The serologic diagnosis of celiac The serologic ... - UZ Leuven

Critically Appraised TopicThe serologic diagnosis of celiacdisease in adultsDr. P. VermeerschDr. G. Mariën and Prof. Dr. X. Bossuyt

Introduction- Celiac disease is an autoimmune disorder characterized by animmunologic responsiveness to ingested gluten- Diagnosis is based on intestinal biopsy (Marsh classification)- Genetic susceptibility (HLA DQ2 and DQ8)- Prevalence in the general population estimated at 0.5-1%- Most patients are diagnosed as adults and rarely present with overtceliac disease

Clinical PresentationHopper, A. D et al. BMJ 2007;335:558-562

PathogenesisLumenPartially digested gluten peptidesEpithelial barrierIncreased permeability of mucosaSubepithelial region Gln GlutTGDQ2 / DQ8Antibodies to- GliadinAPC- Deamidated gliadin- tTG- Neo-epitopeB-cellIFN-γT-cell

Marsh ClassificationMarsh 1 Intra-epithelialMarsh 2lymphocytosisCrypthyperplasiaMarsh 3bSubtotal villous atrophyMarsh3cTotal villous atrophy

Serology- Serologic testing is traditionally performed as a screening assay inpatients suspected of celiac disease- Current methods include the detection of antibodies direct against:1. Endomysium (indirect immunofluorescence)2. Tissue transglutaminase (ELISA)3. Gliadin (ELISA)- Detection of IgA antibodies is considered the most sensitive andspecific- IgA anti-endomysial antibodies and IgA anti-tTG tTG antibodies areconsidered the best screening assay in adults (sens. and spec. >90%).

Meta-analysis (2005, Rostom et al)Studies in Sensitivity (95% Specificity (95%Nadults CI) CI)Prev PPVIgA-AGA 11 0.75–0.90 (H) 0.80–0.90 (H) 0.36 H HIgG-AGA 7 0.17-1.00 (H) 0.70-0.80 (H) 0.37 H HNPVIgA-EMA (ME) 11 0.974 (0.957-0.985) 0.996 (0.988-0.999) 0.40 0.974 0.996IgA-EMA (HUC) 6 0.902 (0.859-0.934) 1.000 (0.991-1.000) 0.33 0.902 1.000IgA-tTG (GP) 5 0.859 (0.808-0.898) 0.953 (0.930-0.969) 0.31 0.859 0.953IgA-tTG (HR) 3 0.0.981 (0.901-0.997) 0.997) 0.981 (0.958-0.991) 0.991) 0.16 0.981 0.981H: Heterogeneous; ME: monkey oesophagus; HUC: human umbilical cord; GP: guinea pig;HR: human recombinantRostom et al. Gastroenterology 2005;128:S38-S46

Meta-analysis (2005, Rostom et al)Studies in Sensitivity (95% Specificity (95%NAdults CI) CI)Prev PPVIgA-AGA 11 0.75–0.90 (H) 0.80–0.90 (H) 0.36 H HIgG-AGA 7 0.17-1.00 (H) 0.70-0.80 (H) 0.37 H HNPVIgA-EMA (ME) 11 0.974 (0.957-0.985) 0.996 (0.988-0.999) 0.40 0.974 0.996IgA-EMA (HUC) 6 0.902 (0.859-0.934) 1.000 (0.991-1.000) 0.33 0.902 1.000IgA-tTG (GP) 5 0.859 (0.808-0.898) 0.953 (0.930-0.969) 0.31 0.859 0.953IgA-tTG (HR) 3 0.0.981 (0.901-0.997) 0.997) 0.981 (0.958-0.991) 0.991) 0.16 0.981 0.981H: Heterogenous; ME: monkey oesophagus; HUC: human umbilical cord; GP: ginea pig;HR: human recombinant1) Performance of anti-gliadin antibodies is inferior to anti-endomysiumor anti-tTG

Meta-analysis (2005, Rostom et al)Studies in Sensitivity (95% Specificity (95%NAdults CI) CI)Prev PPVIgA-AGA 11 0.75–0.90 (H) 0.80–0.90 (H) 0.36 H HIgG-AGA 7 0.17-1.00 (H) 0.70-0.80 (H) 0.37 H HNPVIgA-EMA (ME) 11 0.974 (0.957-0.985) 0.996 (0.988-0.999) 0.40 0.974 0.996IgA-EMA (HUC) 6 0.902 (0.859-0.934) 1.000 (0.991-1.000) 0.33 0.902 1.000IgA-tTG (GP) 5 0.859 (0.808-0.898) 0.953 (0.930-0.969) 0.31 0.859 0.953IgA-tTG (HR) 3 0.0.981 (0.901-0.997) 0.997) 0.981 (0.958-0.991) 0.991) 0.16 0.981 0.981H: Heterogenous; ME: monkey oesophagus; HUC: human umbilical cord; GP: ginea pig;HR: human recombinant1) Performance of anti-gliadin antibodies is inferior to anti-endomysiumor anti-tTG2) Sensitivity appears lower when more patients are included with lowgrade histologic damage

