Critically Appraised Topic. Dr. P. Vermeersch Dr. G. Mariën and Prof. Dr. X. Bossuyt

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1 Critically Appraised Topic The serologic diagnosis of celiac disease in adults Dr. P. Vermeersch Dr. G. Mariën and Prof. Dr. X. Bossuyt

2 Introduction - Celiac disease is an autoimmune disorder characterized by an immunologic 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 overt celiac disease

3 Clinical Presentation Hopper, A. D et al. BMJ 2007;335:

4 Pathogenesis Lumen Partially digested gluten peptides Epithelial barrier Increased permeability of mucosa Subepithelial region Gln Glu ttg DQ2 / DQ8 Antibodies to - Gliadin APC - Deamidated gliadin - ttg - Neo-epitope B-cell IFN-γ T-cell

5 Marsh Classification Marsh 1 Intra-epithelial Marsh 2 lymphocytosis Crypt hyperplasia Marsh 3b Subtotal villous atrophy Marsh3c Total villous atrophy

6 Serology - Serologic testing is traditionally performed as a screening assay in patients 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 and specific - IgA anti-endomysial antibodies and IgA anti-ttg ttg antibodies are considered the best screening assay in adults (sens. and spec. >90%).

7 Meta-analysis (2005, Rostom et al) Studies in Sensitivity (95% Specificity (95% N adults CI) CI) Prev PPV IgA-AGA (H) (H) 0.36 H H IgG-AGA (H) (H) 0.37 H H NPV IgA-EMA (ME) ( ) ( ) IgA-EMA (HUC) ( ) ( ) IgA-tTG (GP) ( ) ( ) IgA-tTG (HR) ( ) 0.997) ( ) 0.991) H: Heterogeneous; ME: monkey oesophagus; HUC: human umbilical cord; GP: guinea pig; HR: human recombinant Rostom et al. Gastroenterology 2005;128:S38-S46

8 Meta-analysis (2005, Rostom et al) Studies in Sensitivity (95% Specificity (95% N Adults CI) CI) Prev PPV IgA-AGA (H) (H) 0.36 H H IgG-AGA (H) (H) 0.37 H H NPV IgA-EMA (ME) ( ) ( ) IgA-EMA (HUC) ( ) ( ) IgA-tTG (GP) ( ) ( ) IgA-tTG (HR) ( ) 0.997) ( ) 0.991) H: Heterogenous; ME: monkey oesophagus; HUC: human umbilical cord; GP: ginea pig; HR: human recombinant 1) Performance of anti-gliadin antibodies is inferior to anti-endomysium or anti-ttg

9 Meta-analysis (2005, Rostom et al) Studies in Sensitivity (95% Specificity (95% N Adults CI) CI) Prev PPV IgA-AGA (H) (H) 0.36 H H IgG-AGA (H) (H) 0.37 H H NPV IgA-EMA (ME) ( ) ( ) IgA-EMA (HUC) ( ) ( ) IgA-tTG (GP) ( ) ( ) IgA-tTG (HR) ( ) 0.997) ( ) 0.991) H: Heterogenous; ME: monkey oesophagus; HUC: human umbilical cord; GP: ginea pig; HR: human recombinant 1) Performance of anti-gliadin antibodies is inferior to anti-endomysium or anti-ttg 2) Sensitivity appears lower when more patients are included with low grade histologic damage

10 Meta-analysis (2005, Rostom et al) Studies in Sensitivity (95% Specificity (95% N Adults CI) CI) Prev PPV IgA-AGA (H) (H) 0.36 H H IgG-AGA (H) (H) 0.37 H H NPV IgA-EMA (ME) ( ) ( ) IgA-EMA (HUC) ( ) ( ) IgA-tTG (GP) ( ) ( ) IgA-tTG (HR) ( ) 0.997) ( ) 0.991) H: Heterogenous; ME: monkey oesophagus; HUC: human umbilical cord; GP: ginea pig; HR: human recombinant 1) Performance of anti-gliadin antibodies is inferior to anti-endomysium or anti-ttg 2) Sensitivity appears lower when more patients are included with low grade histologic damage 3) PPV is likely lower when applied to a low prevalence population

11 IgA deficiency - Selective IgA deficiency: primary immunodeficiency characterized by a selective deficiency of IgA in patients with normal serum levels of IgG and IgM in whom other causes of hypogammaglobulinemia have been excluded. - The frequency in Western Europe is estimated at 1/400-1/ When IgA antibodies are determined, it is important to rule out selective IgA deficiency since CD occurs times more often in these patients than in the general population. => Determine serum IgA in all patients who have a IgA anti-ttg

12 Current Algorythm Request IgA anti-ttg Serum IgA Decreased (Adults <0.82 g/l) IgG anti-gliadin Normal or increased (Adults 0.82 g/l) IgA anti-ttg 130 patients 2050 patients

13 Critical Appraisal - Questions have been raised regarding the diagnostic performance of IgA anti-ttg testing in routine clinical practice. - The reported high sensitivities and specificities might be related to the use of pre-selected groups of celiac disease patients (e.g. severe histological 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-positive IgA anti-ttg results, but this has not systematically been studied.

14 Questions 1) What is the diagnostic performance of IgA anti-ttg in routine clinical practice? 2) Does taking into account IgA anti-ttg titer and IgA concentration improve clinical interpretation? 3) Is the detection of IgG antibodies against deamidated gliadin peptides (IgG DGP-AGA) a better alternative than IgG anti-gliadin antibodies (IgG AGA) in patients t with aselective IgAdeficiency? i

15 Questions 1) What is the diagnostic performance of IgA anti-ttg in routine clinical practice? 2) Does taking into account IgA anti-ttg titer and IgA concentration improve clinical interpretation? 3) Is the detection of IgG antibodies against deamidated gliadin peptides (IgG DGP-AGA) a better alternative than IgG anti-gliadin antibodies (IgG AGA) in patients t with aselective IgAdeficiency? i

16 Methods - Retrospective analysis over a 42-month period to determine the performance of IgA anti-ttg - Consecutive non-iga deficient ( 0.82 g/l) patients aged 16 years or older who had a IgA anti-ttg and for whom biopsy results were available were identified (558/2050). - Patients who were previously diagnosed with celiac disease or dermatitis herpetiformis, a severe skin manifestation of gluten sensitivity associated with celiac disease, or that were on a glutenfree diet were excluded.

17 Methods Diagnosis of celiac disease by the clinician was considered confirmed when: - Duodenal biopsy showed Marsh 3 - Duodenal biopsy showed Marsh 1 or 2 and the patient responded to a gluten free diet clinically or on duodenal biopsy - Skin biopsy indicated dermatitis herpetiformis and the lesions disappeared with a gluten free diet Patient was diagnosed as non-celiac disease when: - Duodenal biopsy showed Marsh 0 and the clinician did not consider the biopsy to be false-negative

18 Results Celiac disease Non-celiac disease Demographic data Number of patients Male/Female l 15/33 207/351 Duodenal biopsy Marsh Marsh Marsh # Marsh Dermatitis herpetiformis i 4 0 #P Patient twas diagnosed dwith giardiasis i

19 Results Patient results IgA anti-ttg CD Non CD <7 U/mL U/mL Total

20 Results Patient results IgA anti-ttg CD Non CD <7 U/mL U/mL Total Overall sensitivity and specificity IgA anti-ttg CD Non CD <7 U/mL U/mL

21 Results Likelihood ratio IgA anti-ttg CD Non CD LR <7 U/mL U/mL LR Interpretation 1 No clinical value >10 < Clinically important differences in pretest-posttest probability Small difference, may be relevant in certain clinical settings Modest, but substantial difference in pretest-posttest probability

22 Results Likelihood ratio IgA anti-ttg CD Non CD LR <7 U/mL U/mL BUT: Positive predictive value was only 50.5% with a prevalence of 7.9% in patients who had a biopsy

23 Results Likelihood ratio IgA anti-ttg CD Non CD LR <7 U/mL U/mL BUT: Positive predictive value was only 50.5% with a prevalence of 7.9% in patients who had a biopsy The negative predictive value, in contrast, was 99.6%.

24 Summary (1) 1. Overall sensitivity (95.8%) and specificity (91.9%) of our second generation IgA anti-ttg assay were good and comparable to other studies. 2. Given a prevalence of 7.9% in patients who had a IgA anti-ttg ttg test and a biopsy, the positive predictive value (PPV) of a positive IgA anti-ttg result was only 50.5%.

25 Summary (1) 1. Overall sensitivity (95.8%) and specificity (91.9%) of our second generation IgA anti-ttg assay were good and comparable to other studies. 2. Given a prevalence of 7.9% in patients who had a IgA anti-ttg ttg test and a biopsy, the positive predictive value (PPV) of a positive IgA anti-ttg result was only 50.5%. Of note: Since only patients with biopsy results were included, specificity is most likely underestimated and the PPV overestimated due to a selection bias.

26 Selection Bias - Clinicians are more likely to propose an intestinal biopsy in patients with 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- ttg patients who did not require a biopsy were included 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%

27 Questions 1) What is the diagnostic performance of IgA anti-ttg in routine clinical practice? 2) Does taking into account IgA anti-ttg titer and IgA concentration improve clinical interpretation? 3) Is the detection of IgG antibodies against deamidated gliadin peptides (IgG DGP-AGA) a better alternative than IgG anti-gliadin antibodies (IgG AGA) in patients t with aselective IgAdeficiency? i

