Rational application of the new European Society for Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) 2020 criteria for the diagnosis of coeliac disease

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SPECIAL ARTICLE

Rational application of the new European Society for Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) 2020 criteria for the diagnosis of coeliac disease ^夽

Enriqueta Román Riechmann

, Gemma Castillejo de Villasante

, M. Luz Cilleruelo Pascual

, Ester Donat Aliaga

, Isabel Polanco Allué

, Félix Sánchez-Valverde

, Carmen Ribes Koninckx

aUnidaddeGastroenterologíayNutriciónPediátrica,HospitalUniversitarioPuertadeHierro-Majadahonda,Madrid,Spain

bUnidaddeGastroenterologíaPediátrica,ServiciodePediatría,HospitalUniversitariSantJoandeReus,Reus,Spain

cUnidaddeGastroenterología,HepatologíayNutriciónPediátrica,HospitalUniversitarioyPolitécnicoLaFe,Valencia,Spain

dFacultaddeMedicina,UniversidadAutónomadeMadrid,Madrid,Spain

eUnidaddeGastroenterologíayNutriciónPediátrica,ServiciodePediatría,ComplejoHospitalariodeNavarra,Pamplona,Spain

Received15October2019;accepted5December2019 Availableonline31January2020

KEYWORDS Coeliacdisease;

Diagnosis;

Recommendations;

Serology;

HLA;

Intestinalbiopsy;

ESPGHAN

Abstract Coeliacdiseaseisasystemicimmune-mediateddisordertriggeredbytheingestion ofgluten,whichisgiveningeneticallypredisposedsubjects.Itmanifestswithawidevariety ofclinicalsymptoms,specificserologicalmarkers,HLA-DQ2/DQ8haplotype,andenteropathy.

ThecriteriafollowedforthishaveusuallybeenthoseestablishedbytheEuropeanSocietyfor Paediatric Gastroenterology,HepatologyandNutrition(ESPGHAN)since1969. Thesecriteria haveadvancedfromtheneedofseveralintestinalbiopsiesto,thankstothedevelopmentof serologicaltests ofhighsensitivityandspecificity,consideringtheenteropathyasonemore element inthisdiagnosisandmakesitpossibletoperform adiagnosiswithoutthe needof anintestinalbiopsyincertaincircumstances.Theupdatedreviewofthe2012criteriain2019 providesnewevidenceonsomeaspects,suchastheroleofHLA,thediagnosisofasymptomatic patients,andtheeffectivenessoftheserologicalmarkers.Theseaspectsarereviewedindetail, withtheaimoffacilitatingtherationalapplicationofthenew2020criteriaatallcarelevels.

In this sense,Paediatric Primary Careis fundamentalinthe searchfor active casesand to

夽 Pleasecitethisarticleas:RiechmannER,VillasanteGCd,PascualMLC,AliagaED,AlluéIP,Sánchez-ValverdeF,etal.Aplicaciónracional delosnuevoscriteriosdelaEuropeanSocietyforPaediatricGastroenterology,HepatologyandNutrition(ESPGHAN)2020paraeldiagnóstico delaenfermedadcelíaca.AnPediatr(Barc).2020;92:110.

∗Correspondingauthor.

E-mailaddress:[email protected](E.R.Riechmann).

2341-2879/©2019Asociaci´onEspa˜noladePediatr´ıa.PublishedbyElsevierEspa˜na,S.L.U.ThisisanopenaccessarticleundertheCCBY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).

performafirstserologicalstudy,beingrecommendedthatthediagnosisisalwaysestablished byaPaediatricGastroenterologist.

©2019Asociaci´onEspa˜noladePediatr´ıa.PublishedbyElsevierEspa˜na,S.L.U.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/

4.0/).

PALABRASCLAVE Enfermedadcelíaca;

Diagnóstico;

Recomendaciones;

Serología;

HLA;

Biopsiaintestinal;

ESPGHAN

AplicaciónracionaldelosnuevoscriteriosdelaEuropeanSocietyforPaediatric Gastroenterology,HepatologyandNutrition(ESPGHAN)2020paraeldiagnósticodela enfermedadcelíaca

Resumen Laenfermedadcelíacaesunprocesosistémicodecarácterinmunológico,desen- cadenado porelconsumode gluten,que sedaensujetosgenéticamentepredispuestos.Se expresaconunagranvariedaddesíntomasclínicos,marcadoresserológicosespecíficos,hap- lotipoHLA-DQ2/DQ8yenteropatía.Eltratamientoconsisteeneliminardeporvidaelglutende ladieta,porloqueesfundamentalundiagnósticoadecuado.Loscriteriosseguidosparaello hansidohabitualmentelosestablecidosporlaEuropeanSocietyforPaediatricGastroenterol- ogy,HepatologyandNutrition(ESPGHAN)desde 1969.Estoscriterios hanidoevolucionando desdelanecesidaddevariasbiopsiasintestinalesparaeldiagnósticoa,graciasaldesarrollo depruebasserológicasdealtasensibilidadyespecificidad,considerarlaenteropatíacomoun elementomásenestediagnósticoyposibilitar endeterminadascircunstanciasrealizarlosin necesidaddebiopsiaintestinal.Larevisiónactualizada en2019deloscriterios2012aporta nuevaevidenciasobrealgunosaspectos,comoelpapeldelHLA,eldiagnósticodelospacientes asintomáticosylaeficaciadelosmarcadoresserológicos.Estosaspectosserevisanendetalle, conelobjetivodefacilitarlaaplicacióndelosnuevoscriterios2020deunaformaracionalen todoslosnivelesasistenciales.EnestesentidoelpediatradeAtenciónPrimariaesfundamental paralabúsquedaactivadecasosyrealizarunprimerestudioserológico,recomendándoseque eldiagnósticoseasiempreestablecidoporunpediatragastroenterólogo.

