Nomenclature and diagnosis of gluten-related disorders: A position statement by the Italian Association of Hospital Gastroenterologists and endoscopists (AIGO)

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ContentslistsavailableatScienceDirect

Digestive

and

Liver

Disease

j o u r n a l ho me p a g e :w w w . e l s e v i e r . c o m / l o c a t e / d l d

Position

Paper

Nomenclature

and

diagnosis

of

gluten-related

disorders:

A

position

statement

by

the

Italian

Association

of

Hospital

Gastroenterologists

and

Endoscopists

(AIGO)

Luca

Elli

a,∗

_,

_Danilo

_Villalta

b

_,

_Leda

_Roncoroni

c

_,

_Donatella

_Barisani

d

_,

_Stefano

_Ferrero

e,f

_,

Nicoletta

Pellegrini

g

_,

_Maria

_Teresa

_Bardella

a

_,

_Flavio

_Valiante

h

_,

_Carolina

_Tomba

i

_,

Antonio

Carroccio

j,k

_,

_Massimo

_Bellini

l

_,

_Marco

_Soncini

i

_,

_Renato

_Cannizzaro

m

_,

Gioacchino

Leandro

n

a_Center_for_the_Prevention_and_Diagnosis_of_Celiac_Disease,_Fondazione_IRCCS_Ca’_{Granda—Ospedale}_Maggiore_Policlinico,_Milano,_Italy b_Allergology_and_Clinical_Immunology_Unit,_AO_“Santa_Maria_degli_Angeli”,_via_Montereale_24,₃₃₁₇₀_Pordenone,_Italy

c_Department_of_{Pathophysiology}_and_{Transplantation,}_Università_degli_Studi_di_Milano,_Milano,_Italy

d_Dipartimento_di_Medicina_e_Chirurgia_(school_of_Medicine_and_Surgery)_Università_Milano_Bicocca,_Monza,_Italy e_Pathology_Unit,_Fondazione_IRCCS_Ca’_Granda_Ospedale_Maggiore_Policlinico,_Milano,_Italy

f_Department_of_Biomedical,_Surgical_and_Dental_Sciences,_Università_degli_Studi_di_Milano,_Milan,_Italy g_Department_of_Food_Science,_University_of_Parma,_Parma,_Italy

h_{Gastroenterology}_and_Digestive_Endoscopy_Unit,_Santa_Maria_del_Prato_Hospital,_Feltre,_Italy i_{Gastroenterology}_Unit_ASST_–_Santi_Paolo_e_Carlo_–_Milano,_Italy

j_DiBiMIS,_University_of_Palermo,_Palermo,_Italy

k_Internal_Medicine,_Giovanni_Paolo_II_Hospital,_Sciacca,_Italy

l_{Gastrointestinal}_Unit,_Department_of_{Gastroenterology,}_University_of_Pisa,_Pisa,_Italy m_{Gastroenterology,}_CRO_National_Cancer_Institute,_via_Franco_Gallini_2,_33081,_Aviano,_Italy n_{Gastroenterology,}_IRCCS_De_Bellis_Hospital,_Castellana_Grotte,_Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received1August2016 Accepted25October2016 Availableonline4November2016 Keywords:

Celiacdisease Wheatallergy

Non-celiacglutensensitivity Gluten-relateddisorders Foodallergy

a

b

s

t

r

a

c

t

Background:“Gluten-relateddisorders”isatermthatencompassesdifferentdiseasesinducedbythe ingestionofgluten-containingfood.Becauseoftheirincidencethescientiﬁccommunityhasbeen inten-sivelystudyingthem.

Aim:Tosupportgastroenterologistswithacorrectnomenclatureanddiagnosticapproachto gluten-relateddisordersinadulthood.

Methods:TheItalianAssociationofHospitalGastroenterologistsandEndoscopists(AIGO)commissioned apanelofexpertstoprepareapositionstatementclarifyingthenomenclatureanddiagnosisof gluten-relateddisorders,focusingonthoseofgastroenterologicalinterest.Eachmemberwasassignedatask andlevelsofevidence/recommendationhavebeenproposed.

Results:Thepanelidentiﬁedceliacdisease,wheatallergyandnon-celiacglutensensitivityasthe gluten-relateddisordersofgastroenterologicalinterest.Celiacdiseasehasanautoimmunenature,wheatallergy isIgE-mediatedwhilethepathogenesisofnon-celiacglutensensitivityisstillunknownasisthecase ofnon-IgEmediatedallergy.Diagnosisshouldstartwiththeserologicalscreeningforceliacdiseaseand wheatallergy.Incaseofnormalvalues,theresponsetoagluten-freedietshouldbeevaluatedanda conﬁrmatoryblindfoodchallengecarriedout.

Conclusions:Gluten-relateddisordersareclinicallyheterogeneous.Patientsshouldbecarefullymanaged andspeciﬁcprotocolsappliedforacorrectdifferentialdiagnosisingastroenterologicalsetting.

∗ Correspondingauthorat:CenterforthePreventionandDiagnosisofCeliacDisease,FondazioneIRCCSCa’Granda—OspedaleMaggiorePoliclinico,ViaF.Sforza35,20122 Milano,Italy.

