Gastric mucosal status in populations with a low prevalence of Helicobacter pylori in Indonesia

15 Pages·2017·2.85 MB·English

RESEARCHARTICLE
Gastric mucosal status in populations with a
low prevalence of Helicobacter pylori in
Indonesia
MuhammadMiftahussurur1,2,3,4,IswanAbbasNusi3,FardahAkil5,AriFahrialSyam6,I.
DewaNyomanWibawa7,YudithAnnisaAyuRezkitha4,UmmiMaimunah3,
PhawineeSubsomwong1,MuhammadLuthfiParewangi6,I.KetutMariadi7,PangestuAdi4,
TomohisaUchida8,HerryPurbayu3,TitongSugihartono3,LanggengAgungWaskito1,4,
HanikBadriyahHidayati4,MariaIngeLusida4,YoshioYamaoka1,2*
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1 DepartmentofEnvironmentalandPreventiveMedicine,OitaUniversityFacultyofMedicine,Yufu,Japan,
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2 DepartmentofMedicine,GastroenterologyandHepatologySection,BaylorCollegeofMedicine,Houston,
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Texas,UnitedStatesofAmerica,3 DivisionofGastroentero-hepatology,DepartmentofInternalMedicine,
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FacultyofMedicine-Dr.SoetomoTeachingHospital,UniversitasAirlangga,Surabaya,Indonesia,4 Institute
a1111111111 ofTropicalDisease,UniversitasAirlangga,Surabaya,Indonesia,5 CenterofGastroentero-hepatology,
DepartmentofInternalMedicine,FacultyofMedicine,HasanuddinUniversity,Makassar,Indonesia,
6 DivisionofGastroenterology,DepartmentofInternalMedicine,FacultyofMedicine,UniversityofIndonesia,
Jakarta,Indonesia,7 DivisionofGastroentero-hepatology,DepartmentofInternalMedicine,Facultyof
MedicineUniversityofUdayana,Denpasar,Indonesia,8 DepartmentofMolecularPathology,OitaUniversity
FacultyofMedicine,Hasama-machi,Yufu-City,Oita,Japan
OPENACCESS
*[email protected]
Citation:MiftahussururM,NusiIA,AkilF,Syam
AF,WibawaIDN,RezkithaYAA,etal.(2017)
Gastricmucosalstatusinpopulationswithalow
prevalenceofHelicobacterpyloriinIndonesia. Abstract
PLoSONE12(5):e0176203.https://doi.org/
10.1371/journal.pone.0176203 InIndonesia,endoscopyservicesarelimitedandstudiesaboutgastricmucosalstatusby
Editor:NiyazAhmed,InternationalCentrefor usingpepsinogens(PGs)arerare.WemeasuredPGlevels,andcalculatedthebestcutoff
DiarrhoealDiseaseResearchBangladesh(icddr,b), andpredictivevaluesfordiscriminatinggastricmucosalstatusamongethnicgroupsinIndo-
BANGLADESH
nesia.Wecollectedgastricbiopsyspecimensandserafrom233patientswithdyspepsialiv-
Received:December20,2016 inginthreeIndonesianislands.When(cid:21)5.5U/mLwasusedasthebestcutoffvalueof
Accepted:April6,2017 Helicobacterpyloriantibodytiter,8.6%(20of233)werepositiveforH.pyloriinfection.PGI
andIIlevelswerehigheramongsmokers,andPGIwashigherinalcoholdrinkersthanin
Published:May2,2017
theircounterparts.PGIIlevelwassignificantlyhigher,whereasPGI/IIratioswerelowerin
Copyright:©2017Miftahussururetal.Thisisan
H.pylori-positivethaninH.pylori-negativepatients.PGI/IIratiosshowedasignificant
openaccessarticledistributedunderthetermsof
theCreativeCommonsAttributionLicense,which inversecorrelationwiththeinflammationandatrophyscoresoftheantrum.Thebestcutoff
permitsunrestricteduse,distribution,and valuesofPGI/IIwere4.05and3.55fordiscriminatingchronicandatrophicgastritis,respec-
reproductioninanymedium,providedtheoriginal
tively.PGI,PGII,andPGI/IIratiosweresignificantlylowerinsubjectsfromBanglithanin
authorandsourcearecredited.
thosefromMakassarandSurabaya,andconcordantwiththeABCgroupdistribution;how-
DataAvailabilityStatement:Allrelevantdataare
ever,groupD(H.pylorinegative/PGpositive)wasthelowestinsubjectsfromBangli.Incon-
withinthepaperanditsSupportingInformation
files(S1Table). clusion,validationofindirectmethodsisnecessarybeforetheirapplication.Weconfirmed
thatserumPGlevelisausefulbiomarkerdeterminingchronicgastritis,butamodestsensi-
Funding:Thisworkwassupportedinpartby
grantsfromtheNationalInstitutesofHealth tivityforatrophicgastritisinIndonesia.TheABCmethodshouldbeusedwithcautionin
(DK62813)andGrants-in-AidforScientific areaswithalowprevalenceofH.pylori.