© 2019Asociaci´on Espa˜nola dePediatr´ıa. Publicado por Elsevier Espa˜na, S.L.U.Este es un art´ıculoOpenAccessbajolalicenciaCCBY-NC-ND(http://creativecommons.org/licenses/by- nc-nd/4.0/).

Definition of coeliac disease

BasedonthemostrecentdefinitionoftheEuropeanSociety for Paediatric Gastroenterology, Hepatologyand Nutrition (ESPGHAN),¹ coeliac disease (CD) is an immune-mediated systemicdisorderelicitedbytheconsumptionofglutenand relatedprolamins(secalins,hordeinsandpossiblyavenins) in genetically susceptible individuals (human leukocyte antigen[HLA]haplotype).Itmanifestswithavariablecom- binationofclinicalsymptoms,specificserologicalmarkers, aHLA-DQ2/DQ8haplotypeandenteropathy.

Treatment consistsinthe life-longstrictelimination of gluten fromthe diet, which achieves resolution of symp- toms, negative titres of autoantibodies and histological recoveryoftheintestinalmucosa.

Evolution of diagnostic criteria

In 1969, the ESPGHAN established the first stringent cri- teriafordiagnosis of CDin thepaediatric population,the so-calledInterlakencriteria²(Table1).Theyestablishedas mandatorycriteriaevidenceofvillousatrophyintheinitial intestinalbiopsy(IB)whilethepatientconsumesagluten- containingdiet, histological recovery after elimination of glutenandrecurrenceofthelesionafterglutenreintroduc-

tion(challengetest)todifferentiateCDfromother causes ofenteropathy.

Theapplicationofthesecriteriaalongwiththediscov- eryintheearly1980sofamarkerofactiveCD,antigliadin antibodies(AGAs),³promptedtheirrevision,leadingtothe establishmentofthenew1990ESPGHANcriteriafordiag- nosisof CD.⁴These criteriaallowedmakingthediagnosis in older children with symptoms suggestive of CD after a single IB withcharacteristic (albeitnonspecific) abnormal mucosalhistologyinthecontextofagluten-containingdiet andimprovementofsymptomswithexclusionofglutenfrom thediet.Theglutenchallengewastobereservedforchil- drenagedlessthan2yearsatthetimeoftheinitialIB,cases diagnosedwithoutanIBandcasesofuncertaindiagnosis.

Endomysial antibodies (EMAs)⁵ were discovered in the mid-1980s,andtissuetransglutaminase(tTG)wasidentified in1997astheautoantigenofCD,followedbythediscovery of thepresence of circulatinganti-tTG type2antibodies.

Bothtypesofantibodieshadasimilarsensitivityandspeci- ficity and performed better asmarkers of active CD than AGA.⁶

The21stcentury:anewscenario

The 1990 ESPGHAN criteria continued to be in use until numerous studies demonstrated the excellent correlation

Table1 EvolutionofESPGHANcriteriafordiagnosisofCD.^1,2,4,13

1969criteria 1990criteria 2012criteria 2020criteria

•3IBsrequiredfordiagnosis. •AtleastoneinitialIBis necessaryfordiagnosis.

•Insymptomaticpatients withananti-tTGIgAtitre

>10xULN,positiveEMA(in asecondserumsample)and HLADQ2/DQ8,CDcanbe diagnosedwithoutthe initialIB.

•Insomeasymptomatic patientsmeetingthe establishedcriteria,the diagnosisofCDcanbemade omittingtheinitialIB.

•Glutenchallengetest requiredinalmostevery case.

•Theglutenchallengetest isrequiredinallchildren agedlessthan2yearsorin caseofuncertaindiagnosis.

•Theglutenchallengetest isonlyrequiredincaseof uncertaindiagnosis.

•HLAtestingisnot necessaryfortheno-biopsy diagnosis.

InpatientswithIgA deficiencyorT1D,IBis required.

Timeelapsedtoconfirmationofdiagnosisbasedontheappliedcriteria:

•Inallcases,diagnosisis confirmedafter5---6yearsof followup.

•Inmostchildren,diagnosis isconfirmedafter5---6years offollowup.

•Inmostcases,diagnosisis confirmedafter1---3months offollowup.

•Inmostcases,diagnosisis confirmedafter1---3months offollowup.

CD,coeliacdisease;EMA,endomysialantibodies;IB,intestinalbiopsy;T1D,type1diabetesmellitus;tTG,tissuetransglutaminase;ULN, upperlimitofnormal.

betweenanti-tTGantibodytitresandthedegreeofvillous^6,7 andthestrongperformanceofEMAtitresforpredictionof futurelesions.⁸Thisledtoquestioningtheroleofthetra- ditional histological examination asthe gold standard for diagnosis of CD^8,9 and the need for a gluten challengein childrenagedlessthan2years,asmostofthosewithvillous atrophyandpositiveEMAtitresexperiencedarecurrenceof symptomswiththechallenge.⁹

Asaresultofthegatheredevidence,thenewESPGHAN guidelines for the diagnosis for Coeliac Disease in chil- drenand adolescents.Anevidence-based approach¹ were published in 2012,with a novel definitionof CD in which enteropathy was just one of the elements to considerin the diagnosis, and not a requisite criterion. The guide- linescontemplateddiagnosiswithoutperformanceofanIB incertain cases(suggestivesymptoms,high titres ofanti- tTGantibodies,positiveEMAantibodiesinasecondsample and compatibleHLA haplotype). Evidencefrom the years thatfollowedconfirmedthatdiagnosiswithoutabiopsywas safeinthesecases,^10---12 andthelatestupdateofthe2012 guidelines, published in 2020, presents new evidence on certainaspectsofCD,suchastheroleofHLA,thediagno- sisofasymptomaticpatientsandtheefficacyofserological markers.¹³ Wewillnowreviewtheseaspectsindetailwith theaim offacilitatingtherational applicationof thenew 2020criteriaintheoutpatientandinpatientsettings.