E-mailaddresses:lucelli@yahoo.com(L.Elli),danilo.villalta@aas5.sanita.fvg.it(D.Villalta),leda.roncoroni@tiscali.it(L.Roncoroni),Donatella.barisani@unimib.it (D.Barisani),stefano.ferrero@unimi.it(S.Ferrero),nicoletta.pellegrini@unipr.it(N.Pellegrini),ﬂavio.valiante@gmail.com(F.Valiante),tomba.carolina@gmail.com (C.Tomba),acarroccio@hotmail.com(A.Carroccio),mbellini@med.unipi.it(M.Bellini),rcannizzato@cro.it(R.Cannizzaro),leandro@media.it(G.Leandro).

http://dx.doi.org/10.1016/j.dld.2016.10.016

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1. Introduction

Inspiteoftheimportanceofwheatinthehumandiet through-out history, the interaction between its components (gliadin, gluten,amylasetrypsininhibitoretc.)andthehumanbodytriggers anincreasingvarietyofsymptoms,syndromes,allergicreactions, autoimmunediseases[1].Thefirstdescriptionofapatientaffected byadisorderrelatedtogluteningestion(celiacdisease,CD)has beenascribedtoAreteusofCappadocia,whointhe2ndcentury ADreportedacaseofchronicdiarrheaandmalabsorption[2].This scenarioappearsincontrastwiththewidediffusionofthegenetic backgroundsusceptibletoimmunereactionsagainstgluten,the HLADQ2and/orDQ8haplotypes,whichareessentialforthe devel-opmentofCD[3].Thecontinuousworldwidediffusionofdiseases and syndromes recognizingglutenor other wheatcomponents asthedominantenvironmentalfactors,hasinducedthescientific communitytostudythemechanismsunderpinningtheintestinal andsystemicdamagefollowingtheiringestionandtogroupthem intotheumbrelladefinitiongluten-relateddisorders(GRD)[4,5]. GRDaredividedonthebasisoftheirpathomechanism: autoim-mune,celiacdisease[6];allergic,wheatallergy(WA,IgEornon-IgE mediated)andunknownasinthecaseofnon-celiacgluten sensitiv-ity(NCGS)(Fig.1)[5].Takentogether,thesedisorderscanaffectat least3%ofthepopulationandprobablyanevengreaterproportion ofthepatientsattendinggastroenterologicaloutpatientservices [1,7].ForthesereasonstheItalianAssociationofHospital Gastroen-terologistsandEndoscopists(AIGO)considereditimportantand necessarytodevelop apositionstatementonthenomenclature anddiagnosisofGRDinadults,toclarifytheclinicalissuesthat gastroenterologistsandendoscopistsusuallyfacewithduringtheir clinicalpractice.

2. Methods

InMarch2015AIGOcommissionedanexperts’panel,composed ofnutritionists,gastroenterologists,allergologistsandbiologists,to prepareapositionstatementonthenomenclatureanddiagnosis ofGRD.Aspeciﬁctaskwasassignedtoeachmemberofthegroup onthebasisofhis/herexpertise.Inparticular,LRandNPwrote thesectionabout glutenandgluten-free diet (GFD)and super-visedthemanuscriptfromanutritionalpointofview;DBdescribed thegeneticaspects;SFdealtwithduodenalhistology;MTBandLe organizedthesectiononCDandFV,RCandCTtheNCGSpart;DV coveredwheatallergy;ACsummarizedtheconnectionsbetween GRDandfunctionalsymptoms;MBandGLdealtwiththe possi-bleuseofsymptomaticscoresespeciallyinNCGS.LEsupervised themanuscript.Eachpanelmembercarriedoutacomprehensive PubMedresearchforEnglish-writtenarticles,withouttime lim-its,usingappropriate MeSHterms.Regularconferencecallsand web-basedexchangeswerescheduled.

Wheneverpossible,thelevelsofevidenceand recommenda-tionsweredefinedforeach partof thestatementfollowingthe GRADEsystem[8,9].Briefly,thequalityoftheevidenceisgraded fromhightolow.“High”qualityevidenceisassignedwhen fur-therresearchisunlikelytochangetheauthors’confidenceinthe

Fig.1.Aproposedschemeforthediagnosisofgluten-relateddisorders.

estimateofeffect.“Moderate” qualityevidencemeansthat fur-therstudieswouldbelikelytohaveaneffectontheconfidence oftheestimate.Finally,“Low”qualityevidenceindicatesthat fur-therresearchprobablyhasanimportantimpactontheconfidence intheestimateoftheeffectandwouldchangetheestimate.About recommendations,a“Strong”recommendationisassignedincase ofbenefitsclearlyoutweighingthenegativesandtheresultofno actionwhile“Conditional”isusedwhensomeuncertaintyremains. 3. Gluten

Wheat(andothergluten-containingcereals)isoneofthemost importantcropsintheworldanditsdoughcanbeprocessedinto avarietyoffoodstuffs,notablybread,otherbaked productsand pasta. The grainproteins determine the viscoelasticproperties ofdough,inparticular,thestorageproteinthatformanetwork in thedough calledgluten[10]. Glutenis composedbya mix-tureofmonomericgliadinsandpolymericgluteninsubunits (in equal amounts) and representsapproximately the80% of stor-ageproteinsinwheat.Storageproteinscanbesubdividedonthe basisoftheiralcoholsolubility;prolamins,whichmake mostof theproteiccontentinwheat,havebeendefinedonthebasisof theirsolubilityinalcohol–watermixtures,typically60–70%(v/v) ethanol [11]. Theprolaminspresent in rye,barley andoatsare namedsecalin,hordeinandavenin,respectively.Inwheat,these groupsofmonomericprolaminsareknownasgliadins,whichare usuallysubtypedfollowingtheirelectrophoreticmobilityin␣,␥ (sulphurrichandusually harmfulfor CD)and␻(sulphurpoor) [12].Wheatprolaminsarethemajorstorageproteinspresentinthe starchyendospermcellsofthegrain,wheretheyaresynthetized anddepositedviathesecretorysystems.Gliadinconsistsofabout 300aminoacidsandischaracterizedbyahighcontentofprolin andglutamine,andalowcontentoflysineandmethionine[11]. Thesecharacteristicsareresponsiblefortheimmunologic prop-ertiesofgliadinsanditsimmunodominantoligopeptide33-mer. 33-merisresistanttotheintestinalenzymaticdigestionand,after tissue transglutaminase(tTG)deamidation,itbinds theCD spe-cificHLADQ,efficientlyactivatingtheTcells(adaptativeimmune response)[13].Anothergliadinpeptide,the13-merisresponsible forinnateimmunityandcooperateswith33-merinCD pathogen-esis[14].ObscureistheglutenstimulatorymechanisminNCGS, althoughitscytotoxiceffectscouldhavearole[15].

