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Keywords:

  • antibodies;
  • coeliac disease;
  • endomysium;
  • gliadin;
  • screening;
  • transglutaminase

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Subjects and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Abstract. Lagerqvist C, Ivarsson A, Juto P, Persson LÅ & Hernell O (Umeå University, Umeå, Sweden). Screening for adult coeliac disease – which serological marker(s) to use? J Intern Med 2001; 250: 241–248.

Objective. To determine which serological marker(s) to use when screening for coeliac disease.

Design. In a population-based cross-sectional study we compared the use of antigliadin antibodies (AGA) of isotypes IgA and IgG, antiendomysial antibodies (AEA) of isotype IgA and antitransglutaminase antibodies (ATGA) of isotype IgA for detecting coeliac disease amongst adults.

Setting. Northern Sweden.

Subjects. A total of 1850 of 2500 (74%) invited adults (aged 25–74 years) who were randomly selected from the population register after stratification for age and sex.

Main outcome measures. The sensitivity, specificity and predictive values of the AGA, ATGA and AEA tests.

Results. Nine cases of biopsy proven, previously undiagnosed coeliac disease were detected by screening. The sensitivity of both ATGA and AEA was 100% whilst AGA IgA and IgG both had a sensitivity of 89%. The AEA test had a specificity of 100% whereas the specificities of the ATGA, AGA IgA and IgG tests were 97, 96 and 78%, respectively. The positive predictive value for the AEA test was 100%, whereas it was considerably lower for the other tests (ATGA > AGA IgA > AGA IgG), with further decreases for all tests when shifting from a clinical to a screening situation.

Conclusions. When screening for coeliac disease we suggest a serial testing approach, i.e. an initial ATGA test and, when positive, followed by an AEA test, provided that IgA deficiency has been excluded. However, assessment of the small intestinal mucosal morphology is still required to ascertain the diagnosis.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Subjects and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Coeliac disease is a permanent intolerance to gluten, a major protein fraction in wheat, and to related proteins in rye, barley and possibly also oats. In genetically predisposed individuals a gluten-containing diet may lead to small intestinal mucosal lesion, the so-called villous atrophy, which resolves on a strict gluten-free diet [1].

It has become apparent in many European countries and the USA that coeliac disease is a public health problem [2–7]. In older children and adults the symptoms are often vague, and consequently the diagnosis is easily neglected [8]. A correct diagnosis is, however, important, as untreated coeliac disease leads to increased morbidity [1, 8, 9]. Screening for coeliac disease in the general population has been discussed [10–13], but to our knowledge no such programme has yet been initiated in the public health sector of any country.

The coeliac disease diagnosis is based on a small intestinal biopsy demonstrating the mucosal lesion whilst the patient is on a normal, gluten-containing diet, and preferably also a second biopsy showing restitution of the mucosa on a gluten-free diet [9]. In a screening-situation it is necessary to have simple, objective and reliable laboratory tests. The serological tests most commonly used in clinical routine are antigliadin antibodies of isotypes IgA and IgG (AGA IgA and IgG), and antiendomysial antibodies (AEA) of isotype IgA [14]. The AEA test has been the most reliable, with both high sensitivity and specificity [14, 15], but the immunofluorescence technique used is labour intensive, expensive and dependent on an experienced microscopist. Therefore a serial testing approach has been suggested: analysis of AGA and, if that is positive, analysis of AEA [6, 9, 16].

It was recently suggested that the autoantigen recognized by the AEA is tissue transglutaminase (tTG) [17], a calcium-dependent enzyme that catalyses cross-linking and deamidation of proteins. Interestingly, gliadin is an excellent substrate for tTG [18]. It has been suggested that tTG is involved in the pathogenesis of coeliac disease in two different ways: (i) cross-linking of dietary gliadin resulting in gliadin–gliadin or gliadin–tTG complexes, which may contain neoepitopes that trigger the immune response [17], and (ii) deamidation of certain glutamine residues of α-gliadin, creating epitopes that bind to HLA-DQ2 and are recognized by disease specific intestinal T cells [18]. Thus, from a theoretical perspective analysis of anti-tTG antibodies (ATGA) should be extremely useful as a diagnostic tool for coeliac disease. Consequently, enzyme-linked immuno-sorbent assay (ELISA) methods for ATGA have been developed and evaluated in different series of patients. However, the outcome has varied considerably [19–24].

We previously performed a population-based cross-sectional study of coeliac disease amongst Swedish adults revealing a prevalence of 5.3 per 1000, the majority of which had not been diagnosed prior to the screening [6]. The aim of the present study was to assess which serological marker(s) to use; AGA IgA and IgG, AEA or ATGA, alone or in combination, in a tentative future general screening for coeliac disease in adults.

Subjects and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Subjects and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Subjects

This population-based cross-sectional study was performed in the two northernmost counties (Västerbotten and Norrbotten) of Sweden in conjunction with the 1994 WHO MONICA survey (The World Health Organization project of Multinational Monitoring of Trends and Determinants in Cardiovascular Disease) [25]. The study population constituted all the 318 359 inhabitants (December 1994) in the age range 25–74 years. A sample of 2500 individuals, stratified by age and sex, was randomly selected from the population register. From each 10-year age cohort (25–34, 35–44, 45–54, 55–64 and 65–74 years, respectively), 250 men and 250 women were invited to participate.

Out of those 2500 invited, 1850 (74%) were finally included in the study. Noninclusion was the result of nonparticipation (n=579), lack of sera (n=65), already diagnosed coeliac disease (n=2) or IgA-deficiency (n=4), defined as s-IgA below 0.05 g L–1. Blood samples were obtained by venipuncture and sera were stored at –20°C until analysis.

Serological markers

Antigliadin antibodies of isotypes IgA and IgG (AGA IgA and IgG). Antigliadin antibodies IgA and IgG were analysed by ELISA, as previously described [26], with minor modifications. The cut-off value for a positive outcome was defined as the mean value of the optical densities (OD) of six negative sera multiplied by 2.1 (corresponding to the mean +2 SD of the absorbance values). When a cut-off value was below 0.1, it was given the value 0.1. For practical reasons the antibody content of each serum was expressed as an index value, i.e. the OD of the test serum divided by the cut-off value. Index values of 1.0 and above were considered positive. The AGA IgA and IgG tests had intra-assay coefficients of variation (CVs) of 3.6% (n=10) and 6.3% (n=10), respectively, and interassay CVs of 10% (n=13) and 9.5% (n=13), respectively.

Antiendomysial antibodies (AEA) of isotype IgA. Antiendomysial antibodies was analysed by immunofluorescence technique as previously described [6] using monkey oesophagus as antigen source (SciMedx, Denville, NJ, USA). A high positive control was run on every analysis occasion, and all AEA slides were read by the same investigator. Positive slides were reanalysed using serial dilution (1/20, 1/80 and 1/320). Serum samples showing fluorescence when diluted 1/20 or more were considered positive.

Antitransglutaminase antibodies (ATGA) of isotype IgA. Antitransglutaminase antibodies was analysed by ELISA-technique. A stock solution was made by dissolving 2 units of tTG from guinea-pig liver (lot 38H7822, Sigma Chemical Co., St. Louis, MO, USA) in 1 mL of sterile PBS (phosphate-buffered saline) and was then stored at –70°C until use. The stock solution was diluted 100-fold in 50 mmol L–1 Tris–HCl, 150 mmol L–1 NaCl, 5 mmol L–1 CaCl2, pH 7.5, and microtiter plates (Immulon® 1B, Dynex Technologies, Inc., Chantilly, VA, USA) were coated by adding 100 μL to wells in odd columns. Even columns received no antigen and represented control wells. The plates were left at 4°C overnight and were thereafter washed once with deionized water using a microplate washer (Wellwash 4, Denley Instr. Ltd, West Sussex, UK). Unreacted sites were blocked at room temperature for 60 min with 200 μL PBS containing 0.05% Tween 20 and 1% bovine dry milk. The wells were washed once with deionized water, and 200 μL of control or subject serum, diluted 1 : 50 in the blocking buffer, was added in duplicate to antigen coated and control wells, respectively, and incubated for 2 h at 37°C. After four washes with deionized water, 100 μL of alkaline phosphatase conjugated F(ab′)2 fragment of goat antihuman IgA (Sigma Chemical Co.), diluted 1 : 10 000 in the blocking buffer, was added to each well and the plate was incubated for 60 min at 37°C. Unbound antibodies were removed by washing the wells four times with deionized water. Colour was developed by adding 100 μL of 1 mol L–1 diethanolamine, 0.5 mmol L–1 MgCl2, 1 mg mL–1p-nitrophenyl phosphate (Sigma Chemical Co.), pH 9.8, and leaving the plates in the dark at 37°C for 30 min before the reaction was stopped by adding 50 μL of 3 M NaOH. Absorbance was read by the use of an ELISA reader (Multiscan® Plus, Labsystems Oy, Helsinki, Finland) at 405 nm. To correct for unspecific binding (‘sticky’ sera), the absorbance of the sample in the noncoated well was subtracted from that in the coated well. Each analysis included buffer blanks, six negatives, one high positive and one low positive control. Samples were run in duplicate. The cut-off value for a positive outcome was set at the mean value of the OD + 2 SD of all noncoeliac disease samples (n=1841). For practical reasons the antibody content of each serum was expressed as an index value, i.e. the OD of the test serum divided by the cut-off value. Index values of 1.0 and above were considered positive. The intra-assay CVs for the low and high controls were 5.3 and 3.1% (n=10), respectively, and the interassay CVs for the low and high controls were 14 and 15% (n=58), respectively.