Role of clinical manifestations

ClinicalsignsandsymptomsassociatedwithCD(Table2)are thefeaturesthat,onappearing,leadtosuspicionofCDand theneedfordifferentialdiagnosis.

The information available on the clinical presentation of CD hasexperienced drastic changes withthe develop- mentofhighlysensitiveandspecificserologicaltests.New

Table 2 Situations in which CD should be investigated (adaptedfromtheESPGHAN2020guidelines).¹³

Inchildrenandadolescentswithanyofthefollowing symptomsiftheaetiologyisunknown:

•Gastrointestinalsymptoms:

Chronicorintermittentdiarrhoea Abdominaldistension

Recurrentnauseaorvomiting Chronicabdominalpain Chronicconstipation

•Extraintestinalsymptoms

Failuretothrive,weightloss,stuntedgrowth Iron-deficiencyanaemia

Abnormalresultsofliverfunctiontests Recurrentaphthousstomatitis

Shortstature

Delayedpuberty,amenorrhoea Dermatitisherpetiformis Chronicfatigue,irritability

Bonefracturefrommildtrauma/osteopenia/osteoporosis Neuropathy

Arthritis,arthralgia Dentalenameldefects

Inchildrenandadolescentsbelongingtoanyofthe followingat-riskgroups:

•First-degreerelativewithCD

•DiabetesmellitustypeI

•Autoimmunethyroiddisease

•Autoimmuneliverdisease

•Downsyndrome

•Turnersyndrome

•Williamssyndrome

categoriesofpatientshavebeen identifiedthat havemild gastrointestinal symptoms, symptoms considered atypical (extraintestinalmanifestations)ornosymptoms,aswellas factorsthatincreasetheriskofCD,whichhasledtochanges intheclinicalformsofdiseasecurrentlydiagnosed,withan increase in atypical and silent forms. Coeliac diseasehas gonefrombeing considered a paediatric diseaseto being diagnosedthroughoutthelifespan.¹⁴

Whenitcomestothespecificityofitsfeatures,arecent studyinacohortofpatientswithpositiveserologicaltests forCD found thatthecombination of aclinical pictureof malabsorption (chronic diarrhoea, weight loss, failure to thriveand anaemia)and high titres of specific antibodies (anti-tTGantibody titre>10 timestheupperlimitof nor- mal[ULN]andapositive EMAresult)hada 100%positive predictivevalue for intestinalinvolvementcomparedtoa slightly lowervalue ifthe antibody titres were combined withsymptomsotherthanmalabsorption.¹²

Screening for CD should also be performed in spe- cific situations that carry an increased risk for it, such as autoimmune disease, certain chromosomal disorders, IgA deficiency and historyof CD in a first-degree relative (Table2).¹³

Ontheother hand,thenotionofuniversalscreeningin thegeneralpopulationremainscontroversial,asCDdoesnot fulfilthecriteriaestablishedbytheWorldHealthOrganiza- tionforscreeningtests.Differentgroupsofexpertssupport therecommendationoftheESPGHANofactivecasefinding, giventhelackofevidenceontheoutcomesofasymptomatic patientsthatdonotreceiveadiagnosis.^15,16

Role of serology

Giventhehighaccuracyoftheantibodytestsusedfordiag- nosisof CD,serologicaltesting isrecommendedfor initial screeningofpatientswithsuspectedCD.

Antitransglutaminaseantibodies

TheinitialtestintheevaluationofCDshouldbetheanti-tTG antibodytestonaccountofitshighsensitivityandspecificity aswellasitswideavailabilityandtheuseofanautomated andobjectivemethod(enzymeimmunoassay).Arecentsys- tematicreviewwithmeta-analysis¹⁶ found a sensitivityof 92.8% (95% confidence interval[CI],90.3 %---94.8 %)and aspecificity of 97.9% (95 % CI,96.4 %---98.8%). Titres of anti-tTGIgAgreaterthan10timestheULNpredictthepres- enceofvillouslesionswithahighspecificity.¹⁷ Therehave beenreportsofoccasionalfalsepositiveresults,usuallyat lowtitres, due tocross-reactivitywithantibodies present inother clinicalconditions,suchasautoimmunediseases, liverdiseasesorinfections.¹⁸

Endomysialantibodies

Endomysial antibodies react with the endomysium, the perivascularconnectivetissuethatlinessmoothmusclebun- dles.Theytargettransglutaminaseintissuessuchasmonkey oesophagusorumbilicalcordtissue,whichareusedassub- stratesinthetestsdeveloped fortheirmeasurement.The

diagnosticaccuracyofEMAIgAisveryhigh,¹⁹withahighsen- sitivityandspecificity.¹³Themaindrawbackofthismarker isthatitismeasuredbymeansofindirectimmunofluores- cence, a semiquantitative and subjective method that is moreexpensive,requiresexperiencedstaffandtakeslonger thanthemeasurementofanti-tTGantibodies.