Glutenhasanimportant palatability[12].Individualcells of wheatﬂourcontaincomplexnetworksofglutenproteins,brought togetherduringdoughmixing.Nowadays,changesoccuring dur-ingdoughmixingarenotexactlyknown;certainly,anincreasein doughstiffnessoccursasresultofanincreaseand optimization ofprotein-to-proteininteractionswithintheglutennetwork.This optimizationisduetotheformationofdisulphidebondswhile mix-inginair;thepresenceofoxygenandnitrogenresultsindifferent effectsonthesulphydrylanddisulphidecontentsofdough[10,16]. Glutenispresentin:durumwheat(Triticumdurum),common wheatorbreadwheat(Triticumaestivum),usedforbreadandfresh pastaandinbakeryproducts,rye(Secalecereale),barley(Hordeum vulgare),thethreespeciesofspelt(einkorn,Triticunmonococcum, emmer,TriticumdicoccumSchrankandspelta,Triticumspelta), kho-rasanwheat(Triticumturanicum,Kamut®_),_triticale_{(Triticosecale}

Wittmack), a hybrid of rye and common wheat. Other gluten-containingwheatderivatesarebulgurandseitan[10].

4. Gluten-relateddisorders 4.1. Celiacdisease

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Table1

Diagnostictestsforgluten-relateddisorders.

GRD Typeoftest

Serology Histology Skintest Blindfoodchallenge

CD tTGAIgAscreeningtest Duodenalhistologyis

mandatory

Notused Notused

tTGAIgGandDGPIgGincaseofIgAdeﬁciency HLAtypingindoubtfulcases

WA sIgEscreeningtest Notused Reliablebutwithlow

sensitivity

Goldstandard (potentiallydangerous) MApotentiallyusefulbutonlyafewallergensareavailable

Non-IgEWA IgGisnotsupportedbythescientiﬁcsocieties Notused Notused Goldstandard

NCGS Unavailable Notused Notused Goldstandard

CD,celiacdisease;GRD,gluten-relateddisorders;MA,molecular-basedallergydiagnostics;WA,wheatallergy.

predisposedsubjects,bygluteningestion.AstrictGFDusually

nor-malizestheintestinaldamageandclinicalpicture.Theprevalence

ofCDisestimatedfrom0.5%to1%oftheworldwidepopulation

[1,17].The overallprevalenceofCD maybehigherin northern Europe[18]orinspecific“atrisk”groupsasfirst-degreerelativesof patientswithCD[19],patientswithtype-1diabetesmellitus[20], Hashimoto’sthyroiditis[21],geneticdisorders(Down’ssyndrome andTurner’ssyndrome)[22],IgAdeficiency[9].

Thesymptomsarevariousanddifferentclinicalpicturescan bepresent,makingCDlargelyunder-diagnosed[23].Clinical ﬁnd-ings range from severe malabsorption syndrome to a mild or monosymptomatic form characterized by mild gastrointestinal symptomssuchasdyspepsia,constipation,diarrhea,epigastricpain orevenjustextra-intestinalsignssuchasirondeﬁciencyanemia orasthenia.CDcanevenoccurwithoutsymptoms(silent form) [24–26].

Theclassic onsetcan becharacterizedby diarrhea (less fre-quentlysteatorrhea),weightlossorgrowthdeﬁcitduringpediatric age,andsymptomssometimesrelatedtoliposolublevitamins(A, D,E,K)malabsorptionorelectrolytedisorders[27].

Aboutextra-intestinalmanifestations,differentsystems biolog-icalprocessescanbeinvolved[28]suchas:hemopoieticprocess, bonemineralization,muscularfunction,nervoussystem,endocrine homeostasisandfertility[29].

Inadults,CDdiagnosisisperformedbyreferring todifferent parameters:duodenalhistology,serologicantibodies(antitissue transglutaminase,tTG,anti endomisium,EmA,anti gliadin,AGA andantideamidatedgliadin,DGP),genetics,clinicalpictureand GFDresponse[30].

CDscreeningisperformedduringagluten-containingdietand usuallyinvolvestTGIgAantibodiesandtotalIgAdosagestoexclude anyIgAdeficiency. tTG IgAantibodies arehighly efficient: 98% sensitivity,98%specificity,72%positivepredictivevalueand99% negativepredictivevalue;theyareusuallyconsideredsufficient toruleCDoutifnegativeortosuggestduodenalhistologywhen positive[30].IncaseofIgAdeficiency,tTGIgGcanbedosedbut duodenalhistologyshouldalwaysbeconsidered.AGAshouldnot beconsideredinCDdiagnosisduringadulthood[25].Whendealing withmalabsorptionsymptomsorinthepresenceofaCDrisk>5% (first-degreerelatives,Down’ssyndromeortype-Idiabetes),also intheabsenceofcirculatingtTGIgA,theexclusionofaduodenal atrophymustbeconsideredduetothepossibilityofaseronegative CD,presentin5–22%ofthesecases(Table1)[9].

Differentlyfromchildhood,duodenalhistologyremainspivotal intheCDdiagnosticﬂowchartinadults.Histologyisrequiredto evaluatemucosaldamageduringglutenintake.Adequate histologi-calsamplesshouldbeobtainedduringendoscopyfromthesecond duodenalportionandbulb.Atleast4orientedbiopsiesfromthe secondpartoftheduodenumand1or2fromthebulbare manda-tory[31].

Acorrectorientationofsamplescanbeobtainedbypositioning thefragmentsoncelluloseacetateﬁlters.Theseﬁltersallowthe

technicians,witha90◦rotation,toparafﬁn-embedthecombined biopsy-ﬁlterforcorrectevaluationbythepathologist[32].