ResearchfromtheMinistryofEducation,Culture,
Sports,Science,andTechnology(MEXT)ofJapan
(25293104,26640114,and15H02657)(YY).
SupportwasalsoprovidedbytheJapanSocietyfor
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GastricmucosalstatusinIndonesia
thePromotionofScience(JSPS)Institutional Introduction
ProgramforYoungResearcherOverseasVisits
Helicobacterpylorihasauniquecapacitytopersistentlycolonizetheextremelyacidicenviron-
(YY),theStrategicFundsforthePromotionof
ScienceandTechnologyfromtheJapanScience mentofthestomachandcauseprogressivegastricmucosalinflammation.Long-terminfection
andTechnologyAgency(JSTandYY).MMwasa inducesamultistephistologicalcascade,fromchronicnon-atrophicgastritisthatprogressesto
previousPhDstudent,andPSandLAWarePhD chronicatrophicgastritis,intestinalmetaplasia,andadenocarcinoma[1].Currently,chronic
studentssupportedbytheJapanesegovernment
atrophicgastritischaracterizedbychronicinflammationwithlossofgastricglandularcellsis
(MEXT)scholarshipprogramfor2012,2013,and
anestablishedprecursorlesiontogastricadenocarcinoma[2].Althoughgastricmucosal
2015,respectively.
biopsyisthereferencemethodfordeterminingthegradeandtopographicaldistributionof
Competinginterests:Theauthorshavedeclared
gastritis[3],thismethodisuncomfortableandexpensiveforpatients.Incontrast,serologyisa
thatnocompetinginterestsexist.
cheaper,acceptable,andeasilyrepeatedmethod.Recentreportsconfirmedthatserumpepsin-
Abbreviations:PGs,pepsinogens;OLGA, ogens(PGs)areavaluablebiomarkerofthegastricmucosalstatus,includinginflammation,
OperativeLinkforGastritisAssessment;ELISA,
atrophicgastritis,andgastriccancer[4],evenbeforethediscoveryofH.pylori[5].
enzyme-linkedimmunosorbentassay;AUC,area
AlthoughthemostpartofPGsaresecretedbythegastriccellsand,inlowlevels,areperme-
undercurve;ROC,receiver-operating
atedintoserum[6].PGIandPGII,thetwomaintypesofPGsareproducedindifferent
characteristic;NSAID,nonsteroidalanti-
inflammatorydrug;PPV,positivepredictivevalue; regionsofthestomach[7].SerumPGI,whichissecretedpurelyonthefundus,decreasespro-
NPV,negativepredictivevalue;OR,oddsratio;CI, gressively,whereasPGIIleveldecreaseslessmarkedlyorremainstableowingtotheadditional
confidenceinterval.
productioninnon-gastricglandssuchastheduodenalbulb;therefore,PGI/IIratiosserveasa
gastricmucosalbiomarker[7]andcanbeappliedtogastriccancerscreening[4,8,9].Thecut-
offpointsof(cid:20)70μg/Land(cid:20)3forPGIlevelandPGI/IIratio,respectively,wereusedinJapan
foridentifyingtheriskofgastriccancer[10].However,thediscriminativebaselinePGlevels
maynotbeapplicabletoothercountriesandthereforeshouldberecalculated,becauseseveral
factorsincludinggeographicarea,race,age,sex,smokinganddrinkinghabits,andH.pylori
infectionalsocontributetothelevels[11–13].Inaddition,thereisevidencethatPGIIlevel
increasesandthePGI/IIratiodecreaseswiththegradeofinflammation[14],providingan
opportunityfortheapplicationPGlevelsinpatientswithchronicgastritiswhoalsofrequent
incountrieswithalowerprevalenceofgastriccancerthanthatinJapan[10].
Asweexplainedindetailpreviously[15–19],IndonesiaisamultiethnicnationinSoutheast
Asiawitharelativelylowriskofgastriccancer(age-standardizedincidencerate[ASR]:2.8in
100,000;GLOBOCAN2012,http://globocan.iarc.fr/)withtheoverallprevalenceofH.pylori
infectioninthefivelargestislandsofIndonesiawas22.1%[17].Thepredominantethnic,Java-
nese,hasalowprevalenceofH.pyloriinfection(2.4%)[16,17],however,severalethnicgroups
haveamuchhigherriskofH.pyloriinfection[17].AccordingtoIndonesiaasthefourthmost
populouscountryworldwidewithahighprevalenceofH.pyloriantibioticresistance[19,20],
andthestrainsharbormorevirulentH.pylorigenotypes[18],consequently,H.pylori-associ-
ateddiseasesareanimportantprobleminIndonesia.Moreover,althoughdyspepsiawassixth
andfifthofthe10mostprevalentoutpatientandinpatientdiseasesinIndonesia,respectively
[21],hospitalsthatprovidegastrointestinalendoscopyservicesinthecountryareverylimited.