Antibodiesagainstdeamidatedformsofgliadin peptides

Gliadinpeptidesaredeamidatedbytissuetransglutaminase, whichresultsinasignificantincreaseintheirimmunogenic- ity.Thisinspiredthedevelopmentofmethodsfordetection of deamidated gliadin peptide (DGP) antibodies, which have sincereplacedpreviously usedtestsfordetection of antigliadinantibodies.OneadvantageofDGPantibodies is that they are detected by an objective method, enzyme immunoassay. The sensitivityof DGPIgA and IgGantibod- iesis87.8%(95%CI,85.6%---89.9%)and94.1%(95%CI, 92.5%---95.5%),respectively.¹⁶TestingforDGPIgGantibod- iesisusefulfor evaluationofCDinpatientswithselective IgA deficiency.²⁰ However,theyield is lower comparedto positiveanti-tTGIgAtitresandisolatedpositiveDGPresults maygiverisetofalsepositivesthatincreasethefrequency ofunnecessaryendoscopies.^21---23

Rapidtests

They areperformed bymeans of immunochromatography, and therefore arenot quantitative. They involveapplica- tionoffewdropsofcapillarybloodonasolidsupport,and astheblooddiffusesthroughthematrixalinebecomesvis- ibleinthetest windowiftheresultispositive.Thereare rapidtestsfordetectionofanti-tTGtype2,AGAoracombi- nationofseveralantibodies.Thesetestshavebeenusedfor non-invasivescreeningofCDandexhibitedagoodcorrela- tionwithstandardserologicaltestsaslongasthetestsare interpretedbyadequatelytrainedstaff.Thesensitivityand specificityofthesetestsare94.0%(95%CI,89.9%---96.5%) and94.4%(95%CI,90.9%---96.5%)respectively,although currentlytherearenotenoughdatatosupporttheirusein everydayclinical practice,sotheirresultsmust alwaysbe confirmedbystandardserologicaltesting.²⁴

Limitationsofserologicaltests

Althoughthemeasurementofanti-tTGIgAantibodiesisnot standardised,mostcommerciallyavailabletestsarehighly accurate,especiallyathightitres.²⁵However,thereisevi- dence of variability between different tests or different laboratoriesusingthesametestwhenitcomestomoderate anti-tTGtitres.¹² Laboratories mustbeextremely rigorous intheirinternalqualitycontrolmeasures,accuratelycalcu- latingthecalibrationcurve,whichshouldincludethevalue of10timestheULN.¹³

Serologicaltestingmustbeperformedwhilethepatient continues toconsume gluten, asantibody titres decrease after initiation of a low-gluten or gluten-free diet. If a patientwasonagluten-restricteddietduringthediagnos- ticevaluation,expertsrecommendconsumptionofanormal

dietincludingatleast3slicesofbreadadayfor1---3months tothenrepeatthemeasurementofanti-tTGantibodies.²⁶

The initialanti-tTGIgA measurementshouldbeaccom- panied by measurement of total serum IgA and, in case ofdetectionofselectiveIgAdeficiency,thepatientshould befurtherevaluatedwithperformanceofanti-tTG,EMAor PDGIgGtests.^1,13Incontrasttoanti-tTGIgAtitres,nostud- ieshavebeenconductedtoestablishthelevelsofanti-tTG IgGthatcanreliablypredictthepresenceof enteropathy.

Thus,performance of an IB isnecessary in thesechildren to confirm the diagnosis.¹³ Other possible causes of false negatives are immunosuppressive therapy and dermatitis herpetiformis, usually associated with negative serology.

Measurement in haemolysedspecimens mayalsoreflect a falsedecreaseinantibodytitres.²⁷

The evidenceis alsoinsufficientasregardsthecorrela- tionofhighanti-tTGtitreswiththepresenceofenteropathy in children with type 1 diabetes (T1D). In addition, high titres ofanti-tTGantibodies havebeen describedin stage 1 of T1D with spontaneous normalization of CD serology at moderatetitres,^28,29 soperformanceof an IBis recom- mendedfordiagnosisofCDinthesepatients.

Role of genetic testing

Coeliacdiseaseisassociatedwithaspecifichigh-risk vari- ants of the HLA class II region, whose alleles encode molecules involved in the presentation of gluten peptide antigenstoCD4⁺Tcells,thusplayingaroleintheimmune cascadethatresultsinglutenintolerance.Morethan90%of patientswithCDarefoundtobehomozygousorheterozy- gous for the HLADQ2.5 variant encoded by the DQA1*05 (␣-chain) andDQB1*02(␤-chain) genes.The isolatedHLA- DQ2.2haplotype(DQA1*02-DQB1*02) carriesamuchlower risk, for while the resulting heterodimer is homologous to the DQ2.5 heterodimer it forms less stable complexes withthepeptides.PatientsthatdonothavetheDQ2type havetheDQ8type,encodedbytheDQA1*0301-DQB1*0302 alleles.¹Thereareseveralcombinationsofthe2allelesthat carryaspecificriskof developingCD.³⁰ ClassII HLAgenes accountfor40%ofthegeneticriskofCD,whileotherparts ofthegenomehavebeenshowntohaveaninfluenceofless than 10%.³¹ Inrecent years,the complexityof the inter- pretationofHLAtypinghasledtoeffortstostandardisethe nomenclature.³²

High-riskHLAhaplotypesarerelativelyfrequentinsome populations,whichisassociatedwithahigherincidenceof CDifconsumptionofwheatandothergrassesiscustomary inthepopulation,asisthecaseinSpain.³⁰Inthissense,HLA genotypingisoflittleusefordiagnosis,asithasaverylow positive predictive value. However,its high negative pre- dictivevalue makesitveryusefulin rulingoutCD incase ofuncertaindiagnosisandtoestablishtheriskofdevelop- ingCD inpopulationgroups withahigh prevalenceof the disease.12,19,33,34

Recent studieshavereportedthatnearly100%ofindi- viduals with anti-tTG antibody titres above 10 times the ULN, apositive EMAresultand an abnormalmucosahave aDQ2/DQ8haplotype,whichmeansthatHLAtypingwould notbenecessarytoconfirmthediagnosiswithoutanIB,^12,19

asonlyindividuals withDQ2/DQ8 haplotypes canproduce theseantibodies.³⁵

Duetoalloftheabove,itseemsreasonabletoconsider thattheinterpretationoftheHLAhaplotypeandtheassoci- atedriskisjustoneamongmanytoolsinthemanagementof CDandneedstobeperformedbypaediatriciansspecialised inpaediatric gastroenterology andwithexperiencein the managementofCD,^1,13andthereforewedonotrecommend HLAgenotyping for initial screening of CD at the primary carelevel.