Meta-analysis (2005, Rostom et al)Studies in Sensitivity (95% Specificity (95%NAdults CI) CI)Prev PPVIgA-AGA 11 0.75–0.90 (H) 0.80–0.90 (H) 0.36 H HIgG-AGA 7 0.17-1.00 (H) 0.70-0.80 (H) 0.37 H HNPVIgA-EMA (ME) 11 0.974 (0.957-0.985) 0.996 (0.988-0.999) 0.40 0.974 0.996IgA-EMA (HUC) 6 0.902 (0.859-0.934) 1.000 (0.991-1.000) 0.33 0.902 1.000IgA-tTG (GP) 5 0.859 (0.808-0.898) 0.953 (0.930-0.969) 0.31 0.859 0.953IgA-tTG (HR) 3 0.0.981 (0.901-0.997) 0.997) 0.981 (0.958-0.991) 0.991) 0.16 0.981 0.981H: Heterogenous; ME: monkey oesophagus; HUC: human umbilical cord; GP: ginea pig;HR: human recombinant1) Performance of anti-gliadin antibodies is inferior to anti-endomysiumor anti-tTG2) Sensitivity appears lower when more patients are included with lowgrade histologic damage3) PPV is likely lower when applied to a low prevalence population

IgA deficiency- Selective IgA deficiency: primary immunodeficiency characterized bya selective deficiency of IgA in patients with normal serum levels ofIgG and IgM in whom other causes of hypogammaglobulinemia havebeen excluded.- The frequency in Western Europe is estimated at 1/400-1/900.- When IgA antibodies are determined, it is important to rule outselective IgA deficiency since CD occurs 10-15 times more often inthese patients than in the general population.=> Determine serum IgA in all patients who have a IgA anti-tTG

Current AlgorythmRequest IgA anti-tTGSerum IgADecreased(Adults

Critical Appraisal- Questions have been raised regarding the diagnostic performance ofIgA anti-tTG testing in routine clinical practice.- The reported highh sensitivities and specificities iti might be related tothe use of pre-selected groups of celiac disease patients (e.g. severehistological changes of the small bowel) and/or controls.- The specificity of the IgG anti-gliadin assay is not good.- There are reports that increased serum IgA can cause false-positiveIgA anti-tTG results, but this has not systematically been studied.

Questions1) What is the diagnostic performance of IgA anti-tTG in routine clinicalpractice?2) Does taking into account IgA anti-tTG titer and IgA concentrationimprove clinical interpretation?3) Is the detection of IgG antibodies against deamidated gliadinpeptides (IgG DGP-AGA) a better alternative than IgG anti-gliadinantibodies (IgG AGA) in patients t with aselective IgAdeficiency?i

Questions1) What is the diagnostic performance of IgA anti-tTG in routine clinicalpractice?2) Does taking into account IgA anti-tTG titer and IgA concentrationimprove clinical interpretation?3) Is the detection of IgG antibodies against deamidated gliadinpeptides (IgG DGP-AGA) a better alternative than IgG anti-gliadinantibodies (IgG AGA) in patients t with aselective IgAdeficiency?i

Methods- Retrospective analysis over a 42-month period to determine theperformance of IgA anti-tTG- Consecutive non-IgA deficient i (≥0.82 g/L) patients t aged 16 years orolder who had a IgA anti-tTG and for whom biopsy results wereavailable were identified (558/2050).- Patients who were previously diagnosed with celiac disease ordermatitis herpetiformis, a severe skin manifestation of glutensensitivity associated with celiac disease, or that were on a glutenfreediet were excluded.