Therefore,noninvasivemethodssuchasmeasurementofPGvaluesarethebestchoicefor
determiningthegastricmucosalstatusinIndonesia,especiallyinremoteareas.
Toourknowledge,studiesexaminingthegastricmucosalstatusinIndonesiabyusingPG
levelsarerare[22,23].TwopreviousstudiesdeterminedtheaccuracyofPGlevelsinasingle
citythatwaspredominantlyinhabitedbytheJavanesepeople:Purwokerto[22]andJakarta
[23].ThesestudiesalsodidnotcomparethePGdatawithhistologicalgrades(e.g.,updated
Sydneysystem)[24].ThestudyperformedinPurwokertoreportedthatthespecificityofPGI
leveltopredictatrophicchronicgastritiswasonly50%,withasensitivityof70%[22].Incon-
trast,thestudyinJakartasuggestedalowsensitivity(43%)ofPGI/IIasthebiomarkerofH.
pylorichronicgastritis,withaspecificityof83%[23].Inthecurrentstudy,wemeasuredthe
PGlevelsofthreeethnicgroupsfromdifferentislands.Wealsocalculatedthebestcutoffand
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GastricmucosalstatusinIndonesia
predictivevaluesfordiscriminatingchronicandatrophicgastritisbasedonPGlevelsamong
ethnicgroupsinIndonesia.
Materialsandmethods
Studypopulation
WeperformedaprospectivestudybetweenJanuaryandAugust2015inSurabaya,JavaIsland;
Makassar,SulawesiIsland;andBangli,BaliIsland.WeexcludedsubjectswithahistoryofH.
pylorieradicationtherapyandpartial/totalgastrectomy,nonfastedsubjects,andthosewith
contraindicationforupperendoscopy.Experiencedendoscopistsacquiredtwogastricbiopsy
specimensduringeachendoscopyprocedure:onefromthelessercurvatureoftheantrum
approximately3cmfromthepyloricring,andanotherfromthegreatercurvatureofthecor-
pus,whichwereusedforhistologicalexamination.Fastingserumwascollectedonthedayof
endoscopyandthenstoredat-20˚C.Socio-demographicdataincludingbodymassindex,
smokinganddrinkinghabits,anduseofnonsteroidalanti-inflammatorydrugs(NSAIDs)
werecollectedduringtheinterview.Writteninformedconsentwasobtainedfromallpartici-
pants,andthestudyprotocolwasapprovedbytheethicscommitteesofDr.SoetomoTeaching
Hospital(Surabaya,Indonesia),Dr.WahidinSudirohusodoTeachingHospital(Makassar,
Indonesia),andOitaUniversityFacultyofMedicine(Yufu,Japan).
DeterminationofH.pyloriserologyandPGlevels
TheseparatedserawereusedforthemeasurementoftheH.pyloriantibodytitersandPGlev-
els.Theanti-H.pyloriIgGlevelswerequantifiedbyusinganELISAkit(Eiken,Co.Ltd.,
Tokyo,Japan),andthePGIandIIlevelsweremeasuredbyusingPGELISA(Eiken),accord-
ingtothemanufacturer’sinstructions.SubjectswithserumH.pyloriantibodytiters(cid:21)10U/
mLwereclassifiedasH.pyloripositiveperthemanufacturer’sinstructions.ThosewithPGI
level(cid:20)70ng/mLandPGI/IIratio(cid:20)3.0wereclassifiedasPGpositiveaccordingtotheJapa-
neseguidelines[4].AccordingtotheABCmethod,wecategorizedthesubjectsintofour
groups:H.pylorinegative/PGnegative(groupA),H.pyloripositive/PGnegative(groupB),H.
pyloripositive/PGpositive(groupC),andH.pylorinegative/PGpositive(groupD)[4].
Histologyandimmunohistochemistry
Allbiopsymaterialsforhistologicaltestingwerefixedin10%bufferedformalinandembedded
inparaffin.SerialsectionswerestainedwithhematoxylinandeosinandMay–Giemsastain.
Thedegreeofinflammation,atrophy,andbacterialdensitywereclassifiedintofourgrades
accordingtotheupdatedSydneysystem:0,normal;1,mild;2,moderate;and3,marked[24].
Sampleswithbacterialloads(cid:21)grade1wereconsideredpositiveforH.pylori.