Role of intestinal biopsy

Anintestinal biopsy is nolonger required for diagnosis of CDineverypatient,¹ butitunarguablycontinuestobean essentialtool.

The interpretation of histological findings poses some challenges and therefore may vary between pathologists.

Tooptimisetheanalysisoftheintestinalmucosa,werecom- mendobtainingseveralbiopsysamplesthroughendoscopy, withatleast4specimensofduodenalmucosaand1ofthe duodenal bulb, asin some casesthis is the only affected area.^1,13

Astandardisedprotocolmustbeusedtoobtaincorrectly oriented cuttings of duodenal tissue. A villus height and cryptdepthratiooflessthan2inat least1ofthebiopsy samplesisconsidereddiagnosticofCD.³⁶ Theschemeused mostwidelytoclassifythedegreeofmucosalinjuryisthe MarshclassificationmodifiedbyOberhüber³⁷(Table3).The diagnosisof CDrequires thepresenceof Marsh2 or3 his- tological changes, although no degree of enteropathy is pathognomonic.

Itisimportanttoincludethefollowinginformationinthe pathologyreport:

• Whethertheamountoftissueinthesamplewassufficient

• Whetherthe specimenswerewellorientedandallowed assessmentofvillousheight

• Percentageof intraepitheliallymphocytes(IELs)in rela- tiontothetotalepithelialcellcount

• Presenceofabsenceofcrypthyperplasia(mitosis)

• Degreeofvillousatrophy.

The presence of Marsh 1 changes (a density of more than 25 IELs per 100 epithelial cells) is not sufficient to diagnoseCD,asthisamountofinjurycanbeattributedto other causes, suchas foodallergy, infection (viralaetiol- ogy,Giardia,Cryptosporidium,Helicobacterpylori),drugs, autoimmune disease, inflammatory bowel disease, etc.³⁸ Mild changes can also be found in patients with possible CD(patients with mild Marsho or 1 changesand positive anti-tTG and EMA results combined with a high-risk HLA haplotype),in whom prescription of a gluten-free diet is controversial.³⁹ Inthesecases,itmaybeusefultoanalyse thecytometricpatternofIELsubpopulations(elevationof CD3+TCR␣␤ Tcells associatedwitha decreasein NK-like cells)orthepresenceofanti-tTGantibody depositsinthe mucosa.^13,39

The abovenotwithstanding, insomecasesthereis still disagreementbetweentheresultsofanti-tTGantibodytest- ingandhistologicalfindings.Insuchcases,werecommend

Table3 Marsh---Oberhüberclassification.³⁷

Stage0Preinfiltrative Histologicallynormalmucosa

Stage1Infiltrative IncreasedIELswithpreserved

villousarchitecture

Stage2Hyperplastic Hyperplasticcryptsand

increasedIELs

Stage3Destructive Villousatrophywith

hyperplasticcryptsand increasedIELs

SubdivisionstablishedbyOberhüber basedonthedegreeofVA:

3apartialVA 3bsubtotalVA 3ctotalVA

Stage4Hypoplastic Irreversiblevillousatrophy Doesnotrespondtogluten-freediet, screenforestablishmentofmalignant T-cellcloneintheintestinaltract.

IEL,intraepitheliallymphocyte;VA,villousatrophy.

a histological re-evaluation by examination of additional biopsyspecimensorgettingasecondopinionfromanexpert pathologist.¹³

European Society for Paediatric

Gastroenterology, Hepatology and Nutrition 2012 criteria

In light of the excellent correlation found between anti- tTG and EMA IgA antibody test results and the presence ofmucosalchanges,the2012ESPGHANguidelines¹allowed thepossibilityofdiagnosingCDwithoutanIBexclusivelyin patientsmeetingthefollowingcriteria:

• Suggestivesymptoms

• Anti-tTGIgAtitresgreaterthan10timestheULN

• PositiveEMAIgAtest,whichhasthehighestspecificity,in asecondserumsampletoconfirmthediagnosisandmin-

imisetheimpactofpotentialmethodologicalorlabelling errorsintheinitialmeasurementofanti-tTGIgAtitres.

• CompatibleHLA-DQhaplotype(DQ2and/orDQ8).

Theaccuracyofthisapproachwasconfirmedin2large- scale studies published in 2017.^12,19 In everyday clinical practice,thisapproachpreventsatleast35%ofIBs,mainly inchildrenagedlessthan5yearsandinpatientswithsig- nificantsymptoms.⁴⁰

Inrecent years,therole ofserological testingand the 2012ESPGHANcriteriafordiagnosisofCDhasbeenanalysed inscenariosotherthanthesymptomaticpatient.Evidence hasemergedthatanti-tTGIgAtitresgreaterthan10times theULN predictthepresence ofmucosalinjury inasymp- tomatic patients, although the positive predictive value of this finding may be lower compared to symptomatic patients.^12,13

Table4 CorerecommendationsoftheESPGHANfordiagnosisofCDinchildrenandadolescents;2012and2020guidelines.^1,13

•CDshouldbesuspectedinchildrenpresentinganyofthesigns,symptomsofriskfactorslistedinTable2.

•ThefirststepindiagnosisismeasurementofserumlevelsoftotalIgAandanti-tTGIgAantibodies.

•IntheabsenceofCDantibodies(anti-tTGorEMA),theprobabilityofCDinchildrenisextremelylow

•ThediagnosisofCDcanbeestablishedwithoutanIBinchildrenandadolescentswithsymptomssuggestiveofCEandan anti-tTGIgAtitre>10×ULNconfirmedbyapositiveEMAtest(inasecondsample)inapaediatricgastroenterologyunit.

