Journal of Postgraduate Medicine Medknow Publications and Staff Society of Seth GS Medical College and KEM Hospital, Mumbai, India ISSN: 0022-3859 EISSN: 0972-2823 Vol. 49, Num. 1, 2003, pp. 21-24

Journal of Postgraduate Medicine, Vol. 49, No. 1, Jan-March, 2003, pp. 21-24

Brief Report

Association Between Anti-endomysial Antibody and Total Intestinal Villous Atrophy in Children with Coeliac Disease

Ozgenc F, Aksu G, Aydogdu S, Akman S, Genel F, Kutukculer N, Alkanat MB*, Vural Yagci R

Departments of Paediatrics and *Pathology, Ege University, Faculty of Medicine, Izmir, Turkey.
Address for Correspondence: Funda Ozgenc, 1401 sok. No:22/4, Alsancak, Izmir, Turkey, E-Mail: ozgenc@med.ege.edu.tr

Code Number: jp03005

Abstract:

BACKGROUND: There is growing evidence to suggest that detection of anti-gliadin antibody (AGA) and anti-endomysial antibody (EmA) can serve as sensitive markers of the degree of histological abnormalities in patients with coeliac disease. AIM: To evaluate the association between the presence of AGA and EmA and villous atrophy in intestinal biopsies of children with suspected coeliac disease. SETTINGS AND DESIGN: Intestinal samples of 46 children with failure to thrive, chronic diarrhoea, malabsorption and short stature with either AGA and/or EmA positivity were evaluated, retrospectively. The diagnosis of coeliac disease was based on ESPGHAN criteria. METHODS AND MATERIAL: Patients with total villous atrophy who fulfilled the ESPGHAN criteria for the diagnosis of coeliac disease were diagnosed to have coeliac disease. Nine patients without villous atrophy were taken as negative controls for this study. AGA-IgA was measured both by immunoflourescence (IF) and ELISA and EmA-IgA by IF while patients were on normal diet. Relationship between autoantibody positivity and intestinal total villous atrophy was evaluated. RESULTS: Overall positivity for AGA IgA was 85% (39/46) by IF+ELISA and EmA positivity was 85% (39/46) by IF within the study group. Histological examination revealed total villous atrophy with lymphocyte infiltration and crypt hyperplasia in 37 (80%) patients. AGA IgA was positive in 14 (38%) and 31 (84%) of these children by ELISA and IF, respectively. EmA positivity was detected in 35/37 (95%) cases with atrophy and 4/9 (44%) without atrophy (p=0.002). Thirty out of 37 (81%) patients with villous atrophy had both AGA IgA (IF) and EmA positivity (p=0.186). All of the sixteen patients that had both positive AGA IgA (ELISA+IF) and EmA had total villous atrophy (p=0.037). CONCLUSION: A significant association between total villous atrophy and EmA positivity has been documented in this study. (J Postgrad Med 2003;49:21-24)

Key Words: Coeliac disease, anti-endomysial antibody, antigliadin antibody, villous atrophy, immunofluorescence.

Coeliac disease, also called as gluten sensitive enteropathy (GSE) is disease characterised by an immunological intolerance to ingested gluten in genetically susceptible individuals. It usually presents with failure to thrive, diarrhoea and malabsorption.¹ Mucosal flattening also called as total villous atrophy, is the typical histological abnormality in coeliac disease. The atrophy is related to increased apoptosis of the enterocytes, triggered by the release of lymphocyte perforin cytolytic granules and by the Fas/Fas ligand interaction.² The diagnosis of the disease is confirmed by the histological demonstration of villous atrophy with inflammation and hyperplastic crypts and by demonstration of clinical and serological remission after withdrawal of gluten from the diet.³

Markers such as anti-endomysial (EmA), anti-gliadin (AGA) antibodies and tissue transglutaminase (tTG) are widely used to detect and monitor patients with GSE as well as groups of patients considered at high risk to develop coeliac disease.^4-6 There are reports to suggest that these tests are sensitive and specific enough to use them for the diagnostic purposes and there is growing evidence that they can also serve as sensitive markers for histological abnormalities on jejunal biopsy.^7-16

In this study, we evaluated the relationship of EmA and AGA positivity with villous atrophy in intestinal biopsies of children with suspected coeliac disease.