MethodsDiagnosis of celiac disease by the clinician was consideredconfirmed when:- Duodenal biopsy showed Marsh 3- Duodenal biopsy showed Marsh 1 or 2 and the patient responded to a glutenfree diet clinically or on duodenal biopsy- Skin biopsy indicated d dermatitis i herpetiformis i and the lesions disappeareddwith a gluten free dietPatient was diagnosed as non-celiac disease when:- Duodenal biopsy showed Marsh 0 and the clinician did not consider thebiopsy to be false-negative

ResultsCeliac diseaseNon-celiac diseaseDemographic dataNumber of patients 48 558Male/Female l 15/33 207/351Duodenal biopsyMarsh 0 0 522Marsh 1 7 35Marsh 2 3 1 #Marsh 3 34 0Dermatitis herpetiformis i 4 0#P Patient twas diagnosed dwith giardiasisi

ResultsPatient resultsIgA anti-tTG CD Non CD

ResultsPatient resultsIgA anti-tTG CD Non CD

ResultsLikelihood ratioIgA anti-tTG CD Non CD LR10

ResultsLikelihood ratioIgA anti-tTG CD Non CD LR

ResultsLikelihood ratioIgA anti-tTG CD Non CD LR

Summary (1)1. Overall sensitivity (95.8%) and specificity (91.9%) of our secondgeneration IgA anti-tTG assay were good and comparable to otherstudies.2. Given a prevalence of 7.9% in patients who had a IgA anti-tTG tTG testand a biopsy, the positive predictive value (PPV) of a positive IgAanti-tTG result was only 50.5%.

Summary (1)1. Overall sensitivity (95.8%) and specificity (91.9%) of our secondgeneration IgA anti-tTG assay were good and comparable to otherstudies.2. Given a prevalence of 7.9% in patients who had a IgA anti-tTG tTG testand a biopsy, the positive predictive value (PPV) of a positive IgAanti-tTG result was only 50.5%.Of note: Since only patients with biopsy results were included, specificityis most likely underestimated and the PPV overestimated dueto a selection bias.

Selection Bias- Clinicians are more likely to propose an intestinal biopsy in patientswith a high likelihood of celiac disease or who test false-positive.- When IgA anti-tTG+ patients who were clinically diagnosed as non-CD and IgA anti-tTG- tTGpatients who did not require a biopsy wereincluded as true negative:- prevalence decreases from 7.9% to 2.3%- PPV decreases from 50.5% to 40.9%- Specificity increases from 91.9% 9% to 97.8%

Questions1) What is the diagnostic performance of IgA anti-tTG in routine clinicalpractice?2) Does taking into account IgA anti-tTG titer and IgA concentrationimprove clinical interpretation?3) Is the detection of IgG antibodies against deamidated gliadinpeptides (IgG DGP-AGA) a better alternative than IgG anti-gliadinantibodies (IgG AGA) in patients t with aselective IgAdeficiency?i

ResultsIgA anti-tTG100806040200CD CD + DHNon no CD

IgA anti-tTG concentration

IgA anti-tTG concentrationAll patientsPatientswith biopsy

IgA anti-tTG concentrationMarsh 1 Marsh 2 Marsh 3Patientswith biopsy

ResultsLikelihood ratioIgA patients LH+ CD LH+ non CD LR0.82-2.00 g/L 247 0.929 0.034 27.32.00-4.53 g/L 325 0.960 0.097 9.9>4.53 g/L 34 1.000 0.32 3.1- IgA anti-tTG only modestly increases pretest-posttest probability in patientswith an increased IgA concentration- Increased IgA is associated with significantly higher percentage of false-positive results (32% vs. 6.9%)- BUT: prevalence of celiac disease was also higher in patients with anincreased IgA concentration (26% versus 6.8%)

Likelihood ratioLikelihood ratios

Summary (2)Taking into account IgA anti-tTG concentration and serum IgAconcentration improves clinical interpretation.

Questions1) What is the diagnostic performance of IgA anti-tTG in routine clinicalpractice?2) Does taking into account IgA anti-tTG titer and IgA concentrationimprove clinical interpretation?3) Is the detection of IgG antibodies against deamidated gliadinpeptides (IgG DGP-AGA) a better alternative than IgG anti-gliadinantibodies (IgG AGA) in patients t with aselective IgAdeficiency?i

IgG anti-deamidated gliadinPreva alenceTestMetho odSensi itivitySpeci ificityVolta et al., 2008Selected CD (M3) and diseasedcontrol patientsVillalta et al., 2007Consecutive patients with completeIgA deficiency and 113 controlsNiveloni i et al., 2007Unselected consecutive patientsattending small bowel clinicAnkelo et al., 2007Selected CD patients and healthycontrolsNANAIgA anti-tTGIgA DGP-AGAIgG AGAIgG DGP-AGAIgG anti-tTGIgG AGAIgG DGP-AGA43% IgA anti-tTGIgA DGP-AGAIgG DGP-AGANAIgA anti-tTGIgA DGP-AGAIgG AGAIgG DGP-AGAEurospitalInovaEurospitalInovaInovaRadimInovaInovaInovaInovaBiofileIn-houseBiofileIn-house96.8%83.6%73.4%84.4%95%40%80%95.0%98.3%96.7%90%92%78%75%91.0%90.3%76.9%98.5%99%87%98%97.5%93.8%100%90%90%64%98%IgG DGP AGA In house 75% 98%