Screening procedure and case ascertainment

In a first stage AGA IgA and IgG, AEA and ATGA were analysed in all 1850 subjects included in the study. All individuals with elevated levels of AEA were recommended to undergo a small intestinal biopsy. In a second stage subjects with elevated levels of AGA IgA but negative AEA were encouraged to allow a second blood sample for reanalysis, and if still positive they were recommended to undergo a small intestinal biopsy.

The diagnosis of coeliac disease was considered verified when assessment of the small intestinal mucosa demonstrated an enteropathy compatible with untreated classical coeliac disease, i.e. villous atrophy, epithelial cell disarray, crypt hyperplasia and lymphocytic infiltration of the epithelium and lamina propria [27].

Ethics

The study was approved by the Ethical Committee, Faculty of Medicine and Odontology, Umeå University. Consent was obtained after the participants had received written and verbal information.

Statistics

The coefficient of variation (CV) for an assay was calculated by dividing the standard deviation by the average result for a sample given on repeated analysis. The sensitivity and specificity of the serological tests were calculated and the 95% confidence intervals (CI) estimated using the exact binomial method. Receiver operating characteristic (ROC) analyses were carried out to compare the predictive abilities of AGA IgG, AGA IgA and ATGA measurements. The model plots sensitivity vs. 1-specificity for each possible value of a test. The area under the curve (AUC) shows the ability of a test to discriminate between disease and nondisease, with increasing discriminatory ability, with increasing area. The predictive values (PV) of the tests were calculated for different prevalences.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Subjects and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Screening outcome

Results of the screening procedure for coeliac disease are summarized in Fig. 1. The diagnosis of coeliac disease was verified by small intestinal biopsy and assessment of the mucosa in eight subjects, all of whom had elevated levels of AGA IgA and/or IgG, ATGA and AEA. However, it is most probable that there were actually nine cases of coeliac disease. One female who refused biopsy had elevated levels of all serological markers and symptoms compatible with the disease, i.e. flatulence, diarrhoea and depression, that diminished with reduced consumption of gluten-containing foods. Thus the screening detected nine cases of coeliac disease, revealing a prevalence of previously undiagnosed disease of 4.8/1000.

image

Figure 1.  Results of the screening procedure for coeliac disease. AGA, antigliadin antibodies; ATGA, antitransglutaminase antibodies; AEA, antiendomysial antibodies; IgA, antibodies of isotype IgA; IgG, antibodies of isotype IgG; +/–, above and below cut-off level, respectively; biopsy, a small intestinal biopsy performed; CD, coeliac disease; atypic, nonspecific intestinal mucosa changes including one subject with borderline findings; normal, normal intestinal mucosa.

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All individuals with positive AEA had a small intestinal mucosa compatible with coeliac disease. For all the other tests there were also positive results in some noncoeliac disease individuals; AGA IgG 21.8% (401/1841), AGA IgA 3.8% (70/1841) and ATGA 2.8% (51/1841).

Of the 70 subjects with elevated levels of AGA IgA, but negative AEA, a new blood sample was obtained from 58 (83%). In 12 of these (21%), AGA IgA was negative and no further examination was done. AGA IgA levels were still elevated in 46 subjects, seven of whom also had elevated levels of ATGA. Of these 46 subjects, 32 agreed to a small intestinal biopsy (70%). None of these had a small intestinal mucosa compatible with coeliac disease.

