ORIGINAL ARTICLE: GASTROENTEROLOGY
Celiac Disease Can Be Predicted by High Levels of Anti-Tissue Transglutaminase Antibodies in Population-Based Screening
Charlotta Webb, yFredrik Norstro¨m, yAnna Myle´us, yAnneli Ivarsson, zBritta Halvarsson, § Lotta Ho¨gberg, ôCarina Lagerqvist, yAnna Rose´n, yOlof Sandstro¨m, § Lars Stenhammar, and Annelie Carlsson
ABSTRACT Objectives: The aim of the present study was to evaluate any potential correlation between anti-tissue transglutaminase antibodies of type immunoglobulin A (tTG-IgA) and the degree of gluten-induced enteropathy in children participating in a screening study for celiac disease (CD) and to assess to what extent the revised European Society for Pediatric Gastroenterology, Hepatology, and Nutrition (ESPGHAN) guidelines cover this group of patients. Methods: The present study is a substudy of a cross-sectional CD screening study, Exploring the Iceberg of Celiacs in Sweden, a 2-phased study performed during 2005 to 2006 and 2009 to 2010. The 13,279 participating children had a blood test obtained, and those with positive tTG-IgA were recommended a small intestinal biopsy. The tTG-IgA levels at the time of biopsy were compared with those at the assessment of the biopsy. Results: There were 267 children included, of whom 230 were diagnosed as having CD. Of all of the children, 67 children had low tTG-IgA levels (100 U/mL) have a high specificity for Marsh 3 lesions (19,21–26), which underpins the revision of the ESPGHAN diagnostic guidelines. To the best of our knowledge, however, there are yet no data on the correlation between tTG-IgA levels and the degree of enteropathy in screening-detected patients with CD from the general childhood population. The primary aim of the present study was to evaluate a potential correlation between the level of tTG-IgA and the degree of gluten-induced enteropathy in screening-detected patients with CD from a general child population and to assess to what extent the revised ESPGHAN guidelines can be extended to cover this group of patients.
METHODS The present study is a substudy of a cross-sectional CD screening study entitled ‘‘Exploring the Iceberg of Celiacs in Sweden,’’ which is part of the European Prevent CD project. The screening was school-based and included 2 birth cohorts of 12-year-old children born in 1993 or 1997 with the screening taking place in 2005 to 2006 and 2009 to 2010, respectively. Five pediatric departments collaborated in the present study (Umea˚, Norrta¨lje, Norrko¨ping, Va¨xjo¨, and Lund), and each of the sites included a major city with municipalities in the surrounding suburbs and countryside. A total of 18,325 children were invited, and 13,279 consented to CD screening (Fig. 1). The proportion of girls was similar in both cohorts (48% and 49%, respectively). Positive serological markers were found in 291 children, and these children were invited to a further investigation with a small intestinal biopsy. Inclusion criteria for the present substudy were that a small intestinal biopsy had been performed and a second blood sample drawn at the time of the biopsy had been analyzed for tTG-IgA. The Exploring the Iceberg of Celiacs in Sweden study was approved by the regional ethical review board of Umea˚ University, Umea˚, Sweden.
Volume 60, Number 6, June 2015
Participants (n = 13,279) Total blood samples (n = 12,632)
Positive serology in school (n = 291)
Inclusion criteria (n = 268) Small intestinal biopsy (13 children excluded) and renewed blood sample (10 children excluded)
Histological evaluation (n = 268) Marsh 0
=
Marsh 1 no spt
=
3
Marsh 1 and spt Marsh 2
= =
11 2
=
217
Marsh 3 Noninterpretable
34
=
1
Included in the study n = 267
Screening Strategy Participating children were asked for a blood sample at their school, and a second serum sample was obtained at the time of biopsy (Fig. 1). The samples were analyzed at the same laboratory for tTG-IgA by enzyme-linked immunosorbent assay (Celikey, Phadia GmbH, Freiburg im Breisgau, Germany). The manufacturer’s cutoff for positive tTG-IgA was 5 U/mL, but to increase the sensitivity of the screening we chose to consider all of the values >4 U/mL to be elevated. Serum samples were analyzed in duplicate within the measuring range 0 to 100 U/mL, and the mean value was used. Serum samples with intermediate values of tTG-IgA (2–4 U/ mL) were additionally analyzed for EMA-with a 1:5 dilution as a cutoff. EMA were determined by indirect immunofluorescence using monkey esophagus tissue (Binding Site, Birmingham, UK). All of the children (n ¼ 291) with elevated serological markers at the screening occasion were referred to a pediatric clinic for a follow-up with a second celiac serology analysis and a small intestinal biopsy. A total of 278 children accepted the follow-up screening procedure, which has previously been described in detail (27,28). Of the 278 children who had a biopsy performed, 267 children met the inclusion criteria for the study. Five children in the 1993 cohort had IgA deficiency and tTG-IgG >6 U/mL and were excluded, and another 5 children were excluded because no serum samples were taken at the time of the biopsy. One additional patient was also excluded because of an inconclusive biopsy. Of the 267 children in the present study (156 girls and 111 boys), 230 were
788
CD Diagnosis Children = 230 Girls/boys 133/97
FIGURE 1. Flowchart summarizing the results from the screening for celiac disease in 2 birth cohorts. CD ¼ celiac disease.
diagnosed as having CD. There were no significant differences between the 2 birth cohorts with respect to age or sex, so the data for the 2 birth cohorts were combined.
Diagnostic Process In the 1993 cohort, the small intestinal biopsy was performed either with a suction capsule (n ¼ 54) or by endoscopy (n ¼ 125) and has previously been described in detail (29). In the 1997 cohort, all of the biopsies were performed using endoscopy (n ¼ 99). Biopsies were taken both proximally and more distally in the duodenum, including the bulb, following a recommendation of 4 to 6 specimens per procedure. The mucosal specimens were classified according to the revised Marsh-Oberhuber classification (11). All of the biopsies were subjected to a second histopathological evaluation by a pathologist blinded to the previous results. In case of disagreement, www.jpgn.org
Copyright 2015 by ESPGHAN and NASPGHAN. Unauthorized reproduction of this article is prohibited.
Volume 60, Number 6, June 2015
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We found a wide variation in tTG-IgA levels and the degree of enteropathy among the 267 children who were participating in the present study (Fig. 2). There was, however, a tendency toward a gradual increase in the tTG-IgA levels predicting mucosal damage (Table 1), and Marsh 3 lesions were seen in 56%, 53%, 86%, 87%, and 98% of the children with tTG-IgA levels 50 U/mL, respectively. There was no significant difference in Marsh 3 lesions between boys and girls—85% versus 79%, respectively (P ¼ 0.23). There were 2 interesting subgroups of children when comparing the results from the serology and the biopsy. The first group consisted of children with low tTG-IgA levels (50 U/mL). There were 64 children in this group and the majority of them had Marsh 3 lesions, but there was 1 child with a biopsy graded as Marsh 1 who was EMA positive. In the group of children with tTG-IgA levels 30 to 50 U/mL, there was 1 child with Marsh 0 lesions, who consequently was not given a CD diagnosis, and 3 children who had Marsh 1 lesions. All of the children in this group were EMA positive. All of the patients with CD were HLA DQ2 and/or DQ8 positive. All of the children with Marsh 1 lesions and high tTG-IgA levels (>30 U/mL) were considered to have CD based on additional criteria according to the study design.
ar
Level of tTG-IgA and the Degree of Enteropathy and CD Diagnosis
DISCUSSION To the best of our knowledge, the present study is the first one to investigate any potential association between the levels of tTG-IgA and enteropathy in a screening for CD in a general population of children. We found that levels of tTG-IgA >50 U/ mL predicted gluten-induced enteropathy, but we also found an uncertainty to predict enteropathy in lower levels of tTG-IgA. Several studies have found a strong correlation between tTGIgA >100 U/mL and Marsh 3 lesions (21,24–26). These studies, however, were performed in children who had symptoms and signs suggestive of CD in a clinical setting. They also reported a poor correlation between the level of tTG-IgA and the severity of enteropathy. In our study, all of the children who had levels exceeding 10 times the upper level of normal values at the recommended cutoff from the manufacturer (>5 U/mL), that is, 50 U/mL, were diagnosed as having CD. Recent studies using the same serological test in a clinical setting, however, recommended a cutoff of >3 U/ mL. Among those with tTG >30 U/mL, 1 child did not have CD. Interestingly, Marsh 3 lesions were found more frequently in the group with tTG-IgA >50 U/mL (98%) than in the group with tTG-IgA between 30 and 50 U/mL (87%), which could be a reason
M
RESULTS
We assessed the 2012 ESPGHAN diagnostic criteria based on the serological levels of tTg-IgA exceeding 10 times the upper levels of normal. The ESPGHAN diagnostic criteria for the serology would have given 64 CD diagnoses of whom 63 (98%) were graded with Marsh 3 lesions for a cutoff of 5 U/mL and 101 CD diagnoses of whom 96 (95%) were graded with Marsh 3 lesions for a cutoff of 3 U/mL. One child would not have been considered to have CD in our study for the cutoff of 3 U/mL, whereas all of the children were considered to have CD for the higher cutoff of 5 U/mL.
