ht t p : / / w w w . b j m i c r o b i o l . c o m . b r /
Medical
Microbiology
Investigation
of
Helicobacter
pylori
antigen
in
stool
samples
of
patients
with
upper
gastrointestinal
complaints
Zeki
Calik
a,
Murat
Karamese
a,∗,
Osman
Acar
a,
Selina
Aksak
Karamese
b,
Yalcin
Dicle
c,
Fatih
Albayrak
d,
Serpil
Can
e,
Bulent
Guvendi
f,
Alpgiray
Turgut
g,
Mustafa
Cicek
h,
Halil
Yazgi
iaDepartmentofMicrobiology,FacultyofMedicine,KafkasUniversity,Kars,Turkey
bDepartmentofHistologyandEmbryology,FacultyofMedicine,KafkasUniversity,Kars,Turkey cDepartmentofNursing,SchoolofHealth,MusAlparslanUniversity,Mus,Turkey
dDepartmentofInternalMedicine,FacultyofMedicine,AtaturkUniversity,Erzurum,Turkey eDepartmentofPhysiology,FacultyofMedicine,KafkasUniversity,Kars,Turkey
fDepartmentofGenerealSurgery,FacultyofMedicine,KafkasUniversity,Kars,Turkey gDepartmentofBiochemistry,FacultyofVeterinary,AtaturkUniversity,Erzurum,Turkey hDepartmentofAnatomy,FacultyofMedicine,GaziOsmanPasaUniversity,Tokat,Turkey iDepartmentofMicrobiology,FacultyofMedicine,AtaturkUniversity,Erzurum,Turkey
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Articlehistory:
Received8January2015 Accepted23August2015
AssociateEditor:AnaLúciadaCosta Darini
Keywords: Helicobacterpylori
Uppergastrointestinalcomplaints Rapidureasetest
H.pyloricardtest
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Helicobacterpyloriinfectionisusuallyacquiredinearlychildhoodanditcanpersist through-out lifewithoutantibiotic treatment.Thisstudyaimed tocomparetheaccuracyofthe noninvasiveH.pyloriStoolAntigenTest-appliedonthestoolsampleswiththeinvasivegold standartRapidUreaseTest-appliedonthegastricbiopysamplesofpatientswithupper gastrointestinalcomplaints.Afterendoscopy,biopsyandstoolspecimensweretakenin122 patients.Theinfectionwasdetectedwithrapidureasetestwhichisacceptedasgoldstandart test.Rapid,one-stepH.pyloricardtestwasappliedtoallpatientsstoolspecimens.Inthis study106ofthe122patients(86.8%)werepositiveforH.pyloriinfection,while16ofthe122 patients(13.2%)werenegative.H.pyloricardtestwasnegativein13ofthe16patientsand waspositivein98ofthe106.Thesensitivity,specifity,positiveandnegativepredictive val-ueswere92.45%,81.25%,97.02%,and61.90%,respectively.H.pyloricardtestisrapid,easy, noninvasiveandinexpensivemethodsfordetectionH.pyloriinfection.Thistestshowed highsensitivityandspecificity.Additionally,itmaybeagoodalternativetoinvasivetests forthedetectionofH.pyloriinfectionsespeciallyinchildren.
©2015SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
∗ Correspondingauthor.
E-mail:[email protected](M.Karamese).
http://dx.doi.org/10.1016/j.bjm.2015.11.022
1517-8382/©2015SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
Helicobacterpylori (H.pylori)isclassifiedasa gram-negative, spiral-shaped bacterium and a microaerophilic, fastidious, humanpathogen.H.pyloriinfectionisusuallyacquiredinearly childhoodanditcanpersistthroughoutlifewithoutantibiotic treatment. Itaffectsabout 20% ofthe populationin devel-opedcountriesandmorethan90%inthedevelopingworld.1–4 Oral–oraland fecal–oral are the mostcommon methodsof transmission.5
H.pylorispecificallycolonizesonthegasticmucuslayer, andithasdevelopedavarietyofmechanismstosurvivein theharshacidicenvironmentofthegastricmucosa.6H.pylori
containsmanyvirulencefactorsthatcausetheinfectionand contributestogastricinflammation.7Itisamajorcauseof
gas-tricandduodenalulcerandgastritis,andtheorganismhas beenetiologicallyassociatedwithMucosal-Associated Lym-phoidTissue(MALT)lymphomaandgastriccarcinoma.8,9
Invasiveandnon-invasivetestsareusedinthediagnosis ofH.pyloriinfection.Theinvasivemethodsincludeculture, histology,andureasetests.Biopsyspecimensobtainedwith upper gastrointestinal endoscopy are necessary for these tests.10–12Thenoninvasivemethodsincludestoolantigentest
(SAT),ureabreathtestandserology.13
Allthetestshaveadvantagesanddisadvantages.Therapid ureasetest(RUT)isagoldstandardmethodforthedetection ofH.pylori,anditisfasterandcheaperthanother invasive tests.14,15 Protonpumpinhibitors(PPIs),bismuth-containing
compoundsandantimicrobialagentsmayaffectthe perfor-manceofthistestbyinhibitingureaseactivity.Inaddition, otherurease-producingmicroorganismsinthegastricmucosa can cause false positiveresults.12,16 SATsare non-invasive
and inexpensive methods to detect active H. pylori infec-tion.Thistesthastwoversions:enzymeimmunoassayand immunochromatography.EradicationofH.pyloriinfectionis evaluatedbySATs.Thereforethistestisusefulbeforeandafter
H.pyloritherapy.2,16,17
Thisstudyaimedtocomparetheaccuracyofthe nonin-vasiveH.pyloriStoolAntigenTest(SAT)appliedonthestool sampleswiththe invasivegold standartRapidUrease Test (RUT)appliedonthegastricbiopysamplesofpatientswith uppergastrointestinalcomplaints.
Materials
and
methods
Patientselectionandcollectionofsamples
This study was approved by the Local Ethics Committee ofAtaturk University, Institute of Health Sciencewith the number of 1466.The subjects were selected from patients with upper gastrointestinal complaints admitted to the AtaturkUniversity,MedicalFacultyandDepartmentof Gas-troenterology. Of those referred to the endoscopyunit for gastrointestinalendoscopytoevaluatedyspepticcomplaints, 122(49male,73female)wereincludedinthisstudy.Patients takingbismuthpreparations,PPIs,H2receptorantagonistsor antibioticsforthelastmonthortakinganti-acidsforthelast
twodayswereexcludedfromthestudy.Thefirststoolsamples ofallpatientswerecollectedimmediatelyaftertheendoscopy. DetectionofH.pyloriinbiopsysamples
TheRUT(SalubrisHelicheck,Boston,USA)wasusedforthe detectionofH.pylorionbiopsysamplesinthisstudy.H.pylori
produceanabundanceofurease.Theureaseenzyme hydrol-yses ureatorelease CO2and NH3.Thereleaseofammonia
increasesthepHofthemedium.Theureaseactivitycausesa changeinthepHindicatorcolorforpositiveH.pyloriresults. Allthebiopsyspecimensweretakenfromthepatients dur-ingendoscopyandtheRUTswereperformedbytheclinicians accordingtothemanufacturer’sprotocol.2,18
InvestigationofH.pyloriantigensinstoolsamples
Therapid, one-stepH. pylori card test(H+RH. pylori CARD, Madrid, Spain) was used toinvestigate the presenceof H. pylori antigens in the stool samples. This test is a quali-tativeimmunochromatographicassayforthedetermination of H. pylori in stool samples. The membrane is precoated withmonoclonalantibodies,onthetestbandregion,against
H. pylori antigens.During testing,the sampleisallowedto react withthe colored conjugate(anti-H.pylori monoclonal antibodies-redpolystyrenemicrospheres)predriedonthetest strip.Themixturethenmovesupthemembranebycapillary action.Asthesampleflowsthroughthetestmembrane,the colouredparticlesmigrate.Forapositiveresult,thespecific antibodiespresentonthemembranewillcapturethecolored conjugate.Themixturecontinuestomoveacrossthe mem-branetotheimmobilizedantibodyplacedinthecontrolband region,wherearedbandalwaysappears.Thepresenceofthis redbandservesas;aninternalcontrolforthereagentsand verificationthatsufficientvolumewasaddedandproperflow wasobtained.
The stool samples were evaluated by the card test accordingtothemanufacturer’sprotocol.Asingleredband appearingacrossthecentralwindowinthesitemarkedwith thecontrollinewasconsiderednegative.Aredbandappearing inthesitemarkedwiththeresultlineandinthesitemarked withthecontrollinewasconsideredpositive.Atotalabsence ofthecontrolband,regardlessoftheappearanceoftheresult sitewasconsideredinvalid.
Statistical
analysis
ThestatisticalanalysiswasperformedusingSPSSfor Win-dows Version 17.0 (Statistical Package for Social Sciences version 17.0). Positive predictive value, negative predictive value,sensitivityandspecificitywereevaluatedwiththe fol-lowingformulas.
Positivepredictivevalue;
Truepositives/(Truepositive+Falsepositive)×100
Negativepredictivevalue;
20 0 18-27 28-37 38-47 48-57 Age groups % Male Female 58-67 68-77 78+ 2 4 6 8 10 12 14 16 18
Fig.1–Theagegroupsofpatientswithupper gastrointestinalcomplainments.
Table1–Comparisonofendoscopicfindingsandrapid ureasetestresults.
Diagnosis Rapidurease testpositive Rapidurease testnegative Total Gastritis 91 15 106 Ulcer 15 1 16 Total 106 16 122
Sensitivity;Truepositives/(Truepositives+Falsenegatives)×100 Specificity;Truenegatives/(Falsenegatives+Truenegatives)×100
Results
Themeanageofthe122patients’was45.02±15.134years.The malepatients’rangedfrom18to84yearsoldwithameanage of44.41±16.075years.Thefemalepatients’rangedfrom18to 80yearsoldwithameanageof45.42±14.568years.
The RUT detected H. pylori in 63 of the 73 female patients(59.4%)and43ofthe49malepatients(40.6%)with uppergastrointestinalcomplaints.TheRUT-positivepatients withuppergastrointestinal complaints had amean age of 45.21±15.641yearsandtheRUT-negativepatientswithupper gastrointestinalcomplaintshadameanageof43.75±11.538 years.Therelationshipbetweenpatientswithupper gastroin-testinalcomplaintsandtheiragesare showninFig.1. The uppergastrointestinalcomplaintsaremostseenin28–37and 48–57ageranges.Itmeansthatmiddle-agepatientsareunder riskofuppergastrointestinaldiseases.
Ofthesubjects, 106(86.8%)were diagnosedwith gastri-tisand16(13.2%)werediagnosedwithulcerbyendoscopic
Table3–EvaluationofH.pyloricardtestwithrapid ureasetest.
H.pyloriCard
Test
Rapidureasetest
Positive Negative Total
Positive 98 3 101
Negative 8 13 21
Total 106 16 122
Sensitivity;98/(98+8)×100=92.45% Specificity;13/(3+13)×100=81.25%
Positivepredictivevalue;98/(98+3)×100=97.02% Negativepredictivevalue;13/(8+13)×100=61.90% Falsepositiverate;1−0.812=0.188=18.8% Falsenegativerate;1−0.924=0.076=7%.
biopsy.Forthe106RUT-positivepatients,91(85.8%)were diag-nosedwithgastritisand15(14.2%)werediagnosedwithulcer. Forthegastritispatientsdiagnosedbyendoscopy,91(85.8%) were H. pylori positive accordingto theRUT. TheRUTalso testedpositivefor15(93.75%)ofthepatientswithulcers diag-nosedbyendoscopy(Table1).
H.pylori infectionwaspositivein106patientsaccording totheRUT.Amongthesepatients,98(93.75%)werepositive forH.pyloriaccordingtotheimmunochromatographicassay (rapidone-stepH.pyloricardtest).Inaddition,3(18.75%)ofthe RUT-negativepatientswerepositiveaccordingtothismethod (falsepositive).Inregardstotheendoscopydiagnosis,85ofthe 91(93.4%)H.pylori-positivegastritispatientsand13ofthe15 (86.6%)H.pylori-positiveulcerpatientswerepositiveaccording totheimmunochromatographicassay.
Inaddition,12 ofthe15(80%)H.pylori-negativegastritis patientsandthesingleH.pylori-negativeulcerpatientswere negativeaccordingtothecardtest(Table2).
TheH.pyloricardtestandRUTresultswerecomparedand positivepredictivevalue,negativepredictivevalue,sensitivity andspecificitywereevaluatedforthecardtest(Table3).
Discussion
Approximatelyhalfoftheworld’spopulationisinfectedwith
H.pylori.H.pyloriinfectionisespeciallyapublic-health prob-lemindevelopingcountries.Itistheleadingcauseofvarious uppergastroduodenaltractdiseasessuchasgastricand duo-denal ulcer, gastritis,MALT and gastric carcinoma.9,13,15 H.
pyloriinfectioncanbedetectedbyinvasiveandnoninvasive methods. Endoscopyisnecessaryfortheinvasivetests. All thesemethodshaveadvantagesanddisadvantages.14,16
Table2–Allthedataaboutthetestresultsanddiagnosisofpatients.
Diagnosis Rapidureasetestresults H.pyloriCardTestPositive H.pyloriCardTestNegative Total
Gastritis PositiveNegative 853 126 9115
Ulcer PositiveNegative 130 21 151
The RUT is an invasive method and considered a gold standard.Thistestisinexpensive and allowsforthe rapid detectionofH.pylori.Thetestmayresultinfalsenegativeifthe patientrecentlyusedantimicrobialagents,PPIsor bismuth-containing compounds or a heterogeneous distribution of bacteria ispresent in the gastric mucus layer. In addition, contaminationofthebiopsywithsalivaorotherurease pro-ducingmicroorganismsinthegastricmucosacancausefalse positive results. Thistest’s sensitivity,specificity, and pos-itive predictive valuesare more than >98%,99%, and 99%, respectively.12,15,19 TheRUTwas usedasthe goldstandard
method and compared with the immunochromatographic assay(H.pyloricardtest)inthisstudy.
Thestudyexamined122patientswithupper gastrointesti-nalcomplaints especiallyulcer and gastritis. Thepatients’ biopsyspecimensandstoolsampleswereexaminedwiththe RUTand one-step H.pylori card test, respectively. Ofthese patients,106(86.8%)werepositiveforH.pyloriinfection;while 16(13.2%)werenegative.Thesensitivity,specificity,positive predictivevalueandnegativepredictivevalueoftheH.pylori
card testwere 92.45%, 81.25%, 97.02%, and 61.90%, respec-tively.Incurrentliterature,thesensitivityandspecifityofthe cardtestwere87.8%,93.8%and75–100%,respectively.11,20–25 Theseresultsaresimilartoourstudyandshowedthat per-formanceofthecardtestwasexcellentforthenoninvasive detectionofH.pylori.Inaddition,previousstudieshaveshown thattheH.pyloricardtestexhibitsgoodperformancebefore andaftereradicationtherapy.22,26
Inour study,3ofthe 16 (18.75%) RUT-negativepatients werefoundpositiveaccordingtotheH.pyloricardtest(false positive). A cross-reaction with an antigen from Helicobac-ter species or other microorganism of the intestinal flora maycausefalsepositiveresults.27,28Ontheotherhand,the
irregular distributionofbacteria inthe gastricmucosacan causesuspectfalsenegativeresultswiththeRUT.19
Among the 106 RUT-positive patients, 8 (7.25%) were considered as negative (false negative) according to the immunochromatographic assay of the stool samples. Card testcharacteristics orthe applicationofthe testmay have causedfalsenegativeresults.Additionally,contaminationof thebiopsywithsalivaorotherureaseproducing microorgan-ismsinthegastricmucuslayercancausefalsepositiveresults
withtheRUT.12,19
Infections caused by H. pylori usually affect adults. In many previous studies,patient’s age ranged from 23 to89 years.20,29,30 Inthecurrentstudy,patient’sagerangedfrom
18to84years.Nochildrenwereincluded;however,previous studies have shown that the rapid one-step immunochro-matographiccardtestperformswellforthedetectionofH. pyloriinfectioninchildren’sstoolsamples.26,30
Conclusions
Inconclusion,theH.pyloricardtestisrapid,easy,noninvasive andinexpensivemethodforthedetectionofH.pyloriinfection. Thistestshowedhighsensitivityandspecificity.Nosignificant differencewasfoundinsensitivityorspecificitybetweenthe RUT(thegoldstandardmethod)andtheone-stepH.pyloricard test.Ontheotherhand,H.pyloriinthestoolisnotadiagnosis
ofulcerand/orgastritis.So,invasiveexaminationshouldalso bedonetoconfirmthediagnosis.However,thistestcanbe usedinsomeinsufficientconditionssuchasifendoscopyis notavailable.Additionally,theH.pyloricardtesthasexhibited goodperformancebeforeandaftereradicationtherapyandit isagoodalternativetoinvasivetestsforthedetectionofH. pyloriinfectioninchildren.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
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e
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c
e
s
1.BonifácioBV,dosSantosRamosMA,daSilvaPB,BauabTM.
AntimicrobialactivityofnaturalproductsagainstHelicobacter
pylori:areview.AnnClinMicrobiolAntimicrob.2014;13:54.
2.PatelSK,PratapCB,JainAK,GulatiAK,NathG.Diagnosisof
Helicobacterpylori:whatshouldbethegoldstandard?WorldJ Gastroenterol.2014;20:12847–12859.
3.RoeslerBM,Rabelo-Gonc¸alvesEM,ZeituneJM.Virulence
factorsofHelicobacterpylori:areview.ClinMedInsights
Gastroenterol.2014;7:9–17.
4.RheeKH,ParkJS,ChoMJ.Helicobacterpylori:bacterialstrategy
forincipientstageandpersistentcolonizationinhuman
gastricniches.YonseiMedJ.2014;55:1453–1466.
5.YangK,LiY,ZhouX.[OverviewofresearchesforHelicobacter
pyloriinoralcavityandstomach].HuaXiKouQiangYiXueZa
Zhi.2014;32:314–318.
6.CidTP,FernándezMC,BenitoMartínezS,JonesNL.
PathogenesisofHelicobacterpyloriinfection.Helicobacter.
2013;18(Suppl.1):12–17.
7.KalaliB,Mejías-LuqueR,JavaheriA,GerhardM.H.pylori
virulencefactors:influenceonimmunesystemand
pathology.MediatorsInflamm.2014;2014:426309.
8.TadesseE,DakaD,YemaneD,ShimelisT.Seroprevalenceof
Helicobacterpyloriinfectionanditsrelatedriskfactorsin
symptomaticpatientsinsouthernEthiopia.BMCResNotes.
2014;7:834.
9.OsawaH.GhrelinandHelicobacterpyloriinfection.WorldJ
Gastroenterol.2008;14:6327–6333.
10.TamadonMR,SaberiFarM,SoleimaniA,etal.Evaluationof
noninvasivetestsfordiagnosisofHelicobacterpyloriinfection
inhemodialysispatients.JNephropathol.2013;2:249–253.
11.PourakbariB,GhaziM,MahmoudiS,etal.Diagnosisof
Helicobacterpyloriinfectionbyinvasiveandnoninvasivetests.
BrazJMicrobiol.2013;44:795–798.
12.MégraudF,LehoursP.Helicobacterpyloridetectionand
antimicrobialsusceptibilitytesting.ClinMicrobiolRev.
2007;20:280–322.
13.DiRienzoTA,D’AngeloG,OjettiV,etal.13C-Ureabreathtest
forthediagnosisofHelicobacterpyloriinfection.EurRevMed
PharmacolSci.2013;17(Suppl.2):51–58.
14.LopesAI,ValeFF,OleastroM.Helicobacterpyloriinfection–
recentdevelopmentsindiagnosis.WorldJGastroenterol.
2014;20:9299–9313.
15.HuntRH,XiaoSD,MegraudF,etal.Helicobacterpyloriin
developingcountriesWorldGastroenterologyOrganization
GlobalGuideline.JGastrointestinLiverDis.2011;20:299–304.
16.CheyWD,WongBC,PracticeParametersCommitteeofthe
AmericanCollegeofGastroenterology.AmericanCollegeof
Gastroenterologyguidelineonthemanagementof
Helicobacterpyloriinfection.AmJGastroenterol.
17.ShimoyamaT.Stoolantigentestsforthemanagementof
Helicobacterpyloriinfection.WorldJGastroenterol.
2013;19:8188–8191.
18.MayoClinic.MayoMedicalLaboratories;2014.Availableat:
http://www.mayomedicallaboratories.com/articles/ hottopics/transcripts/2010/2010-8a-hpylori/8a-11.html
[accessed3.12.14].
19.RamisIB,deMoraesEP,FernandesMS,etal.Evaluationof
diagnosticmethodsforthedetectionofHelicobacterpyloriin
gastricbiopsyspecimensofdyspepticpatients.BrazJ
Microbiol.2012;43:903–908.
20.ChisholmSA,WatsonCL,TeareEL,SaverymuttuS,OwenRJ.
Non-invasivediagnosisofHelicobacterpyloriinfectioninadult
dyspepticpatientsbystoolantigendetection:doestherapid
immunochromatographytestprovideareliablealternative
toconventionalELISAkits?JMedMicrobiol.2004;53(Pt
7):623–627.
21.GattaL,PernaF,RicciC,etal.Arapid
immunochromatographicassayforHelicobacterpyloriinstool
beforeandaftertreatment.AlimentPharmacolTher.
2004;20:469–474.
22.VeijolaL,MyllyluomaE,KorpelaR,RautelinH.Stoolantigen
testsinthediagnosisofHelicobacterpyloriinfectionbefore
andaftereradicationtherapy.WorldJGastroenterol.
2005;11:7340–7344.
23.WuIC,WangSW,YangYC,etal.Comparisonofanew
office-basedstoolimmunoassayand(13)C-UBTinthe
diagnosisofcurrentHelicobacterpyloriinfection.JLabClin
Med.2006;147:145–149.
24.OzdemirM,BaykanM.EvaluationoftheHelicobacterpylori
stoolantigentest(HpSA)inthediagnosisofHelicobacter
pyloriinfectionindyspepticpatients.GenelTıpDerg.
2005;15:65–70.
25.LiYH,GuoH,ZhangPB,ZhaoXY,DaSP.Clinicalvalueof
Helicobacterpyloristoolantigentest,ImmunoCardSTAT
HpSA,fordetectingHpyloriinfection.WorldJGastroenterol.
2004;10:913–914.
26.AntosD,CroneJ,KonstantopoulosN,KoletzkoS.Evaluation
ofanovelrapidone-stepimmunochromatographicassayfor
detectionofmonoclonalHelicobacterpyloriantigeninstool
samplesfromchildren.JClinMicrobiol.2005;43:
2598–2601.
27.KrausseR,MüllerG,DoniecM.Evaluationofarapidnew
stoolantigentestfordiagnosisofHelicobacterpyloriinfection
inadultpatients.JClinMicrobiol.2008;46:2062–2065.
28.SilvaJM,VillaresCA,MonteiroMDS,ColaútoC,dosSantos
AF,MattarR.Validationofarapidstoolantigentestfor
diagnosisofHelicobacterpyloriinfection.RevInstMedTropSao
Paulo.2010;52:125–128.
29.TrevisaniL,SartoriS,RossiMR,etal.Evaluationofanew
rapidimmunoassayforthedetectionofHelicobacterpyloriin
faeces:aprospectivepilotstudy.AlimentPharmacolTher.
2005;21:485–489.
30.Kulo ˘gluZ,KansuA,Kirsac¸lio ˘gluCT,etal.Arapidlateralflow
stoolantigenimmunoassayand(14)C-ureabreathtestfor
thediagnosisanderadicationofHelicobacterpyloriinfection
inchildren.DiagnMicrobiolInfectDis.2008;62:
