Coeliac disease (CD) is an immunologically mediated intolerance against gluten from wheat and related cereal proteins in genetically predisposed individuals with a prevalence close to 1%. The ethanol-extractable fraction of wheat gluten is called gliadin (Gli). Ingestion of the cereal proteins by coeliac patients induces small intestinal inflammation, villous atrophy, and crypt hyperplasia with typical symptoms of malabsorption to atypical symptoms and conditions that can affect any organ system. Because of atypical symptoms and even silent forms, the disorder often remains undiagnosed and carries the risk of long-term complications. The diagnosis of CD is based on the assessment of the highly variable clinical status, the histological evaluation of intestinal biopsy material, antibody measurements, and response to gluten-free diet (1).
Until now, the histological evaluation of the duodenal mucosal tissue has been regarded as the reference standard for diagnosis. The tissue has to be obtained bioptically and is evaluated according to strict criteria (2). However, although generally regarded as safe, the procedure to obtain biopsy is stressful for the patients, especially at a young age, time consuming, and expensive. Some patients are reluctant to undergo intestinal biopsy. The interpretation of histological findings is subjective, requires experience, and is not completely free from failure (3). Finally, the histological damage of the mucosal tissue is not specific for CD, but clinical and serological recovery after gluten-free diet has to be demonstrated.
Serological diagnosis is based on estimation of antibodies against native Gli (anti-nGli) and of autoantibodies against tissue transglutaminase (anti-tTG) and against endomysium (EmA). However, test systems using native Gli have a low diagnostic sensitivity and specificity for CD. Since the discovery of tissue transglutaminase (tTG) as an autoantigen (4), sensitive and specific diagnostic assays for estimation of anti-tTG have been developed (5–8), and anti-nGli assays have lost importance. However, it was shown that antibodies of patients with CD specifically recognize deamidated Gli peptide (9,10). The validity of gliadin antibodies in the diagnosis of CD can be increased strongly if short dGli are applied as antigens for detection of antibodies. Such dGli are considered to be produced in vivo by the action of tTG under the inflammatory conditions prevailing in the small intestinal mucosa of patients with coeliac disease, who are under a normal gluten-containing diet. Several nonapeptide sequences containing a proline-glutamate-glutamine (PEQ) tripeptide core were found to be useful in a highly specific and highly sensitive luminescence assay for CD diagnosis (11,12). Recently, an assay was described applying dGli (the sequence of which is kept proprietary) for estimation of antibodies in the blood of patients with CD. This test correlated well with assays for EmA and anti-tTG. Most sera negative for autoantibodies but false positive for conventional anti-nGli were true negative for antibodies against dGli (anti-dGli) (13). From a study with sera from biopsy-controlled adult patients, it was concluded that the new “high performance anti-gliadin antibody assay” was superior in sensitivity and specificity compared with standard Gli antibody assays and agreed much better with the determination of anti-tTG and EmA (14). Meanwhile, further publications appeared on the use of anti-dGli in the diagnosis of CD (12–20).
We investigated whether the new tests for anti-dGli reach a similar high accuracy as described for the autoantibody assays. We studied children, in whom the diagnosis of CD was confirmed or excluded by means of intestinal biopsy. Until now, there are only rare data on the performance of tests for anti-dGli in a population of children. Furthermore, we raise the question whether the new antibody assays may replace biopsy at least for some of the patients.
