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Richter et al: Antibodies in celiac diagnosis in children aged up to two years

JPGN Journal of Pediatric Gastroenterology and Nutrition Publish Ahead of Print DOI: 10.1097/MPG.0b013e31824678fc Determination of IgG and IgA antibodies against native gliadin is not helpful for the diagnosis of coeliac disease in children up to 2 years of age Thomas Richter1, Xavier Bossuyt2, Pieter Vermeersch2, Holm H. Uhlig3, Martin Stern4, Almuthe Hauer5, Klaus-Peter Zimmer6, Luisa Mearin7, Johanna Hendrika Clementina de Roo7, Cornelia Dhnrich8, and Thomas Mothes9

From the

Childrens Hospital of the Clinical Centre Sankt Georg Leipzig, Germany,

Dept. Laboratory Medicine of University Hospital Leuven, Belgium, 3 University Childrens Hospital Leipzig, Germany,
4

University Childrens Hospital Tbingen, Germany,

Univer7 8

sity Childrens Hospital Graz, Austria, 6 University Childrens Hospital Gieen, Germany, Department of Paediatrics, Leiden University Medical Centre, The Netherlands,

EUROIMMUN Medizinische Labordiagnostika GmbH Lbeck, Germany, 9 Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Germany

Address for correspondence: Professor Dr. Thomas Mothes, Department of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University of Leipzig, Liebigstrae 27, D-04103 Leipzig, Germany (e-mail: mothes@medizin.uni-leipzig.de)

T. Mothes, H. H. Uhlig, and C. Dhnrich submitted patents on methods for CD diagnosis (T. Mothes, A. Osman, H. H. Uhlig, T. Gnnel, A. Dietl: Peptide und Verfahren zur Diagnostik von Zliakie und Dermatitis herpetiformis and C. Probst, C. Dhnrich, W. Schlumberger, W. Stcker, L. Komorowski, T. Mothes: Verfahren und Immunabsorbentien

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Richter et al: Antibodies in celiac diagnosis in children aged up to two years

zur spezifischen Detektion und Absorption Zliakie- und Dermatitis herpetiformis assoziierter Antikrper). H. H. Uhlig was supported by the German Coeliac Society.

Key Words: antibodies, coeliac disease, deamidated gliadin, diagnosis, tissue transglutaminase

Abbreviations: AUC area under curve, CD coeliac disease, anti-dGli antibodies against deamidated gliadin, anti-nGli antibodies against native gliadin, anti-tTG antibodies against tissue transglutaminase, DOR diagnostic odds ratio, EmA endomysium antibodies, GFD gluten-free diet; NLR negative likelihood ratio, PLR positive likelihood ratio, ROC receiveroperating characteristics

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Richter et al: Antibodies in celiac diagnosis in children aged up to two years

ABSTRACT Objective: Assays for antibodies against native gliadin (anti-nGli) are still often assumed to perform better in the diagnosis of coeliac disease (CD) in young children than tests for antibodies to deamidated gliadin (anti-dGli), tissue transglutaminase (anti-tTG) and endomysium (EmA). We compared the performance of assays for anti-nGli, anti-dGli, anti-tTG and EmA in this age group. Methods: We investigated retrospectively 184 children (42 coeliacs under normal diet and 142 controls) up to 2 years of age. IgA and IgG-anti-dGli, IgA and IgG-anti-nGli, IgA and IgG-anti-tTG, and IgA-EmA were measured in serum. Areas under receiver-operating characteristics curves (AUCs), sensitivities, specificities, positive (PPV) and negative predictive values (NPV), positive and negative likelihood ratios (PLR and NLR), as well as diagnostic odds ratios (DOR) were calculated. Results: From all tests investigated, only assays for IgG-anti-dGli, IgA-anti-tTG and IgAEmA had high specificity ( 0.96) connected with high sensitivity ( 0.86), with high positive predictive values ( 0.52 and 0.69 at pre-test probabilities of 0.05 and 0.1, respectively), and with high negative predictive values ( 0.99 and 0.98 at pre-test probabilities of 0.05 and 0.1, respectively). These assays also showed high PLR ( 24) at low NLR ( 0.15) and high DOR ( 136). Conclusions: Our results do not support the use of assays of anti-nGli to diagnose CD in young children. IgA-anti-tTG, IgA-EmA and IgG-anti-dGli perform better than anti-nGli.

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Richter et al: Antibodies in celiac diagnosis in children aged up to two years

Assays for IgA antibodies against tissue transglutaminase (anti-tTG), endomysium (EmA) as well as for IgG antibodies against deamidated gliadin peptides (anti-dGli) in serum have a high sensitivity and specificity for coeliac disease (CD) in children (1,2). However, in young children (up to two years of age), antibodies against native gliadin (anti-nGli) are still often assumed to have the best performance. At young age, IgA-EmA have been reported to have a low sensitivity (3-8), with maximum values of 89 %. The sensitivity of IgA-anti-tTG ranged between 83 % and 90 % (1,6,8,9). Specificity of IgG-anti-nGli was only 77 % at high sensitivity (5). IgA-anti-nGli were claimed to be best in finding CD in young children (6) with sensitivity between 82 and 97 % and specificity between 88 and 94 % (1,5-8). The diagnostic performance of anti-dGli in very young children still needs to be established. Recently, it was shown that in children younger than 2 years with signs of chronic enteropathy and high serum levels of anti-nGli, but normal values of anti-tTG and of EmA, CD can be predicted by high serum levels of anti-dGli (10). The aim of our study was to compare the diagnostic performance of assays for IgA and IgGanti-dGli, for IgA and IgG-anti-nGli, for IgA and IgG-anti-tTG, and for IgA-EmA in the diagnosis of CD at young age.

MATERIALS AND METHODS Patients Sera of 184 children below two years of age with and without CD were retrospectively investigated. The patients were recruited from the Childrens Hospital of the Clinical Centre Sankt Georg Leipzig, (Germany), University Hospital Leuven (Belgium), University Childrens Hospitals of Leipzig, Tbingen, Mnchen and Gieen (Germany), Childrens University Hospital Graz (Austria), and from the Department of Paediatrics of the University Medical Centre Leiden (The Netherlands). The patients comprised 42 children with CD and 142 con-

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Richter et al: Antibodies in celiac diagnosis in children aged up to two years

trols (100 females and 84 males, mean age 1.34 years, range 0.4 to 2.0 years). Sera were withdrawn at the time of the diagnostic duodenal biopsies. All patients were biopsied under normal diet due to suspicion of CD or other gastrointestinal disorders. Intestinal pathology of all CD patients was in accordance with Marsh 2 or Marsh 3 criteria (11,12). All CD patients improved under gluten-free diet (GFD). The study was approved by the Ethical committee of the University of Leipzig as well as of the local Ethical committees of the participating centres. Antibody assays IgA and IgG antibodies against deamidated gliadin analogous fusion peptides (anti-dGli), anti-nGli, anti-tTG, and IgA-EmA were measured (blinded to the histological diagnosis) with test kits from EUROIMMUN Medizinische Labordiagnostika Lbeck, Germany. EmA were estimated by indirect immunofluorescence analysis using a combination of primate oesophagus, primate small intestine, and primate liver. The analyses were performed by EUROIMMUN. Cut-offs were as suggested by the manufacturer (Table 1). Statistics The data were evaluated by receiver operating characteristic (ROC) analysis. The area under the ROC-curves (AUC) was calculated. Differences between ROC-curves were evaluated by pairwise comparison according to Chi-Square analysis. An error probability P of less than 0.05 was considered statistically significant. Non-inferiority testing was performed if there was no statistically significant difference. For non-inferiority testing, the lower end of 90 per cent confidence intervals of differences between AUCs was considered. Non-inferiority was assumed if the lower end of this confidence interval was not below a zone of diagnostic indifference of 0.01. For the cut-offs suggested by the manufacturer, diagnostic accuracies, sensitivities, and specificities were calculated. Significance of differences (P < 0.05) was evaluated applying

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Richter et al: Antibodies in celiac diagnosis in children aged up to two years

McNemars test. For non-inferiority testing (see above), the proportions of children being false-negative or false-positive were compared between two tests. The z-test was applied for calculation of the 90 per cent confidence intervals of differences in proportions and for comparison of sensitivities and specificities in the different age groups. RESULTS From all tests investigated, only assays for IgG anti-dGli, IgA anti-tTG and IgA EmA had high specificity ( 0.96) connected with high sensitivity ( 0.86), with high positive predictive values ( 0.52 and 0.69 at pre-test probabilities of 0.05 and 0.1, respectively), and with high negative predictive values ( 0.99 and 0.98 at pre-test probabilities of 0.05 and 0.1, respectively, Table 1). The IgG-anti-tTG assay showed the highest specificity (0.993). The specificity of the two anti-dGli tests (0.972 and 0.965) was comparable with that of the assays for IgA-anti-tTG (0.972) and IgA-EmA (0.958). The specificity of the IgG-anti-nGli test was significantly lower than that of all other tests. The IgG-anti-tTG-test had a significantly higher specificity than the IgA-anti-nGli assay. The 2 anti-dGli assays were non-inferior to the IgG-anti-nGli test. The IgG-anti-nGli test exhibited the highest sensitivity (0.929). The sensitivity of the IgGanti-dGli test (0.857) was as high as that of the IgA-anti-tTG and the IgA-EmA assay, and higher than that of IgA-anti-dGli (0.810), IgA-anti-nGli (0.785), and IgG-anti-tTG (0.476) assays. The low sensitivity of the latter test differed significantly from that of all other tests. Positive likelihood ratios (PLR) were lowest for IgG-anti-nGli. These antibodies, however, showed the also lowest negative likelihood ratio (NLR). The diagnostic odds ratios (DOR) as an integrated measure of sensitivity and specificity were lowest for IgA and IgG-anti-nGli (48.4 and 26.3, respectively) and highest for IgA-anti-tTG

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Richter et al: Antibodies in celiac diagnosis in children aged up to two years

and IgG-anti-dGli (207 and 164, respectively). Similarly, ROC analysis revealed that from all antibody tests examined, the AUCs of the IgA-anti-tTG and of the IgG-anti-dGli assay were highest (0.951 and 0.950, respectively). Statistical evaluation of the ROC analysis showed that the IgG-anti-dGli assay was non-inferior to that of IgA-anti-nGli, IgG-anti-nGli, IgGanti-tTG and IgA-EmA. The IgA-tTG assay was noninferior to the IgA-anti-dGli, the IgAanti-nGli and the IgA-EmA assay. Mean concentrations of IgA and IgG-anti-dGli, of IgA-anti-tTG and of IgA-EmA were 50 fold higher in CD patients than in controls. Median concentrations were even increased at least 200 fold. For IgA and IgG-anti-nGli and for IgG-anti-tTG, the factor of increase was lower (Table 1). Positive and negative predictive values of the tests in the children up to two years of age are shown in Figure 1 as a function of pre-test probability. Positive predictive values of the antidGli, anti-tTG, and EmA tests were higher ( 0.52 at a pre-test-probability of 0.05 and 0.69 at a pre-test probability of 0.1) than that of anti-nGli assays ( 0.37 at a pre-test probability of 0.05 and 0.55 at a pre-test probability of 0.1). The negative predictive value of the IgG-antitTG test was lowest (0.97 at a pre-test probability of 0.05 and 0.94 at a pre-test probability of 0.1). Only IgA and IgG-anti-dGli, IgA-anti-tTG and IgA-EmA showed a high positive predictive value in combination with a high negative predictive value. In 19 (45 %) of our CD patients up to two years of age, all 7 antibody tests were positive (Table 2). In 12 further CD patients (29 %) all but the IgG-anti-tTG antibodies were above the cut-offs. In 3 of the CD patients antibody positivity was restricted only to IgG-anti-nGli antibodies. In further 2 patients none of the tests was positive. There were 6 children (14 %) with negative IgA-anti-tTG among the CD patients. Details of these patients are shown in Table 3. In 89 (63 %) of the controls up to 2 years of age, none of the tests was positive (Table 2). In further 35 controls (25 %) only the concentration of IgG-anti-nGli was elevated. In 3 controls

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Richter et al: Antibodies in celiac diagnosis in children aged up to two years

(2 %), only IgA- and IgG-anti-nGli were positive. In 2 controls (1 %) IgA-anti-tTG together with IgG-anti-nGli were elevated. In 2 further controls (1 %), all but IgA-anti-tTG and IgGanti-tTG were positive. In the remaining 11 controls, different combinations of antibody positivity were observed, which occurred only once. Altogether, there were 4 controls with increased IgA-anti-tTG. In all of them, concentration of IgA-anti-tTG was higher than twofold the cut-off. In 2 of them, also an elevated concentration of IgG-anti-dGli was found. In one of these 2 controls, the antibody concentration could be controlled after 19 months (both antibodies positive) and after 23 months (both antibodies negative).

DISCUSSION

Except for IgG-anti-nGli, the specificity of all antibody tests was high for CD for patients up to 2 years. However, the assays with high specificity (IgA- and IgG-anti-dGli and anti-tTG, IgA-anti-nGli and EmA) exhibited a low sensitivity, whereas the low specificity of the IgGanti-nGli test was associated with a high sensitivity. Using paired indicators of diagnostic performance such as sensitivity and specificity or PLR and NLR can be a disadvantage in comparing the performance of competing tests, especially if one test does not outperform the other on both indicators. The DOR can be used as a single indicator of diagnostic performance and combines the strengths of sensitivity and specificity as well as of PLR and NLR and is independent on prevalence (13). Regarding the DOR, the performance of the IgA and IgG anti-nGli was worst. For further evaluation of the tests, calculation of predictive values at defined pre-test probability is useful. In contrast to screening conditions with a prevalence of about 1 %, in paediatric gastroenterology centres a higher pre-test probability has to be assumed. At a pre-test probability of CD of about 0.05, all tests except the assays for IgA- and IgG-anti-nGli result in

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Richter et al: Antibodies in celiac diagnosis in children aged up to two years

post-test probabilities of at least 0.52 (Figure 1). The post-test probability of IgA- and IgGanti-nGli tests was below 0.4. A high positive predictive value reduces the number of patients who need to undergo endoscopy. The negative predictive value is very high for all tests ( 0.99) except for IgG-anti-tTG (0.97). A high negative predictive value assures that the number of CD patients among the children with negative test results is minimum (at most 1 %). Thus, from all tests investigated, only assays for IgG anti-dGli, IgA anti-tTG and IgA EmA had high specificity ( 0.96) connected with high sensitivity ( 0.86), with high positive predictive values ( 0.52 and 0.69 at pre-test probabilities of 0.05 and 0.1, respectively), and with high negative predictive values ( 0.99 and 0.98 at pre-test probabilities of 0.05 and 0.1, respectively). Therefore, these assays should be preferred over tests measuring anti-nGli. A selection bias in our study cannot be excluded. First of all, biopsies are more likely to be performed in symptomatic patients with positive results for CD specific antibodies such as IgA-EmA and IgA-anti-tTG, which favours these tests. Furthermore, most children in this study had not been re-challenged with gluten in order to confirm the diagnosis of CD, which was required according to the previous ESPGHAN criteria on the diagnosis of CD (14). Recently, it was suggested that routine gluten challenge in patients below two years of age is not necessary when patients have villous atrophy in combination with positive EmA (7). According to forthcoming ESPGHAN guidelines, a later gluten challenge should only be performed if villous atrophy was found in children below to years of age, who are negative for CD specific antibodies in order to confirm CD as a cause of the enteropathy (15). Six of our patients were classified as coeliacs in the absence of increased IgA-anti-tTG (however, one of them positive for IgA-EmA and this patient and another positive for routine assay of IgA-anti-tTG outside of this study). The latter finding shows that occasionally diverse results may be obtained when tests of different suppliers are compared (16). In fact, none of the remaining four patients was re-challenged with gluten until now, mainly due to their young age. According to

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Richter et al: Antibodies in celiac diagnosis in children aged up to two years

the ESPGHAN suggestions (15) such a provocation should not be performed before the age of 5 years. Among the 4 IgA-anti-tTG negative CD patients there was one child with selective IgA-deficiency. Interestingly, in this child not only IgA-anti-tTG, but also IgG-anti-dGli were negative. Another of the 4 patients was positive 64 days before but not at the day of endoscopy. The latter patient is suspected to have started too early the gluten-free diet. Due to the histological findings and to recovery of the children under GFD we have to assume, however, cannot guarantee that the IgA-anti-tTG negative CD patients are correctly classified. Mucosal lesions due to infections or allergy might have been considered as CD. Confirmation or exclusion of the diagnosis of CD is therefore needed and should be performed using clinical follow-up, exclusion of IgA deficiency, HLA-analysis and / or gluten provocation. On the other side, our finding of a high percentage (14 %) of IgA-anti-tTG negative children with CD (see Table 1) is in line with the observation of a lower sensitivity of antibody assays in young children (1,6,8,9). Although we included one of the largest number of very young children published so far, the study is not powered to detect potential differences between the tests with both high sensitivity and specificity (IgA-anti-tTG, IgA-EmA, IgG-anti-dGli). In conclusion, our results do not support the use of anti-nGli assays in the diagnosis of CD in children up to 2 years of age. IgA-anti-tTG, IgA-EmA and IgG-anti-dGli perform better than anti-nGli. Despite the good performance of these antibody tests, duodenal biopsy still represents the gold standard, to which these tests have to be compared, especially in young children, with the prospect of a life-long and life-altering diagnosis.

Acknowledgements: H. H. Uhlig was supported by the German Coeliac Society. Sibylle Koletzko (University Childrens Hospital, Mnchen, Germany) contributed patient samples and provided critical intellectual input.

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Richter et al: Antibodies in celiac diagnosis in children aged up to two years

Conflicts of interest: T. Mothes, H. H. Uhlig, and C. Dhnrich submitted patents on methods for CD diagnosis (T. Mothes, A. Osman, H. H. Uhlig, T. Gnnel, A. Dietl: Peptide und Verfahren zur Diagnostik von Zliakie und Dermatitis herpetiformis and C. Probst, C. Dhnrich, W. Schlumberger, W. Stcker, L. Komorowski, T. Mothes: Verfahren und Immunabsorbentien zur spezifischen Detektion und Absorption Zliakie- und Dermatitis herpetiformis assoziierter Antikrper).

REFERENCES 1. Prause C, Richter T, Koletzko S, et al. New developments in serodiagnosis of childhood celiac disease: assay of antibodies against deamidated gliadin. Annals N Y Acad Sci 2009a;1173:28-35. 2. Prause C, Ritter M, Probst C, et al. Antibodies against deamidated gliadin as new and accurate biomarkers of childhood coeliac disease. J Pediatr Gastroenterol Nutr 2009b;49:52-8. 3. Brgin-Wolff A, Gaze H, Hadziselimovic F, et al. Antigliadin and antiendomysium antibody determination for coeliac disease. Arch Dis Child 1991;66:941-7. 4. Ghedira I, Sghiri R, Ayadi A, et al. Anticorps anti-endomysium, anti-rticuline et antigliadine, intrt dans le diagnostic de la maladie coeliaque chez lenfant. Pathol Biol 2001;49:47-52. 5. Tonutti E, Visentini D, Bizzaro N, et al. The role of antitissue transglutaminase assay for the diagnosis and monitoring of coeliac disease: a FrenchItalian multicentre study. J Clin Pathol 2003;56;389-93.

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Richter et al: Antibodies in celiac diagnosis in children aged up to two years

6. Lagerqvist C, Dahlbom I, Hansson T, et al. Antigliadin immunoglobulin A best in finding celiac disease in children younger than 18 months of age. J Pediatr Gastroenterol Nutr 2008;47:428435. 7. Wolters VM, van de Nadort C, Gerritsen SAM, et al. Is gluten challenge really necessary for the diagnosis of celiac disease in children younger than age 2 years? J Pediatr Gastroenterol Nutr 2009;48:56670. 8. Maglio M, Tosco A, Paparo F, et al. Serum and intestinal celiac disease-associated antibodies in children with celiac disease younger than 2 years of age. J Pediatr Gastroenterol Nutr 2010;50:43-8. 9. Basso D, Guariso G, Fogar P, et al. Antibodies against synthetic deamidated gliadin peptides for celiac disease diagnosis and follow-up in children. Clin Chem 2009;55:150-7. 10. Barbato M, Maiella, G, Di Cam illo C, Guida S, Valitutti F, Lastrucci G, Mainiero F, Cucchiara S. The anti-deamidated gliadin peptide antibodies unmask celiac disease in small children with chronic diarrhoea. Dig Liver Dis 2011;43:465-469 11. Marsh MN. Gluten, major histocompatibility complex, and the small intestine. A molecular and immunobiologic approach to the spectrum of gluten sensitivity (celiac sprue). Gastroenterology 1992;102:33054. 12. Oberhuber G, Granditsch G, Vogelsang H. The histopathology of coeliac disease: time for a standardized report scheme for pathologists. Eur J Gastroenterol Hepatol 1999;11:1185 94. 13. Glas AS, Lijmer JG, Prins MH, Bonsel GJ, Bossuyt PMM. The diagnostic odds ratio: a single indicator of test performance. J Clin Epidemiol 2003;56:11291135.

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Richter et al: Antibodies in celiac diagnosis in children aged up to two years

14. Walker-Smith JA, Guandalini S, Schmitz J, et al. Revised criteria for diagnosis of coeliac disease. Report of Working Group of European Society of Paediatric Gastroenterology and Nutrition. Arch Dis Child 1990;65:909-11. 15. Husby S, Koletzko S, Korponay-Szab IR, et al. ESPGHAN guidelines for the diagnosis of coeliac disease in children and adolescents. An evidence-based approach. J Pediatr Gastroenterol Nutr (ahead of print), DOI: 10.1097/MPG.0b013e31821a23d0 16. van Meensel B, Hiele M, Hoffman I, et al. Diagnostic accuracy of ten second-generation (human) tissue transglutaminase antibody assays in celiac disease. Clin Chem 2004;50:2125 2135.

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Richter et al: Antibodies in celiac diagnosis in children aged up to two years

FIGURE LEGENDS

FIGURE 1. Predictive values of the different antibody assays in the diagnosis of CD in children up to two years of age in dependence on pre-test probability.The thick grey line in the lower diagram collectively indicates negative predictive values of IgG-anti-dGli, IgA-anti-tTG as well as IgA-EmA, which are very similar. The vertical dotted line indicates a pre-test probability of 0.05.