Patients and Methods

AGA and EmA results were compared with histological findings in 46 children with suspected coeliac disease over a four-year-period, retrospectively. Intestinal biopsy samples were obtained from second to fourth part of the duodenum by endoscopy in 40 children while for the remaining 6 cases, per-oral capsule biopsies from jejunum were performed. Failure to thrive, chronic diarrhoea, malabsorption and short stature with positivity for either antigliadin antibody and/ or anti-endomysium antibody were accepted as indications for intestinal sampling. All biopsies were double-checked by the same pathologist blinded to the results of serology. Villous atrophy with accompanying lymphocyte infiltration and cryptic hyperplasia were defined as characteristic histological findings of coeliac disease. The diagnosis of coeliac disease was based on these histological findings in association with clinical remission and normalisation of autoantibodies on a "gluten-free-diet" according to the criteria proposed by the European Society for Paediatric Gastroenterology and Nutrition (ESPGHAN).³

All serum samples had been collected at the time of diagnosis when the patients were receiving normal diet without any restrictions. Total serum IgA level assessment was made to exclude selective IgA deficiency. AGA IgA was measured by immunofluorescence (IF)[Cut-off for positivity is 1/200^th dilution] and ELISA (Euroimmun, Lubeck, Germany) and EmA by IF (Euroimmun, Lubeck, Germany) on snap frozen monkey small intestine as described by Chorzelsky et al¹⁷ in all the patients. ELISA results were considered to be positive if they were >100μ/ml in 0-4 age range or >50RU/ml over 4 years of age. Patients who fulfilled the ESPGHAN criteria for coeliac disease with total villous atrophy were diagnosed as having coeliac disease, whereas 9 patients without total villous atrophy were taken as negative controls for this study.

Mann-Whitney U test and Fisher's exact test were used for statistical evaluation.

Results

The median age of patients in the study group was 6.5 (1-18) years and male/female ratio was 13:33. Villous atrophy with lymphocyte infiltration and crypt hyperplasia was documented in 37 intestinal samples.

Serum IgA levels of the patients were found to be within the normal range. Overall positivity for AGA IgA was 85% by either IF or ELISA (39/46) and EmA IgA positivity was 85% by IF within the study group (39/46). Thirty-eight patients had positive AGA by only IF, 16 patients were positive for AGA both with IF and ELISA, 17 were positive for AGA by ELISA only, 39 patients had positive AGA measurements either with IF or ELISA (Figure 1). Thirty-nine patients were positive for EmA IgA by IF (Table 1). Histological examination revealed total villous atrophy with lymphocyte infiltration and crypt hyperplasia in 37 patients (80%). AGA IgA was positive in 14 (38%) and 31 (84%) of these children by ELISA and IF, respectively.

Estimation of AGA IgA by ELISA had been done on 28 patients within the whole study group (mean: 101.28 ± 73.41 RU/ml). Twenty-four of these 28 patients had positive AGA IgA by ELISA (mean: 107.99 ± 77.29 RU/ml) while 4 patients were negative for AGA IgA by ELISA (mean: 61.05 ± 13.76 RU/ml). There were 14 patients with villous atrophy who had positive ELISA levels (mean: 158.08 ± 61.18 RU/ml) for AGA IgA. Three patients without atrophy were positive for AGA IgA by ELISA (mean: 63.73 ± 15.51 RU/ml). EmA positivity was detected in 35 (95%) cases with atrophy and 4 (44%) without atrophy (p=0.002). Thirty out of 37 (81%) patients with intestinal atrophy had both AGA IgA (IF) and EmA (IF) positivity (p=0.186) (Table 1). There was no variation in the presence of anti-gliadin antibodies in patients with and without villous atrophy whatever was the technique used i.e. IF or ELISA or both techniques (p=0.645, p=0.427, p=1.000; respectively). EmA positivity showed a statistically significant increase in patients with villous atrophy (p=0.002). Thirty-five patients had both AGA IgA (IF) and EmA; villous atrophy was documented in 30 of them (65%) while atrophy was not detected in 5 patients who were positive for both (p=0.186). All of the sixteen patients who were positive for EmA IgA (IF) and AGA IgA both with ELISA and IF had total villous atrophy (p=0.037). The statistical analyses and methods are outlined in Table 1.

Discussion

The small intestinal abnormalities characterised by total villous atrophy in histological examination are essential for the diagnosis of coeliac disease. However, serological assays are valuable for taking the decision of intestinal sampling as well as for monitoring the disease under gluten-free-diet. Anti-gliadin and anti-endomysial antibodies are two of the traditional screening tests widely used. EmA IgA assay is 89-100% sensitive and 100% specific in reported series.^9,10 Kaukinen et al¹⁸ found 86% positivity of both of AGA and EmA antibodies in their patients with coeliac disease with villous atrophy. Ninety-five percent of patients (35/37) with severe villous atrophy were positive for EmA IgA in our group (p=0.002). Although the positive predictive value of EmA is reported to be 100%,^10,11 there were four children in our group without severe villous atrophy who were seropositive for EmA. Anti-endomysial antibody does not only support the diagnosis, but also predicts the severity of mucosal damage.^8,12 We have found a significant association between EmA seropositivity and intestinal atrophy in our study. Rostami K et al⁸ have also reported increased EmA positivity in subtotal and total villous atrophic cases. However, they have found that EmA was associated with a lower sensitivity in coeliac disease patients with milder intestinal damage.