Detection of antibodies against dGli type 1 (anti-dGli1): Synthetic DNA coding for Gli analogous fusion peptides (GAF-3X) was cloned into a prokaryotic expression vector and expressed in Escherichia coli. GAF-3X includes repetitive modified copies of the previously described peptides PLQPEQPFP and PEQLPQFEE. The first peptide sequence recognises the main part of patients with CD, whereas the second peptide sequence additionally increases sensitivity (11). Microtitre plates were coated with the GAF-3X preparation (0.5 μg/mL in 100 mmol/L sodium carbonate, pH 9.6), kept overnight at 4°C, washed with phosphate-buffered saline containing 0.05% (w/v) Tween-20, and blocked for 2 hours with phosphate-buffered saline containing 0.1% (w/v) casein. Serum diluted 1:200 in phosphate buffered saline with 0.1% (w/v) casein was added and allowed to react with the peptides for 30 minutes at room temperature. Bound antibodies were detected using anti-human IgA or IgG peroxidase conjugate (codes EV 3011-9601A GAF-3X and EV 3011-9601G GAF-3X, respectively, EUROIMMUN Medizinische Labordiagnostika AG, Lübeck, Germany).
Detection of antibodies against dGli type 2 (anti-dGli2): Synthetic glutamine-glutamate substituted Gli homologous peptides (deamidated gliadin peptides [DGP]), which contain less than 35 amino acids (sequences not disclosed) were fixed on the solid phase (14). Serum was diluted 1:100. After incubation, bound human immunoglobulins (Igs) were detected by goat anti-human IgA or IgG antibodies conjugated with peroxidase (Quanta Lite Gliadin IgA II and Quanta Lite Gliadin IgG II, INOVA Diagnostics, San Diego, CA).
Finally, in both tests tetramethylbenzidine was added as chromogen and the reaction was stopped by the addition of sulphuric acid after 15 minutes (test 1) or 30 minutes (test 2). The absorbance was read in bichromatic mode at 450 nm with 620 nm as reference wavelength.
For comparison the concentration of antibodies against native Gli (IgA-anti-nGli and IgG-anti-nGli, EV3011-9601A/G, EUROIMMUN) and of IgA-anti-tTG and IgG-anti-tTG (EA1910-9601A/G, EUROIMMUN) was determined. All tests were run in duplicate and the means of the measurements were reported.
All antibody measurements were performed blinded to the results of the histological analysis of intestinal biopsy and to clinical data.
The sera of the patients were studied retrospectively. The sera were collected in 6 centres for paediatric gastroenterology (Children's Hospitals in Leipzig, Dresden, Tuebingen, Graz, and Giessen) from July 1998 to July 2007. All patients were on a gluten-containing diet. Biopsies were taken as part of routine diagnostic upper intestinal tract endoscopy due to suspicion of gastrointestinal disorders. Routine antibody measurements were performed independently from the assays reported in this article. Experienced pathologists in the different participating centres performed evaluation of intestinal biopsies routinely. The pathologists were informed about clinical findings and/or routine antibody data, but they were unaware of antibody measurements performed in the present study. At least 3 tissue pieces were used for histological evaluation. Blood was taken at the time of biopsy.
The group of 142 patients with CD comprised 86 females and 56 males (mean age 7.7 years) on a normal gluten-containing diet. Histological analysis of duodenal biopsies was performed according to the revised criteria of the European Society of Pediatric Gastroenterology, Hepatology, and Nutrition (2,21,22). Intestinal pathology of all patients was in accordance with Marsh 2 or Marsh 3 criteria. The control group comprised 160 subjects (78 females, 82 males, mean age 9 years) on a normal gluten-containing diet. These children underwent biopsy due to suspect intestinal disorders, but their histological findings did not point to CD. There were 19 patients with inflammatory bowel disease in the control group. Apart from 4 patients with Crohn duodenitis, there was no villous atrophy and no crypt hyperplasia found in this group. A density of more than 30 intraepithelial lymphocytes per 100 epithelial cells was considered enhanced (Marsh ≥1) (19).
The diagnostic performance of the tests was assessed by receiver-operating characteristic (ROC) analysis and calculation of area under the curve (AUC), in percent. The significance of differences between AUC was calculated by using χ2 statistics. Significance of differences between accuracies and between sensitivities was estimated according to McNemar applying 2 × 2 contingency tables containing the number of patients classified correctly or incorrectly.