ToincreasetheaccuracyfordetectingH.pylori,weperformedimmunohistochemicalcon-
firmation,aspreviouslydescribed[25].Briefly,afterantigenretrievalandinactivationof
endogenousperoxidaseactivity,tissuesectionswereincubatedwithanti-α-H.pyloriantibody
(DAKO,Glostrup,Denmark)overnightat4˚C.Afterwashing,thesectionswereincubated
withbiotinylatedgoatanti-rabbitIgG(NichireiCo.,Tokyo,Japan),followedbyincubation
withanavidin-conjugatedhorseradishperoxidasesolution(VectastainEliteABCKit;Vector
LaboratoriesInc.,Burlingame,CA,USA).PeroxidaseactivitywasdetectedbyusinganH O /
2 2
diaminobenzidinesubstratesolution.Tominimizepotentialbias,thesameexperienced
pathologist(TU)whoalsoperformedexperimentsforMyanmar,Vietnam,Bhutan,Domini-
canRepublic,andIndonesia[16,26–30]evaluatedallthespecimensinthisstudy.
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Dataanalyses
Discretevariablesweretestedbyusingthechi-squaretest;continuousvariablesweretested
withtheMann–WhitneyUandKruskal–Wallistest.TheSpearmanrankcoefficients(r)were
determinedtoevaluatetheassociationbetweenPGlevelsandgastricmucosalinflammation
andatrophy.AmultivariatelogisticregressionmodelwasusedtocalculatetheORsoftheclin-
icaloutcomesbyincludingage,sex,NSAIDuse,alcoholdrinking,smoking,H.pyloriinfection,
andgastritistype.AlldeterminantswithP<0.10wereenteredtogetherinthefullmodelof
logisticregression,andthemodelwasreducedbyexcludingvariableswithP>0.10.TheOR
and95%confidenceinterval(CI)wereusedtoestimatetherisk.P<0.05wasacceptedassta-
tisticallysignificant.Receiver-operatingcharacteristic(ROC)curveswereusedtocalculatethe
bestcutoff,includingtheareaundercurve(AUC)andpredictivevaluesfordiscriminating
chronicandatrophicgastritis.TheSPSSstatisticalsoftwarepackageversion18.0(SPSSInc.,
Chicago,IL,USA)wasusedforallstatisticalanalyses.
Results
H.pyloriinfectionratebasedonserology
Werecruited233patientswithdyspepsiacomprising110womenand123menwithamean
ageof46.1±12.3years(range,14–82years),including99patientsfromSurabaya,75from
Makassar,and59fromBangli(S1Table).Therewere19patientswithpepticulcers.Onthe
basisofhistologyconfirmedbyimmunohistochemistry,15patients(6.4%)werepositivefor
H.pyloriinfection.
Byusingthecutoffvaluefromthemanufacturer’sinstructions(positiveif(cid:21)10U/mL),we
foundthesensitivityandspecificityoftheELISAkitforH.pyloriinfectiontobe66.7%and
97.2%,respectively,comparedwithhistologyconfirmedbyimmunohistochemistryasagold
standard.Asthelowsensitivitywasduetoalargenumberoffalsenegatives(5of15,33.3%),
wedeterminedthebestcutoffvaluesofH.pyloriantibodytitersinELISAkitwithanROC
curve.Wefoundthatthecutoffof(cid:21)5.5U/mLwasthebestvaluetodetermineH.pyloriposi-
tivity(sensitivity,specificity,positivepredictivevalue[PPV],andnegativepredictivevalue
[NPV]:86.7%,96.8%,65.0%,and99.1%,respectively;Fig1),withanAUCof0.913(95%CI,
0.811–1.000).Whenweusedthenewcutoffvalueof(cid:21)5.5U/mL,8.6%(20of233)wereposi-
tiveforH.pyloriinfection(S1Table).TheH.pyloriinfectionratebyagegroupwas5.6%(1of
18),5.2%(3of58),10.3%(6of58),7.5%(5of67),and15.6%(5of32)forpatientsaged(cid:20)29,
30–39,40–49,50–59,and(cid:21)60years,respectively.Patientswithpepticulcerhadahigherprev-
alenceofH.pyloriinfectionthanthosewithoutulcer(5of19,26.3%vs.15of214,7.0%;
P=0.004).Accordingtolocation,Bangli(13.6%,8of59)andMakassar(12.0%,9of75)hada
higherprevalenceofH.pyloriinfectionthanSurabaya(3.0%,3of99)(P=0.01and0.02,
respectively).
Socio-demographicdataandPGlevels
WomenhadlowerPGI,PGII,andPGI/IIratiothanmen(P<0.001,P<0.001,P=0.008,
respectively).PGIandPGIIwerealsoincreasedwithage(r=0.34andr=0.32,P<0.001,
respectively),butnotPGI/IIratio.TherewasnodifferenceinthePGIandIIlevelsandPGI/
IIratiosinpatientswithgastritisandthosewithpepticulcer(P=0.10,P=0.08,P=0.85,
respectively).Whenweconsideredpatientswithanyinflammationandatrophyintheantrum
orcorpusonhistologicalexaminationasasymptomaticpatient,PGIIlevelswashigherand
PGI/IIratiowasloweramongsymptomaticthanasymptomaticpatients(15.4±11.3vs.