Progression of mucosal damage from lymphocyte infiltration to total villous atrophy is reported in coeliac disease by Marsh et al.¹³ Tursi and co-workers detected anti-endomysial antibody in none of the patients in Marsh I class of the disease, but these antibodies were detected in 40% and 88.8% of their patients in Marsh II and III classes, respectively.¹⁴ Histological examination of our patients, described likewise with positive serological measurement and absence of villous atrophy, showed intestinal mucosal abnormalities concordant with Marsh I-II. However, it would not be appropriate to claim that these patients had coeliac disease unless follow-up period extends and endoscopic confirmation of disappearance of mucosal lesions are documented under gluten free diet.

In our study, all of the sixteen patients who were positive for EmA IgA (IF) and AGA IgA either with ELISA or IF had total villous atrophy (p=0.037). Hence co-existence of AGA and EmA positivity may also help to predict villous atrophy in patients with suspected coeliac disease. We could not estimate tissue transglutaminase (tTG)antibody; however, tTG is reported with optimum sensitivity, but lower specificity and positive predictive values than those for EmA in patients with coeliac disease in recent studies.^9,15

EmA positivity alone was significantly related to severe mucosal damage in our group. Although we did not detect any association with accompanying AGA IgA measurements either by ELISA or IF alone, using both IF+ELISA techniques together with EmA IF gave us a significant clue to the presence of villous atrophy. We suggest that EmA is a powerful diagnostic tool to predict total villous atrophy in patients with suspected coeliac disease and verification of this analysis by use of AGA IgA measurements is required. However, the emerging need to use both the techniques for AGA IgA assay should be carefully considered since this may increase the overall cost of the evaluation of these patients.

References

1. Troncone R, Auricchio S. Celiac disease. In: Wyllie R, Hyams JS, ed. Paediatric gastrointestinal disease. Philadelphia: WB Saunders Company; 1999. pp. 306-14.
2. Ciccocioppo R, D'Alo S, Di Sabatino A, Parroni R, Rosi M, Doglioni C, et al. Mechanisms of villous atrophy in autoimmune enteropathy and coeliac disease. Clin Exp Immunol 2002;128:88-93.
3. Revised criteria for diagnosis of celiac disease. Report of Working Group of European Society of Paediatric Gastroenterology and Nutrition. Arch Dis Child 1990;65:909-11.
4. Russo PA, Chartrand LJ. Seidman E. Comparative analysis of serological screening tests for the initial diagnosis of celiac disease. Paediatrics 1999;104:75-8.
5. Vitoria JC, Arrieta A, Arranz C, Ayesta A, Sojo A, Maruri N, et al. Antibodies to gliadin, endomysium and tissue transglutaminase for the diagnosis of celiac disease. J Pediatr Gastroenterol Nutr 1999;29:571-4.
6. Carlsson A, Anderson I, Borulf S, Bredberg A, Forslund M, Lindberg B, et al. Prevalence of Ig A-antigliadin and Ig A-antiendomysium antibodies related to celiac disease in children with down syndrome. Paediatrics 1998;101:272-5.
7. Rostami K, Kerckhaert J, Tiemessen R. von Blomberg BM, Meijer JW, Mulder CJ. Sensitivity of antiendomysium and antigliadin antibodies in untreated celiac disease: disappointing in clinical practice. Am J Gastroenterol 1999;94:888-94.
8. Rostami K, Kerckhaert J, Tiemessen R, Meijer JW, Mulder CJ. The relationship between anti-endomysial antibodies and villous atrophy in coeliac disease using both monkey human substrate. Eur J Gastroenterol Hepatol 1999;11:439-42.
9. Carrocia A, Vitale G, Di Prima L, Chifari N, Napoli S, La Russa C, et.al. Comparison of anti-transglutaminase ELISAs and an anti-endomysial antibody assay in the diagnosis of celiac disease: a prospective study. Clin Chem 2002;48:1546-50.
10. Pearce AB, Sinclair D, Duncan HD, Saas M, Poller DN. Use of the anti-endomysial antibody test to diagnose coeliac disease in clinical practice. Clin Lab 2002;48:319-25.
11. Del RMA, Fitzgerald JF, Chong SK, Croffie JM, Gupta SK. Further studies of anti-endomysium and anti-gliadin antibodies in patients with suspected celiac disease. J Paediatr Gastroenterol Nutr 1998;27:191-5.
12. Szaflarska-Szcepanik A. Assessment of correlation between the presence of antiendomysial antibodies and small intestine mucosal villous atrophy in the diagnosis of celiac disease. Med Sci Monit 2002;8:CR185-8.
13. Marsh MN. Gluten, major histocompatibility and the small intestine: a molecular and immunobiologic approach to the spectrum of gluten sensitivity (`coeliac sprue'). Gastroenterology 1992;102:330-54.
14. Tursi A, Brandimarte G, Giorgetti G, Gigliobianco A, Lombardi D, Gasbarrini G. Low prevalence of antigliadin and anti-endomysium antibodies in subclinical/silent coeliac disease. Am J Gastroenterol 2001;96:1507-10.
15. Rossi TM, Kumar V, Lerner A, Heitlinger LA, Tucker N, Fisher J. Relationship of endomysial antibodies to jejunal mucosal pathology: Specificity towards both symptomatic and asymptomatic coeliacs. J Pediatr Gastroenterol Nutr 1988:7;8585-63.
16. Carroccio A, Di Prima L, Falci C, Le Moli C, Soresi M, Montalta G, et al. Predictive value of serological tests in the diagnosis of coeliac disease. Ann Ital Med Int 2002;17:102-7.
17. Calvani M Jr, Parisi G, Miotto AM, Alessandri C, Notarnicola MA. Anti-endomysial antibodies: a new marker for the diagnosis and treatment of coeliac disease. Pediatr Med Chir 1992;14:33-6.
18. Kaukinen K, Collin P, Holm K, Karvonen AL, Pikkarainen P, Maki M. Small-bowel mucosal inflammation in reticulin or gliadin antibody-positive patients without villous atrophy. Scand J Gastroenterol 1998;33:944-9