•Thesameno-biopsydiagnosticprotocolmaybeappliedtoasymptomaticchildren,butthismustbeconsideredindividually ineachcasemustinapaediatricgastroenterologyunit.

•InasymptomaticchildrenwithIgAdeficiencyorT1D,anIBisrequiredtoconfirmthediagnosis.

•IndividualswithHLAhaplotypesotherthanDQ2/DQ8haveaverylowriskofdevelopingCD.HLAtestingisnotrequiredto makethediagnosiswithoutanIBinpatientswithananti-tTGIgAtitre>10×ULNconfirmedbyapositiveEMAtest.HLA typingisindicatedforscreeninginindividualswithriskfactorsoranuncertaindiagnosis.

CD,coeliacdisease;EMA,endomysialantibodies;IB,intestinalbiopsy;T1D,type1diabetesmellitus;tTG,tissuetransglutaminase;ULN, upperlimitofnormal.

Clinical suspicion of CD

Measure serum anti-tTG IgA and total IgA¹

CONFIRMED CD Refer to paediatric gastroenterologist²

Test for EMA CD risk group Positive CD serology

no anti-tTG test (rapid test or DGP)

tTG IgA & total IgA

• Review the results of initial anti-tTG IgA antibodies

•Repeat if previous test performed with rapid or qualitative test

• Discuss diagnostic pathways with the family³

Anti-tTG IgA < 10 ULN⁴

BIOPSY

EMA IgA

Negative EMA IgA

PAEDIATRIC GASTROENTEROLOGY Negative anti-tTG IgA

Anti-tTG IgA ≥10 x ULN Positive anti-tTG IgA

Positive EMA

PRIMARY CARE

Consider anti-tTG IgA titre (x ULN)

Marsh 2-3 Marsh 0-1⁵ Consider the risk of false negative serology :

•IgA deficiency

•Low gluten intake

•Immunosuppressive therapy

•Extraintestinal manifestations (dermatitis herpetiformis)

Risk of false negative serology

Yes No

No CD

Figure1 Diagnosticalgorithm(adaptedfromESPGHAN2020¹³).

1.IgAdeficiency:valuesbelowthethresholdsestablishedbythelaboratoryforageor<0.2g/Linchildrenagedmorethan3years.

2.Askthefamilynottoinitiatealow-glutenorgluten-freedietbeforethepatientisevaluatedinthePaediatricGastroenterology department.

3.Conveythemessagethatregardlessofhowthediagnosisisreached,treatmentwithagluten-freedietislife-long.

4.Incaseofapositiveanti-tTGIgAtestwithalowtitre,confirmsufficientdietaryglutenintake.Considerrepeatingserological testsaddingtheEMAtest.

5.Consider:a.revisionofbiopsyresults;b.falsepositiveanti-tTGresultandtestingforEMA(ifpositiveEMA=potentialCD);c.

additionaltests(HLA,anti-tTGdeposits,cytometry,etc);d.considerfollowupandretestingensuringnormalglutenintake;e.assess therelevanceofsymptoms.

tTG=tissuetransglutaminase antibodies,EMA=endomysialantibodies;DGP=deamidatedgliadinpeptideantibodies,ULN=upper limitofnormal.

Table5 ConsequencesoferrorsinthediagnosisofCD.⁴¹

Underdiagnosis(maintenanceofgluteninthediet) Overdiagnosis(unnecessaryeliminationofgluten) Impactonqualityoflifeduetopersistentsymptoms Impactonqualityoflifeofpatientandfamily Riskofimpairedgrowthanddevelopment Economicimpact

Long-termcomplications: Riskofimbalanceddiet:potentialdeficienciesinvitaminsand traceminerals

Reproductiveproblems/osteopenia Riskofconstipationandoverweight Gastrointestinalmalignancy

Autoimmunedisease

Updated 2020 criteria

Duetoalloftheabove,the2020guidelines¹³:

• Confirmthat theinitial evaluation ofCD shouldinvolve measurementofanti-tTGIgAandtotalserumIgAlevels.

IncaseofIgAdeficiency,thesecondstepshouldbetesting forIgGantibodies.

• Allow the option of diagnosing CD in asymptomatic patientswithout an IBfollowing thesamestepsusedin symptomaticpatients.However,sincethepositivepredic- tivevalueofhighanti-tTGtitresislowerinasymptomatic patients, the decision to use the no-biopsy approach should be made on a case-by-case basis and with the agreement of the parents and also the patient if age allows.

• Donotallowtheno-biopsyapproachfordiagnosisofCD inasymptomaticpatientswithT1D,asthescientificevi- denceinthispatientsubsetiscurrentlyinsufficient.

• MaintaintheneedforanIBinpatientswithIgAdeficiency due to the lack of dataon the predictive value of IgG antibodiesforthepresenceofmucosalinjury.

• Establish that HLA genetic testing is unnecessary in patients that require an IB or with anti-tTG IgA titres greater than10times theULN.This test wouldonlybe indicatedfor screeningofat-riskindividualsandincase ofuncertaindiagnosis.

Current situation

The evolution of the ESPGHAN criteria for diagnosis of CD a in the past 50 years has led to the current recom- mendations, with7 core recommendations (Table 4). The processtofollowincaseofsuspectedCDorpresenceofrisk factorsissummarisedinadiagnosticalgorithm(Fig.1).Pri- marycarepaediatriciansplayanessentialroleinsuspecting thediagnosisandorderingtheinitialserologicaltests.The ESPGHANrecommendsthatthediagnosisalwaysbemadeby apaediatric^1,13giventhatadiagnosisofCDinvolvesthepres- criptionofagluten-freedietforlife,and,inmostcases,the diagnosisisconfirmedwithinashorttimeframe,allwiththe ultimatepurposeofavoidingbothoverdiagnosisandunder- diagnosisandtheirconsequences(Table5).⁴¹Attheprimary carelevel,thiscallsformaintenanceofagluten-containing dietuntilthediagnosisisconfirmed.