0
Frequency tables, cross-tabulations, medians, and quartiles were used to present our data. Student t test was used to test whether there was a difference in the proportion of boys and girls with a high small intestinal damage. Statistical significance was defined as P < 0.05. Microsoft Access was used for data handling, and Stata 11.2 (StataCorp LP, College Station, TX) was used for statistical analyses and figures.
ESPGHAN CD Diagnostic Criteria for ScreeningDetected Patients
sh
Statistical Analyses
There were 34 patients who had normal biopsies, 26 children (76%) had tTG-IgA levels 30 lymphocytes/100 enterocytes), crypt hyperplasia, and villous atrophy. In patients with Marsh 1 lesions (intraepithelial lymphocytes), symptoms and/or signs compatible with CD were required in combination with an HLA-DQ2/ DQ8 haplotype and clinical response to a GFD (27,29). In the present substudy, we evaluated the results of the serological markers at the time of biopsy. In the 2012 ESPGHAN guidelines, biopsy is no longer mandatory when the tTG-IgA levels exceed 10 times the upper limit of normal. The cutoff for positivity recommended by the manufacturer was >5 U/mL (30). Studies have indicated that a cutoff of 3 U/mL would be more correct, and therefore the results were additionally analyzed at a cutoff of 3 U/mL (31–33). Four children with tTG-IgA >30 U/mL in combination with Marsh 1 lesions and 1 child with tTG-IgA >50 U/mL who had Marsh 0 lesions were additionally tested for EMA.
Prediction of Celiac Disease in Population-Based Screening
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tTG-IgA (U/mL)
JPGN
Degree of mucosal damage
FIGURE 2. Degree of mucosal damage in relation to tTG-IgA. NB: In the 1993 cohort, 2 patients with Marsh 2 lesions have been included in the Marsh 2 to 3a group. tTG-IgA ¼ anti-tissue transglutaminase antibodies of type immunoglobulin A.
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Webb et al
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TABLE 1. Serological levels in correspondence with CD diagnosis and biopsy evaluation Marsh classification Serology groups, U/mL 50 U/mL and Marsh 1 lesions and 4 patients with tTG-IgA >30 U/mL and Marsh 1 lesions, were EMA positive. Although there are many pitfalls in the diagnosis of CD, there are some plausible explanations for the inconsistent association between the level of serological markers and intestinal mucosal damage. Every step in the diagnostic process has an impact on the diagnostic outcome. The histopathological evaluation is dependent on different variables, for example, biopsies from several different locations, biopsy method, and the evaluation by an experienced pathologist (29). The biopsy procedure in the present study has been thoroughly validated (29), and we found that multiple biopsies taken from both the proximal and the distal duodenum sharpened the diagnosis; all of the biopsies in the present study were reevaluated by the same experienced pathologist. An interesting result was the subgroup with severe enteropathy in combination with low levels of tTG-IgA, which supports the results of other studies and underlines the necessity of performing a biopsy to obtain a correct diagnosis (16). It is somewhat easier to explain why some children with high levels of tTG-IgA had only mild enteropathy because it is known that the lesions can be patchily distributed in children that can lead to severe lesions being missed by the biopsy (37). Another interesting finding was that the correlation followed the same pattern regardless of sex, suggesting that boys and girls have a similar immunological response in the intestinal mucosa even though CD is more common in girls than in boys.