CDduodenaldamageischaracterizedbyintraepithelial lympho-cytosis,villousatrophyandcrypthyperplasia[24,33].Histological evaluationforCDshouldtakeintoaccountintraepithelialT lym-phocytes (IEL) rate. The normal count has been variably cited in the literature, however a value <25 IEL/100 enterocytes is considerednormalwhileavalue>30IEL/100enterocytes repre-sentsapathological“lymphocytosis”[24].Althoughdebated[34], immunohistochemicalstudiesincludingCD3immunostainingcan highlightIELinﬁltration.

Otherminorhistologicalparameterstoevaluateare:decreased enterocyteheight,intracytoplasmicenterocyte vacuolationwith reductionorabsenceofbrushborder[33].

Moreover,novelﬁndingssuggestthepresenceofdepositsof antitTGantibodies,detectableinduodenalsamplesbymeansof immunoﬂuorescence;thesedepositsseemusefulincaseof uncer-taindiagnosiswithoutevidenthistologicalterations[35].

Atdiagnosistheseverityofthedamageis usuallyevaluated accordingtotheMarsh–Oberhuber[36]orCorazza–Villanacci clas-siﬁcation [37].However,the pitfallsof duodenaldamagein CD remainintra-epitheliallymphocythosis,crypthyperplasiaand vil-lousatrophy[24].

Basedontheaforementionedhistologicalchanges,CDdiagnosis should alwaysbe correlated withclinical and serological ﬁnd-ingsinordertoexcludeothercausesofvillousatrophy(Giardia lamblia,Cryptosporidium,Microsporidium,cytomegalovirus, tro-pherimawhippeli,lymphoma,Crohn’sdisease,commonvariable immunedeﬁciencyandolmesartanenteropathy)[38].

Therecurrence/persistenceofmalabsorptivesymptomsinspite ofastrictadherencetoGFDforatleast6–12monthsandthe persis-tenceofvillousatrophy(intheabsenceofothercauses),suggests refractoryCD(RCD).Insuchacase,furtheranalysisisrequiredto differentiateRCDbetweentypeI(atlowriskofmalignant evo-lution)and typeII(athighriskofenteropathy-associatedT-cell lymphoma,EATL):duodenalTCR␥rearrangementinordertoreveal anymonoclonalpatternandcytoﬂuyorimetryinordertodetect anyaberrantduodenallymphocytes(CD103+,CD3−,CD4−,CD8−, CD3␧+)[39].

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4.1.1. Celiacdiseasegenetics

Different MHC and non-MHC genes concur in the predis-position to CD. The Major Histocompatibility Complex (HLA) region contains a large number of genes with immunologi-cal functions; they are important predisposing factors for CD. The association between CD and the heterodimer DQ2 is well known.HLA-DQ molecules are formedby alpha and beta sub-units,encodedbyHLA-DQA1andHLA-DQB1genes,respectively. About 90% of patients with CD carry the heterodimer DQ2.5 (DQA1*0501−DQB1*0201).Mostoftheremainingpatientscarry theheterodimerDQ8(DQA1*0301−DQB1*0302),whereasthefew patientslackingDQ2.5or DQ8haveone ofthetwoalleles that encode thesubunits of DQ2.5. In this case they carry the het-erodimerDQ2.2(DQA1*0201andDQB1*0202)orDQ7.5(withthe allele DQA1*0505 which encodes a polypeptide that is identi-cal,afterprocessing,tothatgeneratedbythealleleDQA1*0501) [42,43].

Notably,30%ofCaucasianscarrytheheterodimerDQ2;thus, HLAtypingcanbeusefultoexcludeCD(negativepredictivevalue closeto99%).Forthisreason,theremustbeotherpredisposing alle-lesinvolvedinthedevelopmentofCD.Inthelasttenyearsthere hasbeenagreateffortaimingtoidentifyadditionalpredisposing loci,mainlyusing theGenome-WideAssociationStudy (GWAS) approach[44].Uptonowthesestudieshaveidentifiedfivemore alleleswithintheMHCregionand40non-HLAloci(with58variants intotal)localizedonvariouschromosomes.Takentogether,these polymorphismsaccountforabout50%ofthegeneticpredisposition toceliacdisease.Itisinterestingtonotethatmostofthesegenesare involvedintheimmuneresponse,andthatpolymorphismsinthe samegeneshavebeenidentifiedinotherautoimmunedisorders, suchasrheumathoidartritisorCrohn’sdisease[42].

Clinically,HLAtypingcanbeemployedwhenthereisdoubtor inthosepatientsthathavealreadystartedGFD(Table1)whilethe identiﬁcationoftheheterodimersDQ2and/orDQ8canbea use-fulscreeningtoolinfamilieswithanaffectedsubjecttoexclude subjectsfromfollow-up.However,theuseofHLAtypingforthe screeningofat-riskgroupsshouldbeinterpretedwithcaution.

4.1.2. Celiacdiseaserecommendations

• tTGAIgAistheﬁrst-linescreeningtodetectCDinthegeneral population(strongrecommendation,highlevelofevidence). • Duodenalhistologyismandatoryinthoseadultswithpositive

tTGAandinat-riskpatientsindependentlyofserology.Atleast 4biopsiesinsecondpartofduodenumand1–2inthebulbmust betaken(strongrecommendation,highlevelofevidence). • TheMarsh–Oberhuberclassiﬁcationshouldbeusedtoestablish

thepresenceornotofanyduodenalatrophyatdiagnosis;during follow-uptheuseofnewscores(suchasElli–Ferrero’s)should beconsidered(conditionalrecommendation,highlevelof evi-dence).

• TheabsenceofHLAtype-IIDQ2and/orDQ8excludesthe pres-enceordevelopmentofCD(strongrecommendation,highlevel ofevidence).

• Genetictestingshouldbeconsideredinthosecaseswherethere isdoubtandunresponsivenesstothediet(conditional recom-mendation,moderatelevelofevidence).