Six individuals had a small intestinal mucosa with chronic unspecific inflammation. Five of them had an increased number of cells in the lamina propria, but not within the epithelium, and normal architecture of the villi. One individual was classified as ‘borderline’, i.e. villi of both normal and borderline architecture together with an increased number of intraepithelial lymphocytes. All six had elevated levels of AGA IgA whilst only one (not the ‘borderline’) had an elevated level of ATGA and none had a positive AEA.

Sensitivity and specificity

Sensitivity and specificity of the tests are given in Table 1. The AEA and ATGA tests both had a sensitivity of 100%, which should be compared with the sensitivity of 89% for both the AGA IgA and IgG tests. The AEA test had a specificity of 100%, whereas the specificities for the ATGA, AGA IgA and AGA IgG tests were 97, 96 and 78%, respectively. ROC analyses illustrate that this order of discriminatory ability amongst the test methods (ATGA > AGA IgA > AGA IgG) would not be changed by a change in cut-off level (Fig. 2). AUC was 0.998 for ATGA, 0.985 for AGA IgA and 0.932 for AGA IgG. If AEA had been included, the AUC would have been 1.0, i.e. optimal discriminatory ability.

Table 1.   Diagnostic sensitivity and specificity of serological markers for coeliac disease Thumbnail image of
image

Figure 2.  Receiver operating characteristic (ROC) curves, also giving an estimate of the area under each curve (AUC), for ATGA (antitransglutaminase antibodies of isotype IgA), AGA IgA (antigliadin antibodies of isotype IgA) and AGA IgG (isotype IgG).

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Predictive values

The negative predictive value (PV–) was 100% for all tests evaluated. The AEA test had the highest positive predictive value (PV+) of 100%. All the other tests had considerably lower PV+ with ATGA > AGA IgA > AGA IgG, which further decreased for all tests when shifting from hypothetical clinical situations to the screening situation (Table 2). The prevalence of previously undiagnosed coeliac disease of 4.8 per 1000 found in this study represents a general adult population, whilst the tentative 10- and 20-fold higher prevalences (48 and 96 per 1000) were chosen to illustrate hypothetical prevalences in populations seeking medical advice for symptoms indicative of untreated coeliac disease.

Table 2.   Positive predictive value (%) of the tests at different tentative prevalences representing a general population (4.8 per 1000) and hypothetical clinical situations Thumbnail image of

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Subjects and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Major findings in this study is that the AEA test still seems to be the most reliable serological marker for coeliac disease and that the ATGA-test could (at least in adults) replace the AGA tests, provided that IgA-deficiency has been excluded.

The diagnosis of coeliac disease is based on assessment of the small intestinal mucosal morphology. A correct diagnosis is the basis for evaluating other diagnostic tools, e.g. serological markers. However, in a population-based study it is impossible, both for ethical and economic reasons, to perform a small intestinal biopsy on all subjects. Furthermore, many of those who feel healthy will not volunteer. Our strategy was therefore to recommend a biopsy for all subjects with elevated levels of AEA and/or AGA IgA. Many of these also had elevated levels of ATGA. Those with symptoms that might indicate untreated coeliac disease were, as expected, more prone to agree to a biopsy than the other subjects. Further, considering that the AGA test has used a cut-off aimed at giving priority to sensitivity rather than to specificity [26], most cases of coeliac disease in the population were probably to be diagnosed. The accuracy of the coeliac diagnosis was ascertained by assessment of the intestinal mucosa in all but one female, who refused biopsy. In her case we based the diagnosis on elevated levels of all serological markers, including AEA, and relief of symptoms on a gluten-reduced diet.

In our study the AEA test had both a sensitivity and a specificity of 100%. Including only biopsy-verified cases and noncases would decrease the specificity to 97% (95% CI 84–99.9), whilst the sensitivity would remain at 100%. Thus, this study has shown that AEA remains the most reliable serological marker for coeliac disease. However, not all studies have reported such an excellent diagnostic ability for AEA [3, 7, 28, 29], which illustrates the need for standardization of the test methods. Moreover, when coeliac disease is suspected to be based on clinical symptoms and/or signs, a small intestinal biopsy should always be taken to confirm the diagnosis, irrespective of a negative result of any serological marker.