790
One child had a normal biopsy (Marsh 0) in combination with a relatively high tTG-IgA value of 44 U/mL at the time of biopsy. In the blood sample taken at the school, the tTG-IgA value was 33 U/mL. The mucosal specimen was evaluated to be of good quality and classified as Marsh 0, by both the local and the expert pathologist. The child did not obtain a CD diagnosis and therefore continued on gluten-containing food. The child, however, had a genetic susceptibility for CD and family members with CD. The serum samples were studied annually and showed a gradual decline. After 3 years, the tTG-IgA levels were normalized. A possible explanation to the initial high levels may be that the serological markers were transient or that the gluten load unintentionally decreased over time. The child may have potential CD. Applying the 2012 ESPGHAN guidelines, when tTG-IgA levels exceed 10 times the upper limit of normal and biopsy can be omitted, seems reasonable even in screening-detected patients without symptoms suggestive of CD. The guidelines, which suggest that all of the screening-detected patients should be biopsied, may, therefore, be questioned. Notably, for the child with tTG-IgA 83 U/ mL and Marsh 1 lesions, the guidelines would not have been applicable. The local pathologist, however, reported that the mucosal specimen was of poor quality and therefore the preparation and histopathological evaluation was suboptimal in this patient. This child also had positive EMA. The patient was subjected to a 3month gluten challenge that resulted in increased levels of tTG-IgA (120 U/mL), followed by normalization after treatment with GFD. A possible explanation in this case may be a primary false-negative biopsy because of a patchy distribution and the specimen not representative of the true mucosal damage. Approximately 90% of all of the children with type 1 diabetes mellitus have the same high-risk HLA haplotype DQ2 and/or DQ8, but only 10% of these children develop CD (38–40). Thus, this group of children is subjected to frequent CD screening, and omitting the small intestinal biopsy could be beneficial for the quality of life of these children. Popp et al showed that high tTGIgA levels correlated with Marsh 3 lesions in nonsymptomatic children with type 1 diabetes mellitus (41). This observation of screening high-risk individuals is in line with our findings in the general population and suggests that the revised criteria for CD diagnosis not only are applicable in symptomatic patients but may also be of use in screening-detected patients. Also, CD has many different clinical appearances, and in another substudy of this particular screened population we found that symptoms associated with CD did not predict actual CD in a screening situation in the general population (42). To conclude, our results indicate that high levels of tTG-IgA (>50 U/mL) are related to enteropathy in screening-detected patients and that biopsies could be omitted even in this group. Further studies are needed to investigate the need for biopsy confirmation and whether the ESPGHAN guidelines concerning CD diagnosis in screening-detected patients need to be revised. www.jpgn.org
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Acknowledgments: We thank all of the participating children and their families and Phadia GmbH, Germany, for analyzing all of the blood samples.
REFERENCES 1. Lebenthal E, Branski D. Celiac disease: an emerging global problem. J Pediatr Gastroenterol Nutr 2002;35:472–4. 2. Green PH, Cellier C. Celiac disease. N Engl J Med 2007;357:1731–43. 3. Fasano A. Clinical presentation of celiac disease in the pediatric population. Gastroenterology 2005;128:S68–73. 4. Ludvigsson JF, Ansved P, Falth-Magnusson K, et al. Symptoms and signs have changed in Swedish children with coeliac disease. J Pediatr Gastroenterol Nutr 2004;38:181–6. 5. Collin P, Kaukinen K, Vogelsang H, et al. Antiendomysial and antihuman recombinant tissue transglutaminase antibodies in the diagnosis of coeliac disease: a biopsy-proven European multicentre study. Eur J Gastroenterol Hepatol 2005;17:85–91. 6. Reilly NR, Fasano A, Green PH. Presentation of celiac disease. Gastrointest Endosc Clin N Am 2012;22:613–21. 