4.2. Wheatallergy

Wheatallergy(WA)isanimmune-mediatedadversereactionto theproteinscontainedinwheat.Dependingontherouteof aller-genexposure,WAisclassiﬁedintooccupationalasthma(baker’s asthma) and rhinitis; food allergy (FA), affecting the skin, the gastrointestinaltractor therespiratory tract; wheat-dependent exercise-inducedanaphylaxis(WDEIA)andcontacturticaria.Inall

thesedisorderstheimmunoglobulinE(IgE)antibodiesarepivotal [45].

InadultsFAtoingestedwheatisinfrequent:themostcommon variantinadultsisWDEIA,wherethesymptomsresultfromthe combinationofcausativefoodintakeandphysicalexercise(aswell asnon-steroidalanti-inﬂammatorydrugsoralcohol)[46].Patients withWDEIAdisplayarangeofclinicalsymptoms,fromgeneralized urticaria,dyspnea,gastrointestinalsymptomstosevere anaphy-laxiswhentheyperformphysicalexercisewithin3–4hfromwheat consumption.Insomecasessymptomscanalsooccurwhenwheat isconsumedimmediatelyafterexercise.Themostimportantgrain proteinassociatedwithWDEIAis␻5-gliadin,evenifothergliadins, nsLTPandothergrainproteinsmaybetheelicitors[47].

In patients with IgE-mediated wheat FA the clinical cross-reactivity tomultiplecereal grainsrarelyoccurs; therefore, the eliminationofallgrainsfromthedietinthesepatientsisnot rec-ommendedandmaybenutritionallyharmful[48].

ThediagnosisofWAisclassicallybasedontheclinicalpicture, skinpricktests(SPTs),in-vitrospecificIgE(sIgE)assaysand func-tionalassays.SPTsandsIgEin-vitroassaysrepresentthefirst-level tests,althoughtheyareaffectedbyalowpredictivevalue.sIgE in-vitroassaysaremoresensitive(75–80%)thanSPTbutlessspecific (60%),mainlybecauseofthecross-reactivitywithgrasspollens. Molecular-basedallergy(MA)diagnosticscanovercomethelow accuracyofsIgEin-vitroassaysusingwheatflourextracts.Many allergenicproteinsareinvolvedinwheatandthelatestupdateof theWHO/IUISAllergenNomenclatureDatabasedescribes21 dif-ferent well-classified wheat allergens.Unfortunately, until now only␻-5gliadin(Tria19),nsLTP(Tria14)andgliadinmixtures areavailableintheImmunoCAPTM_assay,_whereas_the_ATI_(Tri_a

aA/TI)isavailableonlyinthemicroarrayISACTM_assay._The_sIgE

to␻-5 gliadin assayis highlyreliable and now widelyused to identifypatientswithWDEIA.However,thetestisestimatedto missapproximately20%ofthecases.InTable2molecular aller-gensinvolvedinwheatfoodallergyarereported.IfSPTandthe sIgEassayswithﬂourextractsormolecularallergensare inconclu-sive,thenfunctionalassaysarerequired.Theyareconsideredthe goldstandardforthediagnosisbutareaccompaniedbyariskof severeinducedreactionsandareimpracticalinbusypractice set-tings.Functionaltestsincludethedouble-blindplacebo-controlled foodchallenge(DBPCFC)andtheopenoralfoodchallenge(Table1) [49].

4.2.1. Thestrangecaseofnon-IgEfoodallergy

Foodallergyhasbeendefinedas“anadversehealtheffectarising fromaspecificimmuneresponsethatreproduciblyoccurson expo-suretoagivenfood”[50].Theoretically,thisdefinitionincludesall typesofimmune-mediatedreactions, includingthosecausedby theinnateimmunesystem.Althoughonecansupposethatfood antigen-specificIgGcancauseadversereactionsviatype-IIor type-IIIhypersensitivity,thepositionpapers from theEuropeanand Americanallergysocietiesstronglyadviseagainsttestingforfood antigen-specificIgGinthediagnosisoffoodallergy[51].Onthe otherhand,type-IVhypersensitivityreactionsareinvolvedinsome well-establishedclinicalentitiesininfantsforwhomwheatmayto representoneoftheoffendingfoods[52].

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Table2

Molecularallergensinvolvedinwheatfoodallergy.

Allergen Biochemicalname FA WDEIA

Tria14 Non-speciﬁclipidtranferprotein Yes

Tria18 Agglutininisolectin1 Yes

Tria19 Omega-5gliadin Yes Yes

Tria20 Gammagliadin Yes Yes

Tria25 Thioredoxin Yes

Tria26 Highmolecularweightglutenin Yes Yes

Tria36 Lowmolecularweightglutenin Yes

Tria37 Alphapurothionin Yes

Tria40 Chloroform/metanol-soluble(CM)17protein[alphaamylaseinhibitor] Yes Tria41 MitochondrialubiquitineligaseacticatorofNFKB Yes

Tria42 HypoteticalproteinfroncDNA Yes

Tria43 HypoteticalproteinfroncDNA Yes

Tria44 EndospermtransfercellspeciﬁcPR60precursor Yes

Tria45 Elongationfactor1(EIF1) Yes

FA,immediatewheatfoodallergy;WDEIA,wheatdependentexerciseinducedanaphylaxis.

inﬂammationviathedirectactivationoftheinnateimmunesystem

[54].

The ﬁrst step in the diagnosis of non-IgE mediated FA is undertakinganeliminationdiet.Iftheresolutionofsymptomsis achieved,thesecondstepisrepresentedbyaDBPCFC(Table1)[52].

4.2.2. Wheatallergyrecommendations

• SPTsandsIgEassaysrepresenttheﬁrst-leveldiagnosticsforWA. However,thetestresultsaloneshouldnotbeconsidered diag-nosticforFA;MAdiagnosticsmaybeconsidered,butuntilnow onlyafewwheatallergensareavailable(strong recommenda-tion,moderatelevelofevidence).

• Performinganoralfoodchallengeshouldbeconsideredforthe conclusivediagnosisofwheatFA(strongrecommendation,high levelofevidence).