12.4±9.6,P=0.05and6.0±1.8vs.6.6±2.0,P=0.017,respectively).Therewasnosignificant
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Fig1.Receiver-operatingcharacteristic(ROC)curvefordeterminingtheoptimalcutoffofHelicobacter
pyloriantibodytiters.ThesensitivitydelineatedinX-axis(86.7%)andtheY-axischaracterizedvalueof
1-specificity(96.8%).PPVwasapositivepredictivevalueandNPVwasanegativepredictivevalue.
https://doi.org/10.1371/journal.pone.0176203.g001
associationbetweenthePGlevelsandNSAIDuseandbodymassindex(P>0.05).Smoking
wassignificantlyhigherinsubjectsfromMakassarandBanglithaninthosefromSurabaya
(28.0%[21of75],22.0%[13of59]vs.10.1%[10of99],P=0.002andP=0.04).PGIandII
levelswerehigherinsmokersthaninnonsmokers(114.9±87.2vs.79.1±67.6,P=0.001and
18.9±13.3vs.12.6±9.4,P<0.0001,respectively).Therewasnosignificantdifferencein
drinkinghabitsinallpopulations(P>0.05).PGIlevelwasalsohigherindrinkingthanin
nondrinkingsubjects(111.8±91.8vs.84.0±70.9,P=0.043).
H.pyloriinfectionstatusandPGlevels
PGIleveltendedtobehigherinH.pylori-positivethaninH.pylori-negativepatients
(P=0.056).Additionally,PGIIlevelwassignificantlyhigherinH.pylori-positivethaninH.
pylori-negativepatients(P<0.0001,Table1),whereasthePGI/IIratiosweresignificantly
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Table1. DemographicsandHelicobacterpyloriantibodystatus(mean±SD).
Parameter H.pyloripositive H.pylorinegative P-value
Male/female 10/10 100/113
Age 49.75±12.1 45.80±12.3 0.14
Bodymassindex(kg/m2) 21.82±2.7 23.02±3.8 0.18
PepsinogenI(ng/mL) 113.73±80.1 83.28±71.8 0.056
PepsinogenII(ng/mL) 24.09±13.9 12.83±9.6 <0.0001
PepsinogenI/IIratio 4.70±1.4 6.43±1.9 <0.0001
Chronicgastritis(%) 20/20(100.0) 69/213(32.4) <0.0001
Atrophicgastritis(%) 15/20(75.0) 45/213(21.2) <0.0001
https://doi.org/10.1371/journal.pone.0176203.t001
lowerinH.pylori-positivethaninH.pylori-negativepatients(P<0.0001,Fig2).Whenwe
consideredpatientswithanyinflammationandatrophyintheantrumorcorpusonhistologi-
calexaminationaspositiveforchronicandatrophicgastritis,respectively,theprevalenceof
chronicandatrophicgastritiswashigherinH.pylori-positivethaninH.pylori-negative
patients(bothP<0.0001).OnlyH.pyloriinfectionwasanindependentriskfactorforthe
developmentofchronicgastritisinthecorpusbutnotintheantrum(OR,13.12;95%CI,2.3–
73.8)afteradjustmentforage,sex,NSAIDuse,alcoholdrinking,andsmoking.Afteradjust-
ment,H.pyloriinfectionalsowasanindependentriskfactorforthedevelopmentofatrophy
intheantrum(OR,9.87;95%CI,1.8–54.4).
PGlevelsandchronicgastritis
Inflammationintheantrumhadacorrelationwithatrophy,similartothatinthecorpus(P<0.001,
r=0.382andP<0.001,r=0.384,respectively).Weexaminedthecorrelationbetweentheseverity
ofchronicgastritisandPGlevels(Table2).Overall,inpatientswithchronicgastritis,PGIlevel
wasalmoststableinconjunctionwiththeincreaseofPGIIlevelanddecreaseofPGI/IIratio
(P<0.05).AlthoughPGIleveldidnotcorrelatewiththeinflammationscores(P=0.55inthe
antrumandP=0.31inthecorpus),PGIIlevelshowedaweakcorrelationintheantrumandcor-
pus(bothP=0.01,r=0.16)andtheassociationwasdisappearedinthemultivariateanalysisafter
adjustedwithage,sex,smoking,drinkinghabitsandpositivityofH.pylori(P=0.21).Additionally,
PGI/IIratiosshowedasignificantinversecorrelationwiththeinflammationscoresintheantrum
andcorpus(P<0.0001,r=-0.24andP=0.001,r=-0.22,respectively).Moreover,whenweana-
lyzedonlytheH.pylori-positivepatients,thePGI/IIratiosshowedasignificantinversecorrelation
withtheinflammationscoresintheantrum(P=0.02,r=-0.61)butnotinthecorpus(P=0.52).In
themultivariateanalysis,theinverseassociationofPGI/IIratiosandantralinflammationwasstill
significantalthoughadjustedwithage,sex,smokingconsumptionanddrinkinghabits(OR,0.34;
95%CI,0.21–0.56).