28 Results IgA anti-ttg CD CD + DH Non no CD

29 IgA anti-ttg concentration

30 IgA anti-ttg concentration All patients Patients with biopsy

31 IgA anti-ttg concentration Marsh 1 Marsh 2 Marsh 3 Patients with biopsy

32 Results Likelihood ratio IgA patients LH+ CD LH+ non CD LR g/l g/l >4.53 g/l IgA anti-ttg only modestly increases pretest-posttest probability in patients with 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 an increased IgA concentration (26% versus 6.8%)

33 Likelihood ratio Likelihood ratios

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

35 Questions 1) What is the diagnostic performance of IgA anti-ttg in routine clinical practice? 2) Does taking into account IgA anti-ttg titer and IgA concentration improve clinical interpretation? 3) Is the detection of IgG antibodies against deamidated gliadin peptides (IgG DGP-AGA) a better alternative than IgG anti-gliadin antibodies (IgG AGA) in patients t with aselective IgAdeficiency? i

36 IgG anti-deamidated gliadin Preva alence Test Metho od Sensi itivity Speci ificity Volta et al., 2008 Selected CD (M3) and diseased control patients Villalta et al., 2007 Consecutive patients with complete IgA deficiency and 113 controls Niveloni i et al., 2007 Unselected consecutive patients attending small bowel clinic Ankelo et al., 2007 Selected CD patients and healthy controls NA NA IgA anti-ttg IgA DGP-AGA IgG AGA IgG DGP-AGA IgG anti-ttg IgG AGA IgG DGP-AGA 43% IgA anti-ttg IgA DGP-AGA IgG DGP-AGA NA IgA anti-ttg IgA DGP-AGA IgG AGA IgG DGP-AGA Eurospital Eurospital Radim Biofile In-house Biofile In-house 96.8% 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%

37 IgG anti-deamidated gliadin Preva alence Test Metho od Sensi itivity Speci ificity Volta et al., 2008 Selected CD (M3) and diseased control patients Villalta et al., 2007 Consecutive patients with complete IgA deficiency and 113 controls Niveloni i et al., 2007 Unselected consecutive patients attending small bowel clinic Ankelo et al., 2007 Selected CD patients and healthy controls NA NA IgA anti-ttg IgA DGP-AGA IgG AGA IgG DGP-AGA IgG anti-ttg IgG AGA IgG DGP-AGA 43% IgA anti-ttg IgA DGP-AGA IgG DGP-AGA NA IgA anti-ttg IgA DGP-AGA IgG AGA IgG DGP-AGA Eurospital Eurospital Radim Biofile In-house Biofile In-house 96.8% 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%

38 IgG anti-deamidated gliadin Preva alence Test Metho od Sensi itivity Speci ificity Volta et al., 2008 Selected CD (M3) and diseased control patients Villalta et al., 2007 Consecutive patients with complete IgA deficiency and 113 controls Niveloni i et al., 2007 Unselected consecutive patients attending small bowel clinic Ankelo et al., 2007 Selected CD patients and healthy controls NA NA IgA anti-ttg IgA DGP-AGA IgG AGA IgG DGP-AGA IgG anti-ttg IgG AGA IgG DGP-AGA 43% IgA anti-ttg IgA DGP-AGA IgG DGP-AGA NA Eurospital Eurospital Radim 96.8% 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-ttg IgA DGP-AGA IgG AGA IgG DGP-AGA Biofile In-house Biofile In-house 90% 92% 78% 75% 90% 90% 64% 98% => Detection of IgG DGP-AGA is both more sensitive and more specific than detection of IgG AGA

39 IgG anti-deamidated gliadin Preva alence Test Metho od Sensi itivity Speci ificity Volta et al., 2008 Selected CD (M3) and diseased control patients Villalta et al., 2007 Consecutive patients with complete IgA deficiency and 113 controls Niveloni i et al., 2007 Unselected consecutive patients attending small bowel clinic Ankelo et al., 2007 Selected CD patients and healthy controls NA NA IgA anti-ttg IgA DGP-AGA IgG AGA IgG DGP-AGA IgG anti-ttg IgG AGA IgG DGP-AGA 43% IgA anti-ttg IgA DGP-AGA IgG DGP-AGA NA Eurospital Eurospital Radim 96.8% 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-ttg IgA DGP-AGA IgG AGA IgG DGP-AGA Biofile In-house Biofile In-house 90% 92% 78% 75% 90% 90% 64% 98% => The sensitivity and specificity of IgG DGP-AGA was not superior to the much better characterized IgA anti-ttg

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

41 To Do s 1. Inform clinicians that the performance of the current IgA anti-ttg assay 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 ( and Euroimmune) on the consecutive non-iga deficient patients. 4. Replace IgG AGA with IgG DGP-AGA in patients with selective IgA deficiency. The cost of both assays is comparable.

42 Acknowledgements Dep. of Laboratory Medicine (UZ Leuven) Prof. Dr. X. Bossuyt Dr. G. Mariën Apr. D. Coenen Dep. of Gastroenterology (UZ Leuven) Prof. Dr. M. Hiele Dep. of Pathology (UZ Leuven) Prof. Dr. K. Geboes