7. Uenishi RH, Gandolfi L, Almeida LM, et al. Screening for celiac disease in 1st degree relatives: a 10-year follow-up study. BMC Gastroenterol 2014;14:36. 8. Valitutti F, Barbato M, Cucchiara S. Screening celiac disease in at-risk groups: effect of diagnosis on perceived health of children and their families. J Pediatr Gastroenterol Nutr 2012;55:365. 9. Di Sabatino A, Corazza GR. Coeliac disease. Lancet 2009;373:1480–93. 10. Sollid LM. Coeliac disease: dissecting a complex inflammatory disorder. Nat Rev Immunol 2002;2:647–55. 11. Dickson BC, Streutker CJ, Chetty R. Coeliac disease: an update for pathologists. J Clin Pathol 2006;59:1008–16. 12. Marsh MN. Grains of truth: evolutionary changes in small intestinal mucosa in response to environmental antigen challenge. Gut 1990;31: 111–4. 13. Bonamico M, Thanasi E, Mariani P, et al. Duodenal bulb biopsies in celiac disease: a multicenter study. J Pediatr Gastroenterol Nutr 2008; 47:618–22. 14. Rashid M, MacDonald A. Importance of duodenal bulb biopsies in children for diagnosis of celiac disease in clinical practice. BMC Gastroenterol 2009;9:78. 15. Husby S, Koletzko S, Korponay-Szabo IR, et al. European Society for Pediatric Gastroenterology, Hepatology, and Nutrition guidelines for the diagnosis of coeliac disease. J Pediatr Gastroenterol Nutr 2012;54:136– 60. 16. Barker CC, Mitton C, Jevon G, et al. Can tissue transglutaminase antibody titers replace small-bowel biopsy to diagnose celiac disease in select pediatric populations? Pediatrics 2005;115:1341–6. 17. Villalta D, Bizzaro N, Tonutti E, et al. IgG anti-transglutaminase autoantibodies in systemic lupus erythematosus and Sjogren syndrome. Clin Chem 2002;48:1133. 18. Ferrara F, Quaglia S, Caputo I, et al. Anti-transglutaminase antibodies in non-coeliac children suffering from infectious diseases. Clin Exp Immunol 2010;159:217–23. 19. Tursi A, Brandimarte G, Giorgetti GM. Prevalence of antitissue transglutaminase antibodies in different degrees of intestinal damage in celiac disease. J Clin Gastroenterol 2003;36:219–21. 20. Rostami K, Kerckhaert J, Tiemessen R, et al. Sensitivity of antiendomysium and antigliadin antibodies in untreated celiac disease: disappointing in clinical practice. Am J Gastroenterol 1999;94:888–94. 21. Donaldson MR, Book LS, Leiferman KM, et al. Strongly positive tissue transglutaminase antibodies are associated with Marsh 3 histopathology in adult and pediatric celiac disease. J Clin Gastroenterol 2008;42:256– 60.
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Prediction of Celiac Disease in Population-Based Screening
22. Liu E, Bao F, Barriga K, et al. Fluctuating transglutaminase autoantibodies are related to histologic features of celiac disease. Clin Gastroenterol Hepatol 2003;1:356–62. 23. Fabiani E, Catassi C, International Working Group. The serum IgA class anti-tissue transglutaminase antibodies in the diagnosis and follow up of coeliac disease. Results of an international multi-centre study. International Working Group on Eu-tTG. Eur J Gastroenterol Hepatol 2001; 13:659–65. 24. Mubarak A, Wolters VM, Gmelig-Meyling FH, et al. Tissue transglutaminase levels above 100 U/mL and celiac disease: a prospective study. World J Gastroenterol 2012;18:4399–403. 25. Kotze LM, Utiyama SR, Nisihara RM, et al. IgA class anti-endomysial and anti-tissue transglutaminase antibodies in relation to duodenal mucosa changes in coeliac disease. Pathology 2003;35:56–60. 26. Alessio MG, Tonutti E, Brusca I, et al. Correlation between IgA tissue transglutaminase antibody ratio and histological finding in celiac disease. J Pediatr Gastroenterol Nutr 2012;55:44–9. 27. Myleus A, Ivarsson A, Webb C, et al. Celiac disease revealed in 3% of Swedish 12-year-olds born during an epidemic. J Pediatr Gastroenterol Nutr 2009;49:170–6. 28. Ivarsson A, Myle´us A, Norstro¨m F, et al. Prevalence of childhood celiac disease and changes in infant feeding. Pediatrics 2013;131:e687–94. 29. Webb C, Halvarsson B, Norstrom F, et al. Accuracy in celiac disease diagnostics by controlling the small-bowel biopsy process. J Pediatr Gastroenterol Nutr 2011;52:549–53. 30. Burgin-Wolff A, Dahlbom I, Hadziselimovic F, et al. Antibodies against human tissue transglutaminase and endomysium in diagnosing and monitoring coeliac disease. Scand J Gastroenterol 2002;37:685–91. 