Conflict of interest

Theauthorshavedeclarednoconflictsofinterest.

References

1.HusbyS,KoletzkoS,Korponay-SzaboIR,MearinML,PhillipsA, ShamirR,et al. forthe ESPGHANWorking Groupon Coeliac DiseaseDiagnosis, onbehalf of theESPGHAN Gastroenterol- ogy Committee. ESPGHAN guidelines for the diagnosis for CoeliacDiseaseinchildrenandadolescents.Anevidence-based approach.JPediatrGastroenterolNutr.2012;54:136---60.

2.MeeuwisseGW.Diagnosticcriteriaincoeliacdisease.ActaPae- diatrScand.1970;59:461---3.

3.Ribes-KoninckxC,GiliamsJP,PolancoI,Pe˜naAS.IgAantigliadin antibodiesinceliacandinflammatoryboweldisease.JPediatr GastroenterolNutr.1984;3:676---82.

4.Walker-SmithJA,GuandaliniS,SchmitzJ,ShmerlingDH,Visako- rpiJK.Revisedcriteriafordiagnosisofcoeliacdisease.Report ofworkinggroupofESPGAN.ArchDisChild.1990;65:909---11.

5.Rossi TM, Slbini CH, Kumar V. Incidence of celiac disease identified by the presence of serum endomysial antibodies in children with chronic diarrhea, short stature, or insulin- dependentdiabetesmellitus.JPediatr.1993;123:262---4.

6.Barker CC, Mitton C, Jevon G, Mock T. Can tissue trans- glutaminase antibody titers replace small-bowel biopsy to diagnoseceliacdiseasein selectpediatricpopulations? Pedi- atrics.2005;115:1341---6.

7.DahlbomI, Korponay-SzabóIR,Kovács JB, SzalaiZ, Mäki M, Hansson T. Prediction of clinical and mucosal severity of coeliacdiseaseanddermatitisherpetiformisbyquantification ofIgA/IgGserumantibodiestotissuetransglutaminase.JPedi- atrGastroenterolNutr.2010;50:140---6.

8.KurppaK,AshornM,IltanenS,KoskinenLL,SaavalainenP,Kosk- inenO,etal.Celiacdiseasewithoutvillousatrophyinchildren:

aprospectivestudy.JPediatr.2010;157:373---80.

9.Ribes-Koninckx C, Mearin ML, Korponay-Szabó IR, ShamirR, HusbyS,VenturaA,etal.ESPGHANWorkingGrouponCoeliac DiseaseDiagnosis.Coeliacdiseasediagnosis:ESPGHAN1990cri- teriaorneedforachange?Resultsofaquestionnaire.JPediatr GastroenterolNutr.2012;54:15---9.

10.KlappG,MasipE,BolonioM,DonatE,PoloB,RamosD,etal.

Coeliacdisease:thenewproposedESPGHANdiagnosticcriteria doworkwellinaselectedpopulation.JPediatrGastroenterol Nutr.2012;56:251---6.

11.DonatE, RamosJM,Sánchez-ValverdeF,Moreno A, Martinez MJ,LeisR,etal.SEGHNPWorkingGrouponCoeliacDisease.

ESPGHAN 2012 Guidelinesfor Coeliacdisease diagnosis: val- idation througha retrospective Spanishmulticentricstudy.J PediatrGastroenterolNutr.2016;62:284---91.

12.Werkstetter KJ, Korponay-Szabó IR, Popp A, Villanacci V, SalemmeM,HeiligG,etal.ProCeDEstudygroup.Accuracyin

diagnosisofCeliacdiseasewithoutbiopsiesinclinicalpractice.

Gastroenterology.2017;153:924---35.

13.Husby S, Koletzko S, Korponay-Szabó I, Kurppa K, Mearin ML, Ribes-Koninckx C, et al. European Society Paediatric Gastroenterology,HepatologyandNutritionGuidelinesfordiag- nosing Coeliac disease 2020. J Pediatr Gastroenterol Nutr.

2020;70:141---56.

14.LudvigssonJF,MurrayJA.EpidemiologyofCeliacdisease.Gas- troenterolClinNorthAm.2019;48:1---18.

15.LudvigssonJF,CardTR,KaukinenK,BaiJ,ZingoneF,Sanders DS,etal.Screeningfor celiacdiseaseinthegeneralpopula- tionandinhigh-riskgroups.UnitedEuropeanGastroenterolJ.

2015;3:106---20.

16.ChouR, Bougatsos C,Blazina I,Mackey K, Grusing S, Selph S.ScreeningforCeliacdisease: evidencereportandsystem- aticreviewfortheUSPreventiveServicesTaskForce.JAMA.

2017;31:1258---68.

17.HillPG,HolmesGK.Coeliacdisease:abiopsyisnotalwaysnec- essaryfordiagnosis.AlimentPharmacolTher.2008;27:572---7.

18.Ferrara F,Quaglia S, Caputo I,Esposito C, Lepretti M, Pas- toreS,etal.Anti-transglutaminaseantibodiesinnon-coeliac childrensufferingfrominfectiousdiseases.ClinExpImmunol.

2010;159:217---23.

19.WolfJ,PetroffD,RichterT,AuthMKH,UhligHH,Laass MW, et al. Validation of antibody-based strategies for diagnosis ofpediatric celiacdisease without biopsy.Gastroenterology.

2017;153:410---9.

20.VillaltaD,TonuttiE,PrauseC,KoletzkoS,UhligHH,Vermeersch P,etal.IgGantibodiesagainstdeamidatedgliadinpeptidesfor diagnosisofceliacdiseaseinpatientswithIgAdeficiency.Clin Chem.2010;56:464---8.