4.3. Non-celiacglutensensitivity

CDandWAdonotexplainalltheGRDspectrumandmostofthe patientsreportingsymptomsaftergluteningestion(intheabsence ofCDandWA)usedtobepreviouslydiagnosedwithIBS.Nowadays, theyaresuspectedtohaveNCGS,a“syndromecharacterizedby intestinalandextra-intestinalsymptomsrelatedtotheingestion ofgluten-containingfood,insubjectsthatarenotaffectedbyCDor WA”[5,7].

AlthoughNCGSisconsideredcommon,littleisknownaboutits prevalence.Publishedexperiences showa prevalenceof 0.6%in primarycarecenters[55]and6%inthird-levelones[5].IntheUK theself-reportedNCGSpopulationprevalencehasbeenestimated at13%[56].

NCGSismorefrequentin30–40yearsoldfemales(F:Mratio5:1) [57].Symptomsusuallyappearwithinafewhoursordaysfollowing theingestionofgluten-containingproducts:theyquicklydisappear afterglutenwithdrawal.SymptomsofNCGSarebothintestinaland extra-intestinal.Amongthegastrointestinalsymptoms,abdominal painandbloating,diarrhea,dyspepsiaandoralaphthosisare fre-quent.Themostfrequent(30–40%)extra-intestinalsymptomsare: foggymind,mentalconfusionafterglutenconsumption, paresthe-sia,anxiety,depressionskindisordersandheadaches[57].

ThesuspicionofNCGS(frequentlyreportedbypatients),should complywithadiagnosticprocessaimedatﬁrstlyrulingCD and WAout. Asﬁrst-lineinvestigationsserologicaltestsfor CD and WAshouldachievethisgoal.However,inclinicalpracticepatients seekmedicalevaluationwhilealreadyavoidinggluten.HLA typ-ingcanexcludeCDwhenGFDisongoingincaseofabsenceofHLA DQ2/DQ8.Ifgenetictestingisunavailableorinconclusive,agluten challengeshouldbeplanned.Insuchcases,a two-week period

ofresumedglutenconsumptionisconsideredadequatebeforeCD testing[58].

Aclinicalhistoryofmalabsorptivesymptoms,nutrient deﬁcien-ciesandautoimmunecomorbiditieshasbeendescribedtobeless frequentingluten-sensitivesubjectsthaninCD[59].Thepresence ofsuchclinicalfeaturesorafamilyhistoryofCDinsubjectswith negativeCDserologysuggesttheexecutionofduodenalhistology toruleoutthepresenceofseronegativeCD[25].

Notwithstanding, an overlap between clinical ﬁndings may occuras anemia and weight loss have been reported in NCGS patientstoo;however,sincealterationsofintestinalpermeability and/orabsorptionhavenotbeenprovedinthesecases[60,61],they seemtobeduetopatient-drivenrestrictionsofthediet.

Serologicalbiomarkers arenot availablefor NCGS, sincethe determination of celiac-related antibodies is not sensitive nor speciﬁc to NCGS [62]. However, in selected cohorts of NCGS-suspectedpatients,theserologicalpresenceofIgGclassAGAhas beendescribedinmorethanhalfoftheinvestigatedsubjects[63]. Inviewofthecurrentlackofserologicalorhistological biomark-ersfor NCGS,the3rdInternationalExperts’Meeting onGRD in Salerno,statedthatthediagnosisofNCGSshouldbebasedupon a standardized glutenDBPCFC recording symptomswith visual analogicalscales(VASs)(Table1)[64].Accordingly,the diagnos-ticwork-upshouldstartwithasixweekslongGFD,symptomatic improvementbeingmonitoredthroughperiodicVAS.Incase of GFDresponsiveness,patientswouldaccesstothesecondstepof theprotocolinwhichtheconﬁrmationofdiagnosiscomesfrom aDBPCFC.Theproposedglutenconsumptionis 8gperday(i.e., theusualglutencontentofWesterndiets),foraone-weekperiod. GlutenisadministeredthroughFODMAPs-freebarswitha stan-dardizeddoseofATI.Symptomsvariationsareassessedattheend ofeachtreatmentweekinordertoclassifythepatient’sresponse. 4.3.1. TheNCGS–IBSconnection,theroleofotherenvironmental factorsanduseofsymptomaticscores

TheaetiologyofIBS,averycommonsyndrome,isnotentirely clearandthelong-terminadequacyofthecurrentdrugtreatment leadpatientstoseekavarietyofalternativeremedies,especially ofadietarynature[65]:infact,about50%ofthemattributetheir symptomstoadversefoodreactions[66].Fromthispointofview, animportantrolecanbeplayedbywheatashighlightedinthe cornerstonearticle by Verduet al.in 2009 [67]. Theseauthors referredto“gluten-sensitivity”asaconditionofsome morpholog-ical,immunological,orfunctionaldisorderthatrespondstogluten exclusion.Theyalsoquotedthatsomescientiﬁcarticleshadbeen publishedinthepreviousyearswhichlinkedGStoIBS[68].

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stoolpassingfrequencydecreasedand37%ofthepatientsreported formedstools[68].Inparticular,Wahnschaffeetal.[68]showed anassociationbetweentheeffectivenessoftheGFDandthe pres-enceoftheHLA-DQ2haplotype;infact,afterGFD,thestoolpassing frequencyandgastrointestinalsymptomscorereturnedtonormal valuesin60%ofIBS-Dpatientswhowerepositiveandin12%who werenegativeforHLA-DQ2.

The ﬁrst prospective gluten DBPCFC of IBS patients with self-reportedGSincluded34 patientsadministered withsnacks containing16-ggluten:itdemonstratedasymptomatic worsen-ingintheglutengroupcomparedtoplacebo[60].Patientswere signiﬁcantlyworsewithglutenastooverallsymptoms,abdominal pain,bloating,stooldissatisfactionandtiredness.

Carroccioet al.[69] reviewed theclinical recordsof 920IBS patientswhoundertookaneliminationdiet andwheatDBPCFC with cross-over: 30% of those patients reacted to the wheat challengereportingabdominalpain,bloatingandalteredstool con-sistency. Prospectively,Elli et al.[70] and Di Sabatinoet al.[71] examinedpatientswithfunctionalgastrointestinalsymptomsby glutenDBPCFCandindividuatedasubgroupofgluten-responding patients.

TheNCGS–IBSrelationshipcanbeseenaspartofmultiplefood hypersensitivity,asshownbyFritscher-Ravensetal.viaconfocal laserendomicroscopy[53].Inlinewiththisstudy,Vazquez-Roque etal.[72]havedemonstratedthatgluten-eatingIBShasanincrease ofdailybowelmovementsassociatedwiththeincreased perme-abilityofthesmallbowel.

However,itshouldbenotedthatsomestudieshavequestioned therole of gluten in this scenario suggesting that fermentable oligosaccharides, disaccharides, monosaccharides and polyols (FODMAP)arepossiblytheactualenvironmentalfactorinducing gastrointestinalsymptomsinthesepatients[62].Biesiekierskietal. [62]performedadouble-blindcrossovertrialin37subjectswith suspectedNCGSandIBS;theparticipantswererandomlyassigned togroupsgivena2-weekdietofreducedFODMAPs,andwerethen placedonhigh-gluten(16ggluten/day),low-gluten(2ggluten/day and14gwheyprotein/day),orcontrol(16gwheyprotein/day,used asplacebo)dietsforoneweek,followedbyawash-outperiod.Inall theparticipantsgastrointestinalsymptomssignificantlyimproved intherun-inperiod,duringreducedFODMAPintake,but signif-icantlyworsened toasimilar degreewhentheirdietsincluded glutenorplacebo.Theseauthorsnotedalsoaclearnoceboeffect. Theauthorsconcludedthattherewasnoevidenceofspecificor dose-dependenteffectsofgluteninpatientswithsuspectedNCGS beingplacedondietslowinFODMAPs[73].Alimitationofthe abovestudywastoexcludethesubjectswhohadaHLA haplo-typeDQ2/DQ8orwhoshowedintra-epithelialinflammationinthe duodenalmucosa (Marsh1 lesion).Asthepreviousstudieshad demonstratedthatthesetwocharacteristicswereveryfrequent (>50%ofthecases)inNCGSsubjects,ithasbeensuggestedthat Biesiekierskietal.studiedadifferentNCGScohortandthus,their resultscannotbecomparedwiththepreviousfindings[74].

DuetotheNCGS–IBSoverlap,inNCGSpatientstheassessmentof symptomseverityisofparamountimportanceinordertoevaluate theclinicalresponsetoGFDandtheeffectoftheglutenDBPCFC.

BecauseofthesimilaritiesbetweenIBSandNCGS,someofthe scoresusedtoassesssymptomseverityandtheimpairmentof qual-ityoflifeinIBSpatients,canalsobesuggestedtoassesstheclinical burdenofNCGS.

TheIBSSymptomSeverityScoremayapplytoassesstheclinical severityofintestinalsymptoms.Itisa5-itemquestionnaire gener-atingamaximumscoreof500.Itisveryeasytouseandveryfast tocomplete.Itisconsideredreliablealsoinassessinganychanges ofthegutsymptomsseverityovertime[75].

TheIBSqualityoflifequestionnaireallowspractitionersto fol-lowtheirpatientslongitudinallyandcanbeusedinclinicaltrials

toevaluatepossibleQoLimprovementsduetospeciﬁctherapies [76].

TheGastrointestinalSymptomRatingScale,usedtoevaluate commonsymptomsofgastrointestinaldisorders,hasbeen specif-ically conceived by Catassiet al.: it evaluates lowerand upper digestivesymptomsandextra-intestinalNCGSmanifestationsby meansofVAS.Itisaself-administeredquestionnaire,whichhas beenproposedforuseinclinicalpracticeandforresearchpurposes, evenifitstillneedsscientiﬁcvalidation[64].

In conclusion, the current data point toat least an overlap betweenIBSandNCGS;itislikelythatabout25–30%ofIBSpatients, especiallythose withdiarrhea-typeIBScarryingtheHLA haplo-typesDQ2/DQ8,arewithNCGS.AconcurrentroleofFODMAPsor otherantigenscanbeconsideredandshouldbefurtherevaluated. 4.3.2. NCGSrecommendations

• TheinitialassessmentinNCGSdiagnosismustaimtoexclude CDandWA.WithregardtopatientsrespondingtoGFD,DBPCFC ismandatorytoconﬁrmdiagnosisasdescribedintheSalerno criteria(strongrecommendation,highlevelofevidence). 5. Withdrawalofglutenfromthediet

TheGFDischaracterizedbyacombinationofnaturally occur-ringgluten-free(GF)foods,commonGFcereals(e.g.,riceandcorn), pseudocereals(i.e.,amaranth,quinoaandbuckwheat),andminor cereals(e.g.,milletandsorghum).Besidesthesefoods,GFDis com-monlysupplementedwithGFsubstitutesofbread,cookies,pasta andothercereal-basedfoodsmadebyeitheringredientsthatdonot includegluten-containingcereals(e.g.,wheat,rye,barley)or ingre-dientsfromcerealsthathavebeenspeciﬁcallyprocessedtoremove gluten.FoodsnotallowedinanyGFDtinclude:(a)alltypesofbread andfoodpreparedwithﬂourfromallthevarietiesofwheat;(b)food thatcontainswheat,orderivatesofglutenusedasthickeners;(c) medicinalproductsthatuseglutenasabinderforpillsortablets [77].

Regardingoats,itsuseinGFDremainscontroversialasthe con-taminationwithprolaminsofothercerealsisfrequentandsome clinicalandexperimentalstudiessupporttheviewthatasubgroup ofceliacpatientsmaybeintoleranttopureoats[78].Thus,the sta-tusofoatsintheinternationalGFStandardhasbeenreassessed (CodexAlimentariusCommission,2008).Yet,oatsarekeptinthe category of gluten-containing cereals, but a footnote has been added whichstates: “oatscanbetolerated bymostbut not all peoplewhoareintoleranttogluten.Therefore,theuseofoatsnot contaminatedwithwheat,ryeorbarleyinfoodscoveredbythe standardmaybedeterminedonnationallevel”.