Next,wedeterminedthebestcutoffvaluesfordiscriminatingchronicgastritis.Byusingthe
ROCcurve,PGIIlevelwasnotfoundtobeagooddiscriminatorymarkerforantralchronic
gastritis(datanotshown).Forstages(cid:21)IIintheinflammationscore,consideredaspositivefor
chronicgastritis,thebestcutoffvalueofPGI/IIwas5.65(sensitivity,specificity,PPV,NPV,
andaccuracy:83.3%,63.7%,16.1%,97.9%,and65.2%,respectively).TheAUCwas0.832(95%
CI,0.721–0.942).Forstages(cid:21)IIIintheinflammationscore,thebestcutoffvalueofPGI/II
was4.05(sensitivity,specificity,PPV,NPV,andaccuracy:80.0%,94.3%,23.5%,and99.5%,
respectively).Theoverallaccuracywas94.0%withanAUCof0.857(95%CI,0.664–1.000).
ThePGIbestcutoffvaluewas47.5(theAUCwere0.674[95%CI,0.585–0.762])withsensitiv-
ity,specificity,PPV,NPV,andaccuracywere80.0%,64.9%,4.8%,99.3%,65.2%,respectively
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Fig2.PepsinogenslevelsamongH.pyloripositiveandnegativepatients.PGIIlevelwassignificantlyhigherandPGI/IIratios
weresignificantlylowerinH.pylori-positivethaninH.pylori-negativepatients.
https://doi.org/10.1371/journal.pone.0176203.g002
forstages(cid:21)IIIintheinflammationscore.WhenweusedacombinationPGIandPGI/IIfor
stages(cid:21)IIIintheantralchronicgastritis,theoutcomespracticallythesameaswhenutilizing
justPGI/IIratioalthoughhadahigherPPVthanonlyPGI/IIratiomarker(sensitivity,speci-
ficity,PPV,NPV,andaccuracywere80.0%,97.3%,40.0%,99.6%,and97.0%,respectively.
PGlevelsandatrophicgastritis
AccordingtotheupdatedSydneysystem,wedeterminedthat74.3%ofpatients(173of233)
hadnomucosalatrophyinboththeantrumandcorpus,andthesepatientswerecategorizedas
thenonatrophicgastritisgroup.Inaddition,only21.9%(51of233),0.4%(1of233),and3.4%
(8of233)ofpatientshadantralatrophy,corpusatrophy,andmultifocalatrophy,respectively.
ThereweredifferencesinthePGIIlevelandPGI/IIratioaccordingtothepatternofatrophic
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Table2. Levelsofpepsinogen(PG)I,PGII,andPGI/IIinchronicandatrophicgastritis(mean±SD).
Grade n PGI PGII PGI/II
Chronicgastritis
Antrum 0 147 85.3±77.9 12.7±9.7 6.5±1.9
1 68 89.3±68.3 14.9±11.4* 6.2±2.0*
2 13 89.7±44.9 21.4±13.2* 4.6±1.4*
3 5 46.7±6.4 12.1±2.8 4.1±1.4
Corpus 0 187 84.5±74.5 12.8±9.7 6.4±1.8
1 42 90.2±66.0 17.2±12.7* 5.7±2.4*
2 4 106.5±73.1 22.5±13.5* 4.7±2.0*
3 0 NA NA NA
Atrophicgastritis
Antrum 0 174 83.5±72.0 12.8±9.7 6.5±2.0
1 53 97.4±78.5 17.2±12.8* 5.7±1.5*
2 5 55.4±10.8 13.6±4.6 4.5±1.8
3 1 18 13.8 3.1
Corpus 0 224 84.6±72.4 13.4±10.2 6.3±2.0
1 8 112.9±84.4 22.1±13.8* 5.3±1.9
2 1 168.0 32.3* 5.2
3 0 NA NA NA
*P<0.05vs.grade0.
https://doi.org/10.1371/journal.pone.0176203.t002
gastritis(P=0.009andP=0.023,respectively;Table3).Comparedwithnonatrophicgastritis,
antralatrophyhadsignificantlylowerPG/IIratio(P=0.01).Moreover,PGItendedtohave
higherlevelsandPGI/IIratiotendedtohavelowerlevelsinsubjectswithnonatrophicgastritis
thaninthosewithmultifocalatrophicgastritis(P=0.053andP=0.057,respectively).PGII
hadasignificantlylowerlevelinsubjectswithnonatrophicgastritisthaninthosewithmultifo-
calatrophicgastritis(P=0.002).
AlthoughthePGIleveldidnotcorrelatewiththeatrophyscores(P=0.49intheantrum
andP=0.11inthecorpus),thePGIIlevelandPGI/IIratiosshowedasignificantcorrelation
intheantrum(P=0.02,r=0.16andP=0.002,r=-0.20,respectively;Table2)andonlyPGII
showedasignificantcorrelationinthecorpus(P=0.02,r=0.16).However,therewasnocor-
relationbetweenthePGlevelsandatrophyscoresamongH.pylori-positivepatients.Thismay
beduetothelownumberofH.pylori-positivepatientswithatrophicgastritis.Whenweana-
lyzedonlyH.pylori-negativepatients,thePGIandPGIIlevelsshowedaweakcorrelation
withtheatrophyscoresinthecorpus(P=0.04,r=0.14andP=0.03,r=0.15,respectively).
Table3. Pepsinogenlevelaccordingtoatrophicgastritispattern.
Pepsinogens NoAtrophy Antralatrophy Corpusatrophy Multifocalatrophy P-value
N 173 51 1 8
PepsinogenI(ng/mL) 83.72±72.1 87.42±73.9 133.0 128.41±81.3 0.24
PepsinogenII(ng/mL) 12.79±9.7 15.53±11.7 17.9 25.30±12.6*,** 0.009
PepsinogenI/IIratio 6.51±2.0 5.67±1.5* 7.4 5.01±1.9 0.023
Multifocalatrophymeansthesubjectshadatrophyinboththeantrumandcorpus.
*P<0.05vs.noatrophy.
**P<0.05vs.antralatrophy.
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WhenweusedtheJapanesestandardstodefinethePG-positivestatus(cutoffvalues:(cid:20)70
ng/mLand(cid:20)3.0forPGIlevelandPGI/IIratio,respectively),wefoundthattheoverallpreva-
lenceofPG-positivesubjectswasverylow(1.3%,3of233).Furthermore,whenweusedthe
cutoffvalueonlyforPGI/IIratios(cid:20)3.0,thesensitivity,specificity,PPV,NPV,andaccuracy
were5.1%,99.4%,75.0%,75.6%,and75.5%,respectively,forantralatrophicscores(cid:21)1.In
caseswithanantralatrophicscore(cid:21)2,thevalueswere16.7%,98.7%,25.0%,97.8%,and96.6%,
respectively.
OwingtothelowsensitivityoftheJapanesestandard,wedeterminedthebestcutoffvalue
ofPGI/IIratiowithanROCcurve.Forstages(cid:21)Iintheantralatrophicscore,consideredas
atrophypositive,thebestcutoffvalueofPGI/IIratiowas6.05(sensitivity,specificity,PPV,
NPV,andaccuracy:61.0%,55.2%,31.6%,80.7%,and56.7%,respectively).TheAUCwas0.629
(95%CI,0.544–0.713).Forstages(cid:21)IIintheantralatrophicscore,consideredasatrophyposi-
tive,thebestcutoffvalueofPGI/IIwas3.55(sensitivity,specificity,PPV,NPV,andaccuracy:
66.7%,96.5%,33.3%,99.1%,and95.7%,respectively),andtheAUCwas0.793(95%CI,0.580–
1.000).
ThePGIandPGIIbestcutoffvalueforstages(cid:21)IIintheantralatrophicscorewere65.0
(theAUCwere0.583[95%CI,0.482–0.684]withsensitivity,specificity,PPV,NPV,andaccu-
racywas83.3%,47.1%,96.0%,99.1%,48.1%,respectively.whilePGIIlevelwasnotobserved
tobeasufficientmarkerforantralchronicgastritisbyusingtheROCcurve(datanotshown).
WhenweusedacombinationPGIandPGI/IIratioforstages(cid:21)IIintheantralatrophicscore,
theresultsalmostthesameaswhenusingonlyPGI/IIratio(sensitivity,specificity,PPV,NPV,
andaccuracywere66.7%,97.8%,44.4%,99.1%,and96.9%,respectively).
PGlevelsandtheABCmethodamongethnicgroups
ThecomparisonofPGlevelsamongtheethnicgroupsisshowninTable4.AmongtheH.
pylori-negativesubjects,PGIandIIlevelsweresignificantlylowerinsubjectsfromBangli
thaninthosefromMakassar(bothP<0.001).Furthermore,thePGI/IIratiosweresignifi-
cantlylowerinsubjectsfromBanglithaninthosefromMakassar(P=0.03).Subjectsfrom
BanglialsohadsignificantlylowerPGI/IIratiosthanthosefromSurabaya(P=0.004).
AlthoughthePGIandIIlevelsinsubjectsfromSurabayaweresignificantlylowerthanin
thosefromMakassar,thedifferenceinPGI/IIratioswasnotsignificant(Table4).Thesimilar
resultsalsoshowedamongtheH.pylori-positivesubjects,PGIandIIlevelsweresignificantly
lowerinsubjectsfromBanglithaninthosefromMakassar(56.3±18.8vs.165.4±87.6,
P=0.007and14.5±6.5vs.31.6±14.3,P=0.012,respectively).However,therewasnodiffer-
enceforthePGI/IIratiosandPGslevelsbetweenSurabayaandMakassarsubjects.
Table4. Characteristicsofpepsinogensamongtheethnicgroups.