31. Hill PG, Holmes GK. Coeliac disease: a biopsy is not always necessary for diagnosis. Aliment Pharmacol Ther 2008;27:572–7. 32. Dahlbom I, Korponay-Szabo IR, Kovacs JB, et al. Prediction of clinical and mucosal severity of coeliac disease and dermatitis herpetiformis by quantification of IgA/IgG serum antibodies to tissue transglutaminase. J Pediatr Gastroenterol Nutr 2010;50:140–6. 33. Vivas S, Ruiz de Morales JG, Riestra S, et al. Duodenal biopsy may be avoided when high transglutaminase antibody titers are present. World J Gastroenterol 2009;15:4775–80. 34. Hopper AD, Hadjivassiliou M, Hurlstone DP, et al. What is the role of serologic testing in celiac disease? A prospective, biopsy-confirmed study with economic analysis. Clin Gastroenterol Hepatol 2008;6:314– 20. 35. Bardella MT, Molteni N, Prampolini L, et al. Need for follow up in coeliac disease. Arch Dis Child 1994;70:211–3. 36. Mayer M, Greco L, Troncone R, et al. Compliance of adolescents with coeliac disease with a gluten free diet. Gut 1991;32:881–5. 37. Bonamico M, Mariani P, Thanasi E, et al. Patchy villous atrophy of the duodenum in childhood celiac disease. J Pediatr Gastroenterol Nutr 2004;38:204–7. 38. Gutierrez-Achury J, Coutinho de Almeida R, Wijmenga C. Shared genetics in coeliac disease and other immune-mediated diseases. J Intern Med 2011;269:591–603. 39. Bybrant MC, Ortqvist E, Lantz S, et al. High prevalence of celiac disease in Swedish children and adolescents with type 1 diabetes and the relation to the Swedish epidemic of celiac disease: a cohort study. Scand J Gastroenterol 2014;49:52–8. 40. Larsson K, Carlsson A, Cederwall E, et al. Annual screening detects celiac disease in children with type 1 diabetes. Pediatr Diabetes 2008; 9:354–9. 41. Popp A, Mihu M, Munteanu M, et al. Prospective antibody case finding of coeliac disease in type-1 diabetes children: need of biopsy revisited. Acta Paediatr 2013;102:e102–6. 42. Rosen A, Sandstrom O, Carlsson A, et al. Usefulness of symptoms to screen for celiac disease. Pediatrics 2014;133:211–8.
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Copyright 2015 by ESPGHAN and NASPGHAN. Unauthorized reproduction of this article is prohibited.
Celiac Disease Can Be Predicted by High Levels of Anti-Tissue Transglutaminase Antibodies in Population-Based Screening
Charlotta Webb, yFredrik Norstro¨m, yAnna Myle´us, yAnneli Ivarsson, zBritta Halvarsson, § Lotta Ho¨gberg, ôCarina Lagerqvist, yAnna Rose´n, yOlof Sandstro¨m, § Lars Stenhammar, and Annelie Carlsson
ABSTRACT Objectives: The aim of the present study was to evaluate any potential correlation between anti-tissue transglutaminase antibodies of type immunoglobulin A (tTG-IgA) and the degree of gluten-induced enteropathy in children participating in a screening study for celiac disease (CD) and to assess to what extent the revised European Society for Pediatric Gastroenterology, Hepatology, and Nutrition (ESPGHAN) guidelines cover this group of patients. Methods: The present study is a substudy of a cross-sectional CD screening study, Exploring the Iceberg of Celiacs in Sweden, a 2-phased study performed during 2005 to 2006 and 2009 to 2010. The 13,279 participating children had a blood test obtained, and those with positive tTG-IgA were recommended a small intestinal biopsy. The tTG-IgA levels at the time of biopsy were compared with those at the assessment of the biopsy. Results: There were 267 children included, of whom 230 were diagnosed as having CD. Of all of the children, 67 children had low tTG-IgA levels (100 U/mL) have a high specificity for Marsh 3 lesions (19,21–26), which underpins the revision of the ESPGHAN diagnostic guidelines. To the best of our knowledge, however, there are yet no data on the correlation between tTG-IgA levels and the degree of enteropathy in screening-detected patients with CD from the general childhood population. The primary aim of the present study was to evaluate a potential correlation between the level of tTG-IgA and the degree of gluten-induced enteropathy in screening-detected patients with CD from a general child population and to assess to what extent the revised ESPGHAN guidelines can be extended to cover this group of patients.