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Richter et al: Antibodies in celiac diagnosis in children aged up to two years

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Richter et al: Antibodies in celiac diagnosis in children aged up to two years

Table 1

TABLE 1. Performance of different antibody tests in children aged up to 2 years IgAIgGIgAIgGIgAanti-dGli anti-dGli anti-nGli anti-nGli anti-tTG Test-No. 1 2 3 4 5 Cut-off (U/L) AUC Specificity Sensitivity PLR NLR DOR 95 % CI AbC CD (U/L) Mean Median; range AbC Co (U/L) Mean Median; range 3.46
1.0; 0.0-94.3

IgGanti-tTG 6 1.0 0.912 0.9933,45 67.6 0.528 128


16.4 to 1004

IgAEmA* 7 10 0.925 0.9584 20.3 0.149 136


41.4 to 447

25.0 0.936 0.97234 0.8106 28.7 0.196 147


41.7 to 515

25.0 0.9503,4,6,7 0.96534 0.8576 24.3 0.148 164


47.5 to 569

25.0 0.920 0.930 11.2 0.231 48.4


18.2 to 129
4,6

25.0 0.931 0.669 2.81 0.107 26.3


7.72 to 89.5
1,2,3,5,6,7

20.0 0.9511,3,7 0.972 30.4 0.147 207


55.5 to 773
4

0.7864,6

0.92913,6

0.8576

0.4761,2,3,4,5,7 0.8573,6

437
580; 0.4-711

296
286; 0.8-700

252
200; 1.0-551

241
200; 3.3-520

427
457; 0.5-700

1.13
0.0-5.5

773
320; 0-10000

5.62
1.4; 0.2-173

9.01
1.5; 0.3-191

35.73
10.0; 0.6-220

3.40

0.09

1.06

0.74; 0.0-98.5 0.04; 0.00-1.37 0; 0-100

* For EmA the reciprocal values of the titres are presented. CI: Confidence interval; AbC CD: Antibody concentration in CD patients; AbC Co: Antibody concentration in control patients. Superscripts denote significant differences to tests with the respective numbers. Subscripts denote non-inferiority to the tests with the respective numbers. Results of non-inferiority tests only shown if there was no statistically significant difference.

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Richter et al: Antibodies in celiac diagnosis in children aged up to two years

Table 2

TABLE 2. Antibody profiles in CD patients and controls aged up to 2 years IgAanti-dGli CD + + Controls + IgGantidGli + + + IgAantinGli + + + + IgGantinGli + + + + + + + IgAanti-tTG + + + IgGanti-tTG + IgAEmA + + + Number of patients 19 12 3 2 89 35 3 2 2

For sake of clarity, from the total of 184 patients, profiles are only shown if occurring more than in one patient. Six CD patients and 11 control patients had unique antibody profiles.

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Richter et al: Antibodies in celiac diagnosis in children aged up to two years

Table 3

TABLE 3. Patients with negative IgA-anti-tTG classified as CD No 1 2 3 4 Total IgA sIgAD Normal Normal Normal HLA DQ8 n.d. n.d. n.d. Histology IEL Marsh 3A >40 Marsh 2 >40 Marsh 3C >40 Marsh 3C >40 Other positive Further findings antibodies IgG-anti-nGli Routine test of IgAanti-tTG negative None IgA-anti-tTG, IgA-EmA, IgA- and IgG-anti-dGli positive 64 days before endoscopy. Preterm GFD? None Routine test of IgA-anti-tTG negative IgG-anti-nGli Routine tests of IgA-anti-tTG and IgA-EmA positive. Progressive decrease in total IgA during follow up IgG-anti-nGli Routine test of IgA-anti-tTG negative IgA-anti-nGli Routine tests of IgA-anti-tTG IgG-anti-nGli and IgA-EmA positive IgG-anti-dGli IgA-EmA

5 6

Normal DQ2 Unknown n.d.

Marsh 3B >40 Marsh 3A >40

None of the patients was re-challenged with gluten. sIgAD: secretory IgA-deficiency. n.d.: not determined. Routine tests: performed in clinical routine at the time of endoscopy outside of this study. Lack of IgA improbable since IgA-anti-nGli and IgA-EmA increased.

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