IgG anti-deamidated gliadinPreva alenceTestMetho odSensi itivitySpeci ificityVolta et al., 2008Selected CD (M3) and diseasedcontrol patientsVillalta et al., 2007Consecutive patients with completeIgA deficiency and 113 controlsNiveloni i et al., 2007Unselected consecutive patientsattending small bowel clinicAnkelo et al., 2007Selected CD patients and healthycontrolsNANAIgA anti-tTGIgA DGP-AGAIgG AGAIgG DGP-AGAIgG anti-tTGIgG AGAIgG DGP-AGA43% IgA anti-tTGIgA DGP-AGAIgG DGP-AGANAIgA anti-tTGIgA DGP-AGAIgG AGAIgG DGP-AGAEurospitalInovaEurospitalInovaInovaRadimInovaInovaInovaInovaBiofileIn-houseBiofileIn-house96.8%83.6%73.4%84.4%95%40%80%95.0%98.3%96.7%90%92%78%75%91.0%90.3%76.9%98.5%99%87%98%97.5%93.8%100%90%90%64%98%IgG DGP AGA In house 75% 98%

IgG anti-deamidated gliadinPreva alenceTestMetho odSensi itivitySpeci ificityVolta et al., 2008Selected CD (M3) and diseasedcontrol patientsVillalta et al., 2007Consecutive patients with completeIgA deficiency and 113 controlsNiveloni i et al., 2007Unselected consecutive patientsattending small bowel clinicAnkelo et al., 2007Selected CD patients and healthycontrolsNANAIgA anti-tTGIgA DGP-AGAIgG AGAIgG DGP-AGAIgG anti-tTGIgG AGAIgG DGP-AGA43% IgA anti-tTGIgA DGP-AGAIgG DGP-AGANAEurospitalInovaEurospitalInovaInovaRadimInovaInovaInovaInova96.8%83.6%73.4%84.4%95%40%80%95.0%98.3%96.7%91.0%90.3%76.9%98.5%99%87%98%97.5%93.8%100%IgA anti-tTGIgA DGP-AGAIgG AGAIgG DGP-AGABiofileIn-houseBiofileIn-house90%92%78%75%90%90%64%98%=> Detection of IgG DGP-AGA is both more sensitive and more specificthan detection of IgG AGA

IgG anti-deamidated gliadinPreva alenceTestMetho odSensi itivitySpeci ificityVolta et al., 2008Selected CD (M3) and diseasedcontrol patientsVillalta et al., 2007Consecutive patients with completeIgA deficiency and 113 controlsNiveloni i et al., 2007Unselected consecutive patientsattending small bowel clinicAnkelo et al., 2007Selected CD patients and healthycontrolsNANAIgA anti-tTGIgA DGP-AGAIgG AGAIgG DGP-AGAIgG anti-tTGIgG AGAIgG DGP-AGA43% IgA anti-tTGIgA DGP-AGAIgG DGP-AGANAEurospitalInovaEurospitalInovaInovaRadimInovaInovaInovaInova96.8%83.6%73.4%84.4%95%40%80%95.0%98.3%96.7%91.0%90.3%76.9%98.5%99%87%98%97.5%93.8%100%IgA anti-tTGIgA DGP-AGAIgG AGAIgG DGP-AGABiofileIn-houseBiofileIn-house90%92%78%75%90%90%64%98%=> The sensitivity and specificity of IgG DGP-AGA was not superior tothe much better characterized IgA anti-tTG

Summary (3)1. IgG DGP-AGA appears to be a better alternative than IgG AGA inpatients with selective IgA deficiency, but cannot replace IgA antitTGas routine screening assay in non-IgA deficient patients.2. Further research is needed to determine the diagnosticperformance, especially the false-positive rate, of IgG DGP-AGA inroutine clinical practice.

To Do’s1. Inform clinicians that the performance of the current IgA anti-tTGassay is good.2. To report the likelihood ratio of the relevant interval to clinicians(including confidence intervals).3. Evaluate the 2 currently available IgG DGP-AGA assays (Inova andEuroimmune) on the consecutive non-IgA deficient patients.4. Replace IgG AGA with IgG DGP-AGA in patients with selective IgAdeficiency. The cost of both assays is comparable.

AcknowledgementsDep. of Laboratory Medicine (UZ Leuven)Prof. Dr. X. BossuytDr. G. MariënApr. D. CoenenDep. of Gastroenterology (UZ Leuven)Prof. Dr. M. HieleDep. of Pathology (UZ Leuven)Prof. Dr. K. Geboes