21.HoerterNA,ShannahanSE,SuarezJ,LewisSK,GreenPHR,Lef- flerDA,etal.Diagnosticyieldofisolateddeamidatedgliadin peptide antibody elevation for Celiac disease. Dig Dis Sci.

2017;62:1272---6.

22.GouldMJ,BrillH,MarconMA,MunnNJ,WalshCM.Inscreening forCeliacdisease,deamidatedgliadinrarelypredictsdisease whentissuetransglutaminaseisnormal.JPediatrGastroenterol Nutr.2019;68:20---5.

23.BishopJ,ReedP,AustinP,HurstM,AmeratungaR,CraigieA, etal. Prospectiveevaluation ofthe ESPGHAN Guidelinesfor DiagnosisofCeliacDiseaseinNewZealandChildren.JPediatr GastroenterolNutr.2018;67:749---54.

24.SinghP,AroraA,StrandTA,LeoflerDA,MäkiM,KellyCP,etal.

Diagnosticaccuracyofpointofcaretestsfordiagnosingceliac disease:asystematicreviewandmeta-analysis.JClinGastroen- terol.2019;53:535---42.

25.VermeerschP,GeboesK,MariënG,HoffmanI,HieleM,Bossuyt X.Definingthresholdsofantibodylevelsimprovesdiagnosisof celiacdisease.ClinGastroenterolHepatol.2013;11:398---403.

26.HusbyS,Murray JA,KatzkaDA.AGAclinicalpracticeupdate ondiagnosisandmonitoringofCeliacdisease-changingutility ofserologyandhistologicmeasures:expertreview.Gastroen- terology.2019;156:885---9.

27.WolfJ,HaendelN,RemmlerJ,KutznerCE,KaiserT,MothesT.

HemolysisandIgA-antibodiesagainsttissuetransglutaminase:

when areantibodytestresultsnolongerreliable? JClinLab Anal.2018;32:e22360.

28.Waisbourd-ZinmanO,HojsakI,RosenbachY,Mozer-GlassbergY, ShalitinS,PhillipM,etal.Spontaneousnormalizationofanti- tissuetransglutaminaseantibodylevelsiscommoninchildren withtype1diabetesmellitus.DigDisSci.2012;57:1314---20.

29.Castellaneta S,Piccinno E,Oliva M,Cristofori F,Vendemiale M,OrtolaniF,etal.Highrateofspontaneousnormalizationof celiacserologyinacohortof446childrenwithtype1diabetes:

aprospectivestudy.DiabetesCare.2015;38:760---6.

30.Martínez-OjinagaE,MolinaM,PolancoI,UrcelayE,Nú˜nezC.

HLA-DQdistributionandriskassessmentofceliacdiseaseina Spanishcenter.RevEspEnfermDig.2018;110:421---6.

31.RomanosJ,RosénA, KumarV,TrynkaG, FrankeL, SzperlA, etal.PreventCDGroup.Improvingcoeliacdiseaseriskpredic- tionbytestingnon-HLAvariantsadditionaltoHLAvariants.Gut.

2014;63:415---22.

32.Nú˜nezC,GarroteJA,ArranzE,BilbaoJR,FernándezBa˜nares F,JiménezJ,etal.Recommendationstoreportandinterpret HLAgeneticfindingsin coeliacdisease. RevEspEnferm Dig.

2018;110:458---61.

33.Paul SP, ChopraJ,VainaCL,Mallikarjuna A, BasudeD.HLA- DQ2/DQ8typing for non-biopsydiagnosis of coeliac disease:

isitnecessary?ArchDisChild.2019;2,pii:archdischild-2019- 317297.doi:10.1136/archdischild-2019-317297.[Epubaheadof print]PubMedPMID:31375474.

34.SciurtiM,FornaroliF,GaianiF,BonaguriC,LeandroG,DiMario F,etal.Geneticsusceptibiltyandceliacdisease:whatroledo HLAhaplotypesplay?ActaBiomed.2018;89(9-S):17---21.

35.AndersonRP,HenryMJ,TaylorR,DuncanEL,DanoyP,CostaMJ, etal.Anovelserogeneticapproachdeterminesthecommunity prevalenceofceliacdiseaseandinformsimproveddiagnostic pathways.BMCMed.2013;11:188.

36.TaavelaJ,KoskinenO,HuhtalaH,LähdeahoML,PoppA,Laurika K,etal.Validationofmorphometricanalysesofsmall-intestinal biopsyreadoutsinceliacdisease.PLoSOne.2013;1:8.

37.OberhüberG,GranditschG,Vogelsang H.Thehistopathology ofceliacdisease:timefor astandardizedreportscheme for pathologists.EurJGastroenterolHepatol.1999;11:1185---94.

38.RostamiK,AldulaimiD,HolmesG,JohnsonMW,RobertM,Sri- vastava A, etal. Microscopicenteritis: Bucharestconsensus.

WorldJGastroenterol.2015;21:2593---604.

39.AuricchioR,ToscoA,PiccoloE,GalatolaM,IzzoV,MaglioM, etal.Potentialceliacchildren:9-yearfollow-uponagluten- containingdiet.AmJGastroenterol.2014;109:913---21.

40.Benelli E, Carrato V, Martelossi S, Ronfani L, Not T, Ven- tura A.Coeliac diseaseintheERA ofthenewESPGHAN and BSPGHANguidelines:aprospectivecohortstudy.ArchDisChild.

2016;101:172---6.

41.LernerBA, GreenPHR, LebwohlB. Going againstthegrains:

gluten-freedietsinpatientswithoutceliacdisease-worthwhile ornot?DigDisSci.2019;64:1740---7.