METHODS The present study is a substudy of a cross-sectional CD screening study entitled ‘‘Exploring the Iceberg of Celiacs in Sweden,’’ which is part of the European Prevent CD project. The screening was school-based and included 2 birth cohorts of 12-year-old children born in 1993 or 1997 with the screening taking place in 2005 to 2006 and 2009 to 2010, respectively. Five pediatric departments collaborated in the present study (Umea˚, Norrta¨lje, Norrko¨ping, Va¨xjo¨, and Lund), and each of the sites included a major city with municipalities in the surrounding suburbs and countryside. A total of 18,325 children were invited, and 13,279 consented to CD screening (Fig. 1). The proportion of girls was similar in both cohorts (48% and 49%, respectively). Positive serological markers were found in 291 children, and these children were invited to a further investigation with a small intestinal biopsy. Inclusion criteria for the present substudy were that a small intestinal biopsy had been performed and a second blood sample drawn at the time of the biopsy had been analyzed for tTG-IgA. The Exploring the Iceberg of Celiacs in Sweden study was approved by the regional ethical review board of Umea˚ University, Umea˚, Sweden.
Volume 60, Number 6, June 2015
Participants (n = 13,279) Total blood samples (n = 12,632)
Positive serology in school (n = 291)
Inclusion criteria (n = 268) Small intestinal biopsy (13 children excluded) and renewed blood sample (10 children excluded)
Histological evaluation (n = 268) Marsh 0
=
Marsh 1 no spt
=
3
Marsh 1 and spt Marsh 2
= =
11 2
=
217
Marsh 3 Noninterpretable
34
=
1
Included in the study n = 267
Screening Strategy Participating children were asked for a blood sample at their school, and a second serum sample was obtained at the time of biopsy (Fig. 1). The samples were analyzed at the same laboratory for tTG-IgA by enzyme-linked immunosorbent assay (Celikey, Phadia GmbH, Freiburg im Breisgau, Germany). The manufacturer’s cutoff for positive tTG-IgA was 5 U/mL, but to increase the sensitivity of the screening we chose to consider all of the values >4 U/mL to be elevated. Serum samples were analyzed in duplicate within the measuring range 0 to 100 U/mL, and the mean value was used. Serum samples with intermediate values of tTG-IgA (2–4 U/ mL) were additionally analyzed for EMA-with a 1:5 dilution as a cutoff. EMA were determined by indirect immunofluorescence using monkey esophagus tissue (Binding Site, Birmingham, UK). All of the children (n ¼ 291) with elevated serological markers at the screening occasion were referred to a pediatric clinic for a follow-up with a second celiac serology analysis and a small intestinal biopsy. A total of 278 children accepted the follow-up screening procedure, which has previously been described in detail (27,28). Of the 278 children who had a biopsy performed, 267 children met the inclusion criteria for the study. Five children in the 1993 cohort had IgA deficiency and tTG-IgG >6 U/mL and were excluded, and another 5 children were excluded because no serum samples were taken at the time of the biopsy. One additional patient was also excluded because of an inconclusive biopsy. Of the 267 children in the present study (156 girls and 111 boys), 230 were
788
CD Diagnosis Children = 230 Girls/boys 133/97
FIGURE 1. Flowchart summarizing the results from the screening for celiac disease in 2 birth cohorts. CD ¼ celiac disease.
diagnosed as having CD. There were no significant differences between the 2 birth cohorts with respect to age or sex, so the data for the 2 birth cohorts were combined.
Diagnostic Process In the 1993 cohort, the small intestinal biopsy was performed either with a suction capsule (n ¼ 54) or by endoscopy (n ¼ 125) and has previously been described in detail (29). In the 1997 cohort, all of the biopsies were performed using endoscopy (n ¼ 99). Biopsies were taken both proximally and more distally in the duodenum, including the bulb, following a recommendation of 4 to 6 specimens per procedure. The mucosal specimens were classified according to the revised Marsh-Oberhuber classification (11). All of the biopsies were subjected to a second histopathological evaluation by a pathologist blinded to the previous results. In case of disagreement, www.jpgn.org
Copyright 2015 by ESPGHAN and NASPGHAN. Unauthorized reproduction of this article is prohibited.