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Eventhoughnotallthestudiesreportedadifferentfiberintake betweenceliac patientsand thematchedcontrol group[79,80], it hasbeensuggested that GFDis inadequate in terms offiber content[81,87–89].Thishasbeenattributedtoadecreased con-sumptionofgrainproducts,exacerbatedbythefactthatmanyGF foodsaremadewithstarchesorrefinedflourswithlowfiber con-tent.However,inrecentyears,manufacturershaveimprovedthe fibercontentofbreads,flourmixesandotherGFproducts,as hydro-colloidsandgumshavingcolloidalpropertiesareusedforreplacing theglutennetworkandimprovingthetechnologicalpropertiesof GFproducts[84].

Regardingmicro-nutrients, lowerlevels of vitamins,suchas folate,niacin,vitaminB12,vitaminDweredescribedinceliac indi-vidualsthanincontrolsubjects[79,81,88].Moreover,suchalow intakehastheconsequencethat,inmanycases,celiacpatientsdid notmeettherecommendedintakeofthesevitamins[80].Sucha lowintakecanpartlybeduetothelowcontentoffolatesinstarches andlow-proteinflours(e.g.,cornandrice),commonlyusedasmain componentsofGFproducts[84].Similarlytovitamins,some stud-iesreportedlowerintakes ofseveralmineralsinceliacpatients thaninthecontrolsandinadequateintakes againstthecurrent recommendations.Forinstance,ascomparedtocontrolsubjects, thedailyintakeofironwassignificantlylowerinfemaleandmale celiacpatients[80,89]andthatofcalciumwassignificantlylower infemaleceliacpatients[81,88].Leeetal.[90]suggestedthatthe inclusionofalternativeGFgrains(e.g.,oatsandquinoa)can signif-icantlyincreasethenutritionalqualityofGFDincludingthelevels ofironandcalcium.

5.1. Gluten-freedietrecommendations

• GFDdoesnotpresentsideeffectsandisusuallybalancedandsafe (strongrecommendation,highlevelofevidence).

• Supplementationtherapy isnot routinelyneeded duringGFD (strongrecommendation,highlevelofevidence).

6. Conclusions

GRDcurrently poseasa challengingissue for gastroenterol-ogists, especially when facing with patients with functional symptoms.Insuchacasethedifferentoptionsshouldbeconsidered andaspecificdiagnosticroadmapestablishedwheneverpossible. Thestartingpointistoestablishthedieteticstatusofthepatient and,inparticular,ifheiseatingglutenduetothehighpercentage ofsubjectsfollowingaGFDorapoor-glutendietwithouta med-icaladvise.Sometimethisstepcouldbedifficultandthustheuse ofaspecificalimentaryquestionnaireissuggested[91].Oncethe dieteticstatusofthepatienthasbeenascertained,clinicianshould followtwodifferentroadmaps.Forpatientsfollowingagluten con-tainingdiettheadoptionofscreeningtestsforCD(tTGA)andWA (sIgE)aremandatory.For patientsfollowing aGFD,and usually unwillingtoreintroducegluten,HLAtypingcouldrepresentsafirst steptoestablishtheCDrisk.IncaseofabsenceoftheCDgenetic backgroundandaftertheexclusionofWA,thepatientcouldbe blindlychallengedwithgluteninordertoestablishthepresenceof aNCGS.WhenfacingwithaCDcompatiblegeneticbackgrounda lowdoseglutenchallenge(atleast3gofglutenperdayfor2weeks) shouldbeadministeredinordertoreturntoa“normaldiet” sta-tus[58].IfallthepreliminarytestsforCDandWAresultnegative andsymptomsimproveafteracorrectGFD,NCGScanbesuspected andappropriatelylookedforwithastandardizeddouble(oratleast single)BPCFC.Nowadays,inabsenceofbiomarkers,thedistinction betweenNCGSandnon-IgEallergyappearsmoreacademicthan real.

Ifin caseofpositivityofoneoftheabovementioned testsa GRDisprovenorhighlysuspected,animportantclinicalquestion

Fig.2. Aproposedﬂowchartforpatientswithsuspectedgluten-relateddisorder.

isrepresentedbythosepatients(many)withoutanytangibleproof ofaconnectionbetweentheirsymptomsandgluteningestionbut reportinganimportantimprovementoftheirclinicalpicturewhen followinga GFD.Thesepatientsareusuallydifﬁculttofacedue thelackofanswerstotheirquestions;however,cliniciansshould followtheminordertoavoidtheirappealstounscientiﬁctestsand potentiallydangerousdietswithoutmedicalcontrol.

ApossiblediagnosticﬂowchartforGRDisreportedinFig.2. Conﬂictofinterest

DaniloVillalta,DonatellaBarisani,FlavioValiante,CarolinaTomba, AntonioCarroccio,MassimoBellini,MarcoSoncini,Renato Canniz-zaroandGioacchinoLeandrohavenoconﬂictofinterest.

LucaElliandNicolettaPellegriniarescientiﬁcboardmembers oftheDrSchaerInstitute.

LucaElli,MariaTeresaBardella,LedaRoncoroniandCarolina Tombaaretheinventorsofacommerciallyavailablediagnostickit forNCGS.

Acknowledgments

The authors acknowledge the Italian Association of Hospi-talGastroenterologistsandEndoscopists (AIGO)for thesupport received.

Thelanguageandstyleofthemanuscripthasbeenrevisedby MarcelloHinxman-Allegri,a nativeEnglishspeakerand experi-encedpublishingprofessional.Neithertheresearchcontentsnor theauthors’intentionswerealteredinanywayduringtheediting process.

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