Expert's Comments

The availability of assays for the detection of antibodies that are specific to coeliac disease (CD) has meant an enormous progress in the diagnosis of this disorder. Anti-endomysium antibodies and the more recently described anti-transglutaminase antibodies have great diagnostic capacity and are a good reflection of the situation in the intestinal mucosa. Nevertheless, patient discrimination based solely on such antibody determination is not absolute and intestinal biopsy remains the reference gold standard for ultimate diagnosis of CD. Patients positive for these antibodies but that do not present with a clear lesion of the intestinal mucosa should be judged with caution, since they could be potential or latent forms of the disease.¹ Also, one must consider that 2.6% of patients with CD also suffer from selective IgA deficiency while most of the serologic tests detect only IgA immunoglobulins. IgG autoantibody detection tests have proven very useful in these situations and besides, are as sensitive and specific as IgA assays.¹ Moreover, it should be pointed out that patients of CD have been described who present with IgG class anti-transglutaminase and anti-endomysium antibodies only even when they do not have IgA deficiency.² There are also patients that are discordant for anti-transglutaminase and anti-endomysium antibodies. In consequence, screening strategies for CD should include a combination of both antibodies and, if possible, at least one of them should be of the IgG class. This will, however, increase the cost of diagnosis of CD. On the other hand, these antibody tests can be used to monitor patients regarding the compliance with gluten-free diet. For this purpose, one must know the rate at which these immunoglobulins disappear after removal of gluten. In this sense, anti-gliadin antibody titres decrease very rapidly after gluten is excluded from the diet, whereas anti-transglutaminase and anti-endomysium antibodies seem to persist for a longer period of time, sometimes even more than a year after removal of gluten.

J. C. Vitoria

Department of Pediatrics, Hospital de Cruces and Basque University School of Medicine, Bilbao, Spain.
E-mail: jcvitoria@hcru.osakidetza.net

References

1. Bilbao JR, Vitoria JC, Ortiz L, Corrales A, Hualde I, Preciado E, Casta-OL. Immunoglobulin G autoantibodies against tissue-transglutaminase. A sensitive, cost-effective assay for the screening of celiac disease. Autoimmunity 2002;35:255-9.
2. Picarelli A, Di Tola M, Sabbatella L, Mastracchio A, Trecca A, Gabrielli F, et al. Identificaction of a new coeliac disease subgroup: antiendomysial and anti-transglutaminase antibodies of IgG class in the absence of selective IgA deficiency. J Intern Med 2001;249:181-8.

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