Volume 60, Number 6, June 2015
www.jpgn.org
100
80
60 50 40 30
Median
20
h
3b h
M ar s
h
M ar s
23a
1 sh ar s
3c
0
M
We found a wide variation in tTG-IgA levels and the degree of enteropathy among the 267 children who were participating in the present study (Fig. 2). There was, however, a tendency toward a gradual increase in the tTG-IgA levels predicting mucosal damage (Table 1), and Marsh 3 lesions were seen in 56%, 53%, 86%, 87%, and 98% of the children with tTG-IgA levels 50 U/mL, respectively. There was no significant difference in Marsh 3 lesions between boys and girls—85% versus 79%, respectively (P ¼ 0.23). There were 2 interesting subgroups of children when comparing the results from the serology and the biopsy. The first group consisted of children with low tTG-IgA levels (50 U/mL). There were 64 children in this group and the majority of them had Marsh 3 lesions, but there was 1 child with a biopsy graded as Marsh 1 who was EMA positive. In the group of children with tTG-IgA levels 30 to 50 U/mL, there was 1 child with Marsh 0 lesions, who consequently was not given a CD diagnosis, and 3 children who had Marsh 1 lesions. All of the children in this group were EMA positive. All of the patients with CD were HLA DQ2 and/or DQ8 positive. All of the children with Marsh 1 lesions and high tTG-IgA levels (>30 U/mL) were considered to have CD based on additional criteria according to the study design.
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Level of tTG-IgA and the Degree of Enteropathy and CD Diagnosis
DISCUSSION To the best of our knowledge, the present study is the first one to investigate any potential association between the levels of tTG-IgA and enteropathy in a screening for CD in a general population of children. We found that levels of tTG-IgA >50 U/ mL predicted gluten-induced enteropathy, but we also found an uncertainty to predict enteropathy in lower levels of tTG-IgA. Several studies have found a strong correlation between tTGIgA >100 U/mL and Marsh 3 lesions (21,24–26). These studies, however, were performed in children who had symptoms and signs suggestive of CD in a clinical setting. They also reported a poor correlation between the level of tTG-IgA and the severity of enteropathy. In our study, all of the children who had levels exceeding 10 times the upper level of normal values at the recommended cutoff from the manufacturer (>5 U/mL), that is, 50 U/mL, were diagnosed as having CD. Recent studies using the same serological test in a clinical setting, however, recommended a cutoff of >3 U/ mL. Among those with tTG >30 U/mL, 1 child did not have CD. Interestingly, Marsh 3 lesions were found more frequently in the group with tTG-IgA >50 U/mL (98%) than in the group with tTG-IgA between 30 and 50 U/mL (87%), which could be a reason
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RESULTS
We assessed the 2012 ESPGHAN diagnostic criteria based on the serological levels of tTg-IgA exceeding 10 times the upper levels of normal. The ESPGHAN diagnostic criteria for the serology would have given 64 CD diagnoses of whom 63 (98%) were graded with Marsh 3 lesions for a cutoff of 5 U/mL and 101 CD diagnoses of whom 96 (95%) were graded with Marsh 3 lesions for a cutoff of 3 U/mL. One child would not have been considered to have CD in our study for the cutoff of 3 U/mL, whereas all of the children were considered to have CD for the higher cutoff of 5 U/mL.
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Frequency tables, cross-tabulations, medians, and quartiles were used to present our data. Student t test was used to test whether there was a difference in the proportion of boys and girls with a high small intestinal damage. Statistical significance was defined as P < 0.05. Microsoft Access was used for data handling, and Stata 11.2 (StataCorp LP, College Station, TX) was used for statistical analyses and figures.
ESPGHAN CD Diagnostic Criteria for ScreeningDetected Patients
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Statistical Analyses
There were 34 patients who had normal biopsies, 26 children (76%) had tTG-IgA levels 30 lymphocytes/100 enterocytes), crypt hyperplasia, and villous atrophy. In patients with Marsh 1 lesions (intraepithelial lymphocytes), symptoms and/or signs compatible with CD were required in combination with an HLA-DQ2/ DQ8 haplotype and clinical response to a GFD (27,29). In the present substudy, we evaluated the results of the serological markers at the time of biopsy. In the 2012 ESPGHAN guidelines, biopsy is no longer mandatory when the tTG-IgA levels exceed 10 times the upper limit of normal. The cutoff for positivity recommended by the manufