfortheorganism.
Conclusion: ThisstudysuggeststhatchildrenareexposedtoHpatanearlyageoftheirlifein thisprovince.Hpmayhavearoleinthepathogenesisofchronicadenotonsillitis,especiallyin endemicareas.
© 2015Associac¸ãoBrasileira de Otorrinolaringologiae CirurgiaCérvico-Facial. Publishedby ElsevierEditoraLtda.Allrightsreserved.
夽 Pleasecitethisarticleas:BayindirT,TopluY,OtluB,YakupogullariY,YildirimO,KalciogluMT.Prevalenceofthe
Helicobacterpyloriin thetonsilsandadenoids.BrazJOtorhinolaryngol.2015;81:307---11.
∗Correspondingauthor.
E-mail:mtkalcioglu@hotmail.com(M.T.Kalcioglu).
http://dx.doi.org/10.1016/j.bjorl.2014.08.018
PALAVRAS-CHAVE Helicobacterpylori; Reservatórios; Adenoides; Tonsilapalatina
PrevalênciadoHelicobacterpyloriemamígdalaseadenóides
Resumo
Introduc¸ão:Há um debate atual sobre os efeitos da Helicobacter pylori (HpHp) no tecido adenotonsilar.
Objetivo:Conduzimos um estudo clinico para avaliara existência de Hp nos tecidos ade-noideanose/ou adenotonsilar, os quais foramremovidoscirurgicamente em decorrência de adenotonsilitecrônica.
Método: Nototal,84amostrasdetecidoobtidosde64pacientesforamanalisadasparaogen fosfoglucosaminamutaseparaadetecc¸ãodeHp.Oscasospositivosforamaseguirexaminados paraogenassociadoàcitotoxina,relacionadoavirulência,usando-seométododeReac¸ãode PolimeraseemCadeia(PCR).
Resultados: AIgGdeHpfoidetectadoem57(89%)sorosdepacientes.Seteamostrasdetecido desessentaequatropacientes(10.9%)resultoupositivoparaoDNAdeHp,dasquaiscincoeram adenóideseduaseramtecidotonsilar.NoPCRtodasasamostrasforamtambémpositivaspara ogenassociadoàcitotoxina,oqualéumdeterminantedevirulência.
Conclusão:Esseestudosugerequeascrianc¸assãoexpostasaoHpnosprimeirosanosdevida nessaprovínciaequeoHppodeterumpapelnapatogênesedaadenotonsilitecrônica, princi-palmenteemáreasendêmicas.
©2015Associac¸ãoBrasileira deOtorrinolaringologiaeCirurgiaCérvico-Facial.Publicadopor ElsevierEditoraLtda.Todososdireitosreservados.
Introduction
Helicobacter pylori (Hp) is a microaerophilic, Gram-negative spiral microorganism that colonizes the human gastric mucosa. It is the most frequent cause of gas-tric and duodenal ulcers in humans. The relationship between Hp and some gastrointestinal malignancies has alsobeendemonstrated.1Particularly,cytotoxin-associated
gene (cagA) positive strains are associated with a sig-nificantly increased risk for the development of atrophic gastritis, mucosa-associated lymphoid tissue lymphoma (MALToma),andgastriccancer.Hpisgenerallyacquired dur-ingchildhood(<10years)viathefecal---oral,oral---oral,and gastric---oraltransmission routes,2---5 and can remain silent
foralifetime.3,6ThetransmissionrouteofHpinfectionhas
notbeenclearlyunderstood.
TheWaldeyerringisthefirststepinthemucosaldefense againstinvadingpathogensthroughitsMALTcontent. Ade-noidandtonsiltissuesarepartoftheWaldeyerring.These participateintheimmunesystem,especiallyinchildren.On theother hand,adenoidectomy and/ortonsillectomy rep-resentthemost commonsurgical proceduresin childhood becauseofchronicinfectionand/orhypertrophy.7
ThoughthestomachisanaturalreservoirforHp,various tissuessuch asthegall bladder,gingiva, oral lesions,and thedentalplate have been demonstrated tobepotential extra-gastricreservoirsofthispathogen.8,9Furthermore,Hp
hasbeenfoundinnasalpolyps,nasalmucosa,andsaliva.10
Recent studies have suggested that adenotonsillar tissue couldbeanextra-gastric reservoirfor Hp,but theresults havebeenconflicting.5,8,11---15Detectionofthecolonization
andaccumulationofHpin differentregionsofthebodyis particularlyimportanttobetterunderstandingthemodesof
transmissionandprogressofinfectionofthisorganism.This study aimedtoclarifythe roleofadenotonsillartissueon Hpinfection.Whetheradenotonsillartissueanextra-gastric reservoir for Hp or Hp has a role in the pathogenesis of chronicadenotonsillitis.
Methods
Patients
ThisclinicalstudywasperformedattheUniversity Otorhino-laryngologyandClinicalMicrobiologyDepartmentsbetween Augustof2011andAugustof2012.Surgicallyexcised ade-noid andtonsil tissues and serumsampleswere collected from children who underwentadenoidectomy and adeno-tonsillectomy due to chronic adenotonsillitis. A total of 64 children (34 males, 30 females) were included in the study.Themeanageofthepatientswas5.9years,ranging between 1 and 17 years.A total of 84 clinical specimens were collected.Sixty-two biopsy specimens wereadenoid and22weretonsillartissue.Venousperipheralblood sam-ples(5---6mL)werecollectedfromallpatients.
Inclusioncriteria
Exclusioncriteria
Patientswhohadtakenantibiotics,protonpumpinhibitors, orH2receptorantagonistsandantacidsduringafour-week periodpriortosurgery,aswellaspatientswhohadaclinical historyof dyspepticorgastroesophageal symptomsonthe systematicquestionnairewereexcludedfromthestudy.All patients were operated undergeneral anesthesia. Tissues andserumsampleswerestoredat −70◦Cuntillaboratory
analysis.
Laboratory
Venous peripheral blood samples (5---6mL) were collected fromallpatients.Bloodsampleswerethen centrifugedat 2500rpmfor10min.Theserumwasseparatedandstoredat
−80◦C.SerumsampleswereanalyzedforHp
immunoglob-ulin G (IgG) with a commercial immune assay kit (Dima GmbH---Germany).Briefly,250---500Lofpatientserumwas
droppedonto thetest cassette andthe resultswere read visuallywithin10min,accordingtothemanufacturer’s rec-ommendations.Thisisarapidserologictest. Thetestwas usedtoindicatethehigherprevalenceofHpexposureinthe childhoodinthisregion.
DNAextraction
Adenoidandtonsiltissuespecimensweremechanically dis-rupted using the Tissue Lyser system (Qiagen --- Hilden, Germany), and then incubated overnight in tissue lysis buffer. Bacterial DNA was extracted from these samples aftercompletingdigestionoftissuesusingtheQIAampDNA MiniKit(Qiagen),accordingtothemanufacturer’s instruc-tions.
Polymerasechainreaction(PCR)fordetectionofHpDNA andcagAgene
Fordetection of Hp andits virulencegene in theclinical specimens, phosphoglucosamine mutase gene (glmM) and cytotoxin-associatedgene(cagA)geneswerestudiedwithan in-housePCRmethodwithpreviously-reportedprimers.16,17
A total 25L of amplification mix consisting of 12.5L
TopTaqDNAPCR MasterMix(QIAGEN--- Hilden,Germany), eachprimers1L(10pmol/L),8LH2O,and2.5L/each
sampleextractionproductwasprepared.Amplification con-ditionsandprimersetsareshowninTable1.PCRproducts were electrophoresed in 2% agarose gel, then ethidium bromide-stainedgelwasevaluatedinagelimagingsystem
Figure1 glmM(294bp)isillustratedintheagarosegel(2%). Lanes1, 2, 5, 8,positive samples; lane11, positive control; lane12,negativecontrol;lanes3,4,6,7,9,10,negative sam-ples.LaneM,molecularweightmarkers(DNAmolecularWeight MarkerIX,Roche).
(Gellogic 2200imagingsystem [1708×1280pixels;Kodak
Company---NY,UnitedStates];Fig.1).
PCRanalysiswasperformedonrandomlyselectedtonsil andadenoidtissues.
Ethicalconsiderations
Thisstudy waspreformed afterethicalapproval fromthe HumanEthicsCommitteeof theUniversity.Informed con-sentsweresignedbyparentsof thepatientsbeforeblood sampleandtissuecollection.
Results
Venousperipheral blood samples(5---6mL)were collected fromallpatients.Atotalof57serumsamplesof64patients (89%)werefound tobepositivefor HpIgG.Mean agewas 6.3years(3---17years);sexdistributioncomprised32boys and25girls.
InthePCR analysis,sevenclinical specimensbelonging todifferentpatients among the 64patients (10.9%) were positiveforHpDNA.Arepresentativegelimageisshownin
Fig.1. Ofthese,twoweretonsils,andtheremainingfive wereadenoidsfromdifferentpatients.ThecagA virulence genewaspositiveinallHppositivetissues.Itwasfoundthat 10.9%ofthepatientswerepositiveforHpand90%ofthese patientswerepositiveforHpIgG.
Table1 DNAprimersusedandtheamplificationconditions.
Gene Primersequence PCRproduct(bp) PCRconditions References
glmM 5′-AAGCTTTTAGGGGTGTTAGGGGTTT-3′ 294 94◦C,1min;55◦C,1min; 72◦C,1min(35cycles)
16
5′-AAGCTTACTTTCTAACACTAACGC-3′
cagA ATAATGCTAAATTAGACAACTTGAGCGA 298 94◦C,1min;60◦C,1min;
72◦C,1min(45cycles)
17
TTAGAATAATCAACAAACATCACGCCA
Discussion
Hpinfection iswidespreadthroughout the world,andthe prevalenceoftheinfectionmayvaryaccordingtopatients’ socioeconomic status, geographic area, age, and ethnic-ity.Itsprevalenceisabout30---40%indevelopedcountries, whereastherateisupto90%indevelopingor underdevel-opedcountries.18---21
ThepresenceofHpintheupperaero-digestivetracthas beenreportedindifferentstudies.Minochaetal.9reported
alowHpprevalenceinthegastricmucosaofpatientswitha historyoftonsillectomy.ItissuspectedthatHpcolonizeson tonsillarand/oradenoidtissuesasanextra-gastricreservoir. Nevertheless,DiBonoventuradidnotsupportthisopinion, and claimedthat tonsilsdid not represent a reservoir or sanctuaryforHp.22However,therearestillcontradictionsin
recentinvestigations.Also,somestudiesproposedthatHp mighthavearoleinthepathogenesisofchronic adenoton-sillitis.The pathogenic processofchronicadenotonsillitis, fromsingletochronicinfections,hasnotbeenclearly under-stood.Ithasbeenacceptedthattonsillarcryptobstruction duetoinfectioncauses differentiationof residentflorato some pathogenic bacteria.2,23,24 In the present study,the
cagAvirulencegenewasfoundpositiveinallpatientswho were positive for Hp DNA on PCR analysis. These results support that Hp may have a role in the pathogenesis of chronicadenotonsillitis,especiallyinendemicregions,such asTurkey.
Bacterial culture is a valuablemethod of diagnosis for Hpinfections.But,thefastidiousnatureofHpcausesahigh frequencyoffalsenegativeresultswiththismethodology. Therefore,variousmethodshavebeenusedforthediagnosis ofHpinfection.Particularly,nestedPCRiswidelypreferred whenitcomestodetectingHpinclinicalpracticebecause ofits high sensitivityand specificity.15 The most accurate
methodsofdiagnosishavebeenacceptedas immunohisto-chemistryandPCRanalysis.Thereisstillnosingletestthatis acceptedasthegoldstandardforHpdetection.Theusageof combinedtestsisstillpreferredbothinpediatricandadult studies.1,25 The present studyusedserology for indicating
thehigherprevalenceofHpexposureinthelocalpediatric populationandthenestedPCRmethod(moresensitive)to detectHpintissuesamples.
Bitar et al.12 tested 25 adenoid specimens using the
rapidureasetest(RUT),histologicalevaluation,andnested PCR methods. They reported an 84% positivity rate for Hp in the adenoids with RUT. On histopathological exam-ination, other bacteria were found 68% in RUT-positive tissues,whereasonly16%wereHp-like organisms.Finally, the authors reported all of their samples to be negative forHpDNAonnestedPCR.Therefore,theyconcludedthat nested PCR was the best way to diagnose Hp. Similarly, Torosetal.tested84adenoidandtonsilspecimensbyRUT andhistopathologicalevaluation.13TheydidnotdetectHp
in any of the specimens by either RUT or histopathology. Hence,theynotedthatcolonizationofHpintonsillarand/or adenoidtissuewasunlikely.
Vilarinhoetal.14 aimedtodetermine whetherthe
ade-notonsillartissuemightconstituteanextra-gastricreservoir forHp.Theyusedmanydiagnosticmethods,includingRUT, immunohistochemistrywithapolyclonalanti---Hpantibody, fluorescence in situ hybridization (FISH) with a specific
Hp peptide nucleic acid(PNA) probe, and PCR-DNA ELISA designed for detection of the vacA gene of the organ-ism.Anti---Hpantibodiesweredetectedin39%ofpatients. Immunohistochemistry was found positive in three tonsil tissues whose serologies were negative.RUT was positive in twoadenoid and two tonsil tissues withpositive sero-logy. The PNA-FISH and PCR-DNA ELISA tests, which are considered the most specific and sensitive methods for Hp detection, were found to be negative in all tissues. According to the results, the investigators reported that adenotonsillar tissues did not constitute an extra-gastric reservoirforHpinfectioninchildren.
Incontrasttothe studiesabove,Abdel-Monem etal.11
found Hptobepositive inadenotonsillartissueusingPCR withtheureCgenesequencein16.6%of patients.Atotal of30surgicalspecimens(20tonsilsandtenadenoidsfrom 20 patients) were evaluatedby RUT, blood serology, and PCR.TheyreportedthatRUTwaspositivein16specimens (53.3%)andserologywaspositivefor HpIgGantibodies in fourpatients (20%).They concludedthat the adenotonsil-lar tissues couldbean extra-gastricreservoir area for Hp infectioninchildrenwithchronicadenotonsillardiseases.
ThecagAgenehasbeenshowntobepresentin60---70% ofHpstrains,andthesestrainsareacceptedasvirulent.26
ThecagAproteininteractswithcellularproteins,and conse-quently,maycausemorphologicaldamageintheepithelium andfailureinthesecretionandsignaltransmissioninhuman cells.Therefore,thesestrainsaremuchmorepotentin caus-inggastricmucosaldamage.26,27
Inthisstudy,itwasfoundthat10.9%ofthechildrenwith chronic adenotonsillitis were positive for Hp, and 90% of thesepatientswerepositiveforHp IgG.Althoughitwasa valuablediagnostictoolingastricsamples,useofRUTwas notpreferred,asitgiveshighratesoffalsepositiveresults duetotheintenseaccumulationofurease-positivepresent in the normal bacterial flora of the upper aero-digestive tract.Bacterial culturecan beavaluable diagnostictool, but because of the low growthability of Hp in materials withahighbacterialloadinthefloramembers,itwasnot used.The detectionofthebacterialDNAanditsvirulence genewerechosentoinvestigatethebacteriain adenoton-sillartissue.ItwasobservedthatHpwaspresentin8%and 9%ofadenoidsandtonsils,respectively.Regardingthe pre-viouslypublishedstudies,ahigh rateofHpinthepresent clinical specimenswasfound. This wasmost likelydueto thehighfrequencyoftheorganisminthispopulation. Addi-tionally,itwasobservedthatallHppositivesampleswere alsosimultaneouslypositiveforthecagAgene.
Conclusion
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
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