Pepsinogens Surabaya Makassar Bangli P*value P**value P***value
N 96 66 51
PGI(ng/mL) 79.5±58.1 113.2±96.1 61.3±54.2 0.01 0.07 <0.001
PGII(ng/mL) 12.0±8.1 17.3±13.0 10.6±9.0 0.004 0.36 <0.001
PGI/IIratio 6.6±1.8 6.5±2.4 5.9±1.3 0.56 0.004 0.03
PG,pepsinogen.
*Surabayavs.Makassar.
**Banglivs.Surabaya.
***Banglivs.Makassar.
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GastricmucosalstatusinIndonesia
Table5. ABCmethodamongethnicgroups.
ABCgroups Surabaya(%) Makassar(%) Bangli(%)
GroupA 94/99(94.9) 63/75(84.0) 50/59(84.7)
GroupB 3/99(3.0) 7/75(9.3) 4/59(6.8)
GroupC 0/99(0.0) 2/75(2.7) 4/59(6.8)
GroupD 2/99(0.2) 3/75(4.0) 1/59(1.7)
https://doi.org/10.1371/journal.pone.0176203.t005
Byusing(cid:21)5.5U/mLand(cid:20)3.55asthebestcutoffvaluesofH.pyloriantibodytitersandPG
I/IIratio,respectively,wedeterminedthedistributionofthefourgroupsineachagecategory,
accordingtotheABCmethod.Overall,groupA(207of233,88.8%)wasthemostpredominant,
followedbygroupsB(14of233,6.0%),C(6of233,2.6%),andD(6of233,2.6%).Acomparison
oftheABCmethodamongtheethnicgroupsisshowninTable5.Consistentwiththeresults
obtainedforPGI/IIratios,subjectsfromBanglihadthehighestproportionofgroupCandlow-
estproportionofgroupAcomparedwiththosefromMakassarandSurabaya.Interestingly,the
percentageofgroupDinsubjectsfromBangliwasthelowestamongallgroups.
Discussion
AlthoughtheELISAkitforserology,whichwasdevelopedbyusingJapaneseH.pyloristrains,
itreachedahighsensitivityandspecificitywhenusedinJapanandNepal(95.2–100%and
76.2–80.0%[31]and89.1%and83.5%[32],respectively),weconfirmedthelowaccuracyof
theELISAkitwhenusedintheIndonesianpopulation.Therefore,wecalculatedthebestcutoff
valuesofIgGELISA.ThesamekitsalsoshowedalowsensitivityinMyanmar[33]andalow
specificitywhenusedintheBhutanpopulation[34].H.pyloriantibodytitersvarygreatly
dependingonthetestkitused[35],andingeneralshowedalowerperformanceingeneral
population,butanoutstandingresultontheselectedsamples[36].ThediscrepancyofH.
pyloripositivitynumberusingELISAandhistology-immunohistochemistryassociatedwith
theadvantagesanddisadvantagesofseveraltests,thustheselectionofthetestshouldbeper-
formedbasedontheseconsiderations[37,38].Importantly,thelowPPVofELISAindicated
thatalthoughthekitscouldbecomethefirstdiagnostictest,itshouldbeconfirmedwithother
testinpositivecase,especiallywithahighPPVvaluetest.TheMaastrichtIIIandIVconsensus
suggestedthat“someserologicaltestswithgoodsensitivityandspecificitycanbeusedtoper-
formtheinitialdiagnosisofinfectionwithH.pylori”[39],andonlyvalidatedcommercialtests
shouldbeused[38,40].Thus,anappraisalofvalidationhastobeperformedoneveryserology
testtotheparticularstudypopulation,andtheantigenhastobewiselyselected[41].Itmight
beimportanttodevelopELISAkitsusingnativeH.pyloristrainstothestudypopulation.
Similartoourresults,previousstudies[42,43]reportedthatPGlevelshaveapositivecorre-
lationwithaging,particularlyinmen,whichmayberelatedtotheincreaseofrenalfunction
lossandinfluenceofhormones[44,45].Inconcordancewithourdata,smokingwasreported
tostimulatePGsecretion[13].However,long-termstimulationmayexhaustthechiefcells
andpromoteatrophyofthegastricmucosa,reflectedaslowerPGI/IIratiosinsmokers[46].
Inourstudy,drinkinghabitsincreasedthePGIandIIlevels,incontrasttopreviousresults
[13].OurresultsalsoconfirmedtheinvolvementofH.pyloriasakeyfactorinthedevelopment
ofchronicandatrophicgastritisevenafteradjustmentforotherfactors.H.pyloriinfection
inducesmucosalinflammationandthelipopolysaccharideofbacteriadirectlystimulatesPG
secretion[47],whichsignificantlydecreasesaftereradicationconcomitantwiththeimprove-
mentininflammationscores[48].Thestimulationismediatedbycalcium,whichincreases
histamineanddibutyryl-cyclicadenosinemonophosphate[49].
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