Cutoffs were defined such that the sum of sensitivity and specificity was maximum (equal to cutofflow), or that specificity was >99% (equal to cutoffhigh). Diagnostic accuracy (in %) was calculated as the sum of true positives and true negatives divided by the total number of tested individuals. Confidence intervals (CI) are reported for the 95% range. SigmaPlot 10 and SigmaStat 3.5 software were applied.
Anti-dGli Are Superior to Anti-nGli
From the 8 antibody species measured, IgG-anti-dGli1 and IgG-anti-dGli2 have the largest AUC (≥98.6%) (Table 1). The AUC of anti-dGli (IgG as well as IgA) is significantly larger than the AUCs of IgG- and IgA-anti-nGli (P = 0.008). All anti-dGli tests, irrespective of Ig class, have a significantly higher accuracy than the IgA- and IgG-anti-nGli assays (P = 0.008).
IgG-anti-dGli Are Superior to IgA-anti-dGli and IgG-anti-tTG
Anti-dGli1 and anti-dGli2 of the IgG class have a significantly larger AUC than the corresponding antibodies of the IgA class (P = 0.031). The error probabilities for comparison of accuracies IgG-anti-dGli1 and 2 with IgA-anti-dGli are 0.009 and 0.002, respectively. The AUC and the accuracy of the 2 IgG-anti-dGli assays do not differ significantly (P = 0.584 and P = 0.579). Both IgG-anti-dGli assays have higher AUC (P ≤ 0.001) accuracies than the IgG-anti-tTG test (P < 0.001).
IgG-anti-dGli Are Comparable to IgA-anti-tTG
There is no significant difference between AUC of IgG-anti-dGli1 (P = 0.534) or IgG-anti-dGli2 (P = 0.318) and IgA-anti-tTG, which was until now considered to be the most accurate antibody species for CD. Thus, ROC analysis shows that the IgG antibodies against dGli and the IgA antibodies against tTG are the best indicators for CD (Table 1). The CI of AUC for IgA-anti-tTG is wider than that of IgG-anti-dGli1 and IgG-anti-dGli2 and includes the CIs of both antibody species against dGli completely. For further comparison of these antibodies the CIs of the differences in AUC between IgA-anti-tTG and IgG-anti-dGli were calculated. Furthermore, a zone of diagnostic indifference of 1% around the AUC of IgA-anti-tTG was defined. The lower limit of the 90% CIs are located in the indifference zone (one-sided test). Therefore, IgG-anti-dGli1 and IgG-anti-dGli2 are noninferior with 95% probability to IgA-anti-tTG concerning AUC.
IgG-anti-dGli1, IgG-anti-dGli2, and IgA-anti-tTG are the only assays that reach sensitivity as well as specificity values above 93%. Diagnostic accuracy is highest for IgA-anti-tTG and the 2 IgG-anti-dGli species (≥94.7%). The differences in diagnostic accuracy between IgA-anti-tTG and the 2 IgG-anti-dGli are 1.0 and 2.0%, respectively. The differences between the 3 assays are not significant (P = 0.149).
A Large Proportion of Patients Can Be Diagnosed as Coeliacs With High Specificity Solely by 1 Antibody Assay (IgG-anti-dGli or IgA-anti-tTG)
The specificity of the 2 tests for IgG-anti-dGli and IgA-anti-tTG can be enhanced up to more than 99% by increasing the cutoffs. More than two thirds of the patients with CD remain true positive above cutoffhigh (Table 2). The CI for 99.4% specificity extends from 96.6% to 100.0%, thus leaving a gap of uncertainty of only 3.4%.
Combination of IgG-anti-dGli Assay With IgA-anti-tTG Test Increases the Performance
The accuracies are increased when the results of 2 tests (1 of the IgG-anti-dGli assays together with the IgA-anti-tTG test) are evaluated in combination (Table 3). For this, a straight line with negative slope was calculated for a plot of IgA-anti-tTG versus IgG-anti-dGli separating positives from negatives so that there was none, 1, or a minimum number of outliers (sum of false positives and false negatives).