The ATGA ELISA in our study had a sensitivity of 100% and a specificity of 97%, which should be compared with other studies reporting sensitivities varying from 85 to 98% and specificities from 90 to 98% [19–24]. Thus, although it has both high sensitivity and specificity, the ATGA test is not as reliable as the AEA test. Interestingly, out of six subjects in our study with a chronic unspecific intestinal inflammation, but not classifiable as coeliac disease [27], only one had elevated levels of ATGA, whilst AGA IgA was elevated in all six. Further, with respect to sensitivities and specificities, ATGA seems to be superior to AGA IgA, and even more so compared with AGA IgG.

The cut-off level used for ATGA in our study is based on experience from participating in an international external quality panel (the UK National External Quality Assessment Schemes). However, judged by the present study, a change in cut off level from 1.0 to 2.0 would increase the specificity from 97 to 99% without a reduction in sensitivity. Nevertheless, there would be 12 nonceliac disease individuals with a positive outcome on the ATGA test, compared with none of the AEA test.

Receiver operating characteristic analyses facilitate a comparison of different test methods with respect to the ability to discriminate between disease and nondisease. Such analyses clearly showed that the discriminatory ability was the highest for ATGA, followed by AGA IgA, and then AGA IgG, and that this relationship was not dependent on the cut-off levels used. However, the discriminatory performance of AEA was even higher.

The predictive value of a test depends not only on the sensitivity and specificity but also on the prevalence of the disease in the investigated population. When shifting from a clinical setting, with people seeking medical advice for symptoms indicative of coeliac disease, to screening of a general population, i.e. from a high to a low prevalence situation, this becomes obvious. Thus, in the screening situation serological markers with both high sensitivity and specificity are required. However, the predictive value of serological markers will increase if a serial testing approach is used, and the number of unnecessary small intestinal biopsies can thereby be reduced. We [6] and others [9, 16] have previously suggested serial testing initiated with an AGA test and, if that is positive, followed by an AEA test, and if that is also positive a small intestinal biopsy for confirmation of the diagnosis. In a screening situation this approach still seems useful, although it would be even more efficient to replace the AGA tests with an ATGA-test, provided that IgA deficiency has been excluded, or is excluded by analysing total s-IgA simultaneously.

Human tTG has recently been cloned [30], and ELISA assays to measure IgA antibodies against the human antigen have been developed [31–33]. These tests seem to have greater accuracy than those based on guinea-pig tTG. Our format for ATGA ELISA based on guinea-pig tTG was, however, highly sensitive and specific, indicating that factors other than the source of antigen may also play a role.

In the present population-based study, two individuals were excluded because of already diagnosed coeliac disease. As nine more cases were found in the screening, roughly one out of five cases of adult coeliac disease was clinically silent or, more correctly, these were previously missed cases. Considering this, screening of the general population should be put on the agenda of topics for further discussion. Coeliac disease is common, albeit often undiagnosed, and has long-term health consequences. An effective treatment is available, i.e. a life-long gluten-free diet. Furthermore, we now have serological markers that, if used in combination and followed by a confirmatory small intestinal biopsy, would make screening of the general population feasible. However, some questions remain to be answered before this approach can be justified [10–13]. It has not yet been proved that patients with the so-called silent coeliac disease, i.e. gluten-dependent enteropathy with no symptoms or only minor symptoms, have the same long-term health risks as those with classical symptoms. Furthermore, there is a question as to whether cases detected by screening will be motivated to adhere to a strict gluten-free diet. Based on several studies it seems clear that many – but not all – screening-detected cases are in fact not silent cases but missed cases [4, 6, 13]. In these cases one would expect dietary treatment to be accepted. Other important questions that remain to be answered are at what age screening should be conducted and if need be repeated and, if so, at what intervals. Another question to consider is whether a screening programme, such as the one outlined above, would have an acceptable cost-effectiveness compared with other needs within the health care system.

The need for increased awareness of the wide spectrum of symptoms that should lead one to suspect coeliac disease is indisputable. When the clinical suspicion of coeliac disease is strong, a small intestinal biopsy should be done irrespective of the results of any serological marker. When coeliac disease is considered as one of several alternative diagnoses, the serial testing approach suggested here should be helpful in daily clinical work and for screening of selected risk populations.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Subjects and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We acknowledge the assistance of the Northern Sweden MONICA Project and we thank our colleagues at the Departments of Medicine in Northern Sweden for performing the intestinal biopsies.

Financial support was received from the Swedish Council for Forestry and Agricultural Research, the Swedish Foundation for Health Care Sciences and Allergy Research, the Swedish Foundation for Research on Asthma and Allergy, the Swedish Medical Research Council (05708) and the Västerbotten County Council.

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  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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