A maximum accuracy of 97.4% is reached. This means an increase of 2.7%, 1.7%, and 0.7% for IgG-anti-dGli1, IgG-anti-dGli2, and IgA-anti-tTG, respectively (compare Tables 1 and 3). Thus, the inaccuracy is reduced by 20%. The number of falsely classified patients by the IgA-anti-tTG test is reduced from 10 to 8.
Under the condition of high specificity, an increase in sensitivity of at least 9.8% is found when the IgA-anti-tTG is considered in combination with 1 of the 2 IgG-anti-dGli tests (compare Tables 2 and 3). This increase is significant (P < 0.001). The combination of both IgG-anti-dGli tests does not lead to improved results compared with single parameter consideration.
There were 19 patients with inflammatory bowel diseases among the controls, 4 of them with Crohn duodenitis. None of these controls was false positive in the IgG-anti-dGli or IgA-anti-tTG tests. One of the 3 controls, who tested positive for IgG-anti-dGli1, IgG-anti-dGli2, as well as IgA-anti-tTG, was a 2-year-old male with diabetes mellitus type 1 and diarrhoea, whose duodenal histology did not point to CD (Marsh 0). He was also positive for IgG-anti-tTG, IgA-anti-dGli1, IgA-anti-dGli2, and IgA-EmA. Another control patient, also false positive for IgG-anti-dGli1, IgG-anti-dGli2, and IgA-anti-tTG, had increased numbers of intraepithelial lymphocytes (Marsh 1). There was 1 female coeliac patient (16 year) with IgA deficiency. She was negative for all IgA-based ELISAs, but strongly positive for IgG-anti-tTG, IgG-anti-dGli1, and IgG-anti-dGli2.
The primary aim of our study was to compare diagnostic sensitivities and specificities of several tests for childhood CD diagnosis, summarized as accuracies for a near one-to-one ratio between patients and controls. A similar ratio between patients with CD and controls for the study of the performance of antibody tests was described (14,19). Our conclusions apply for selected patients, who were subjected to endoscopy due to suspicion of gastrointestinal diseases. From CD patients included, most have been preselected by previous antibody assays (particularly by estimation of IgA tissue transglutaminase or endomysium antibodies), which may even lead to overestimation of accuracy of IgA-anti-tTG in our study.
Reports on the performance of the new generation of Gli antibodies in diagnosis of childhood CD are still rare. There were only 2 previous studies on childhood CD, in which only the assay of anti-dGli2 and anti-tTG was applied (15,18). Our investigation compared 2 different deamidated antigens and is the most comprehensive study on the performance of anti-dGli for diagnosis in children until now. One of the above-cited papers reports on sensitivity and specificity of IgG-anti-dGli2 (15), which were comparable to our results.
The tests for anti-dGli clearly overcome the disadvantage of low specificity and low sensitivity, which is known to be inherent in antibodies against native, unmodified Gli (anti-nGli). The 2 IgG assays for anti-dGli perform similarly. The performance of the IgG class of anti-dGli is better than that of the IgA class. This is surprising because in autoantibody tests the IgA class performs better than the IgG class. The reasons for this remain unclear. The 2 IgG tests for anti-dGli perform similarly to the assay of IgA-anti-tTG.
The cutoffs were calculated from ROC analysis under the condition of cutoffhigh and that the groups of patients and of controls have approximately the same size. For a more detailed description of the performance of the IgG-anti-dGli and IgA-anti-tTG tests, we suggest specifying 2 cutoffs, cutofflow and cutoffhigh. For 99.4% specificity the CIs extend over a range of only 3.4%. Above the cutoffhigh, the probability for misdiagnosis of CD is low. It cannot be excluded that difficulties in proper histological evaluation together with other causes, like inappropriate diet at the time of blood withdrawal, mix-up of samples, and pipetting errors, may account for this small range of uncertainty. Recently, a disagreement between independent histological evaluations in about 3% of 271 patients was reported (3). Thus, accuracy values very slightly below 100% cannot be taken per se as an argument of insufficiency of antibodies due to an inadequate biological assay principle or that antibodies are a worse indicator for the disease than histology. In children with IgG-anti-dGli or IgA-anti-tTG concentrations between cutoffhigh and the cutofflow, serological diagnosis should be followed by intestinal biopsy. It should be kept in mind, however, that antibody concentrations in less than 5% of patients with CD are below the cutofflow (Table 1). In our study population, there is a high proportion of patients (>67%) in whom concentrations of IgG-anti-dGli or of IgA-anti-tTG were above the cutoffhigh, so that in these patients, no invasive biopsy seems to be necessary to confirm the diagnosis of CD.
The percentage of patients, in whom no confirmatory biopsy is necessary, can be increased up to more than 78% when the IgA-anti-tTG test is evaluated in combination with one of the IgG-anti-dGli assays. Furthermore, such a combined evaluation enhances accuracy (calculated for maximum sum of sensitivity and specificity) from 96.7 to 97.4, thus, diminishing the fraction of falsely classified subjects by more than 20% from 3.3% to 2.6%.
A diagnostic approach avoiding intestinal biopsy at high antibody concentrations was proposed earlier (23–26); however, it has not become accepted yet. This approach should be discussed anew in the background of the new IgG-anti-dGli tests.
Diabetes mellitus type 1 is a condition highly associated with CD (27). The antibody-positive 2-year-old boy among the controls with diabetes mellitus type 1 and diarrhoea, but without histological changes, may have latent CD (28) and has to be followed up for possible later development of CD. In this patient, IgA-EmA and IgA-anti-tTG were persistently increased over more than 1 year, arguing against transient antibody elevation. Transient elevation of autoantibodies was described in patients with genetic risk for CD (29). There was another antibody-positive control patient with increased numbers of intraepithelial lymphocytes (Marsh 1). Such an increase is typical in early-stage untreated coeliac disease. Unfortunately, this finding is unspecific (30).
To conclude, the 2 tests based on dGli represent a valuable alternative and complement for autoantibody assays like estimation of IgA-anti-tTG. The use of the IgG-anti-dGli tests may be advantagous in case of IgA-deficiency. In either case, the new antibody tests should displace the conventional techniques of determination of antibodies against native Gli. The validity of the 2 tests for IgG-anti-dGli cannot be distinguished from that of the IgA-anti-tTG assay. The diagnostic accuracy can be enhanced by combination of IgG-anti-dGli tests with the IgA-anti-tTG test. There are assays combining estimation of IgA/IgG anti-tTG and of anti-dGli2 within 1 microplate well. Such tests were described as being highly accurate. However, the superiority of such tests above single determinations of IgG-anti-dGli or IgA-anti-tTG has not been proven statistically (15,19). Our findings show that high concentrations of these antibodies per se indicate CD and biopsy will be not mandatory for a significant number of patients. Of course, the value of the IgG-anti-dGli tests alone or in combination with IgA-anti-tTG to replace biopsy in (at least a substantial) some of the patients has to be confirmed in a larger prospective study. Similarly, the use of the new tests for screening purposes, monitoring of diet compliance, follow-up, detection of IgA-deficient patients with CD, and the test performance at low age has to be investigated in further studies.
Thanks to Christian Wittekind (Institute of Pathology of the Medical Faculty of the University of Leipzig, Germany), Antje Werner and Volker Wiechmann (Institute of Pathology of the Municipal Hospital ‘Sankt Georg’ Leipzig) for critical re-evaluation of intestinal biopsy material. The test kits are produced by EUROIMMUN AG. The INOVA testkits are distributed by A. Menarini Diagnostics, Berlin-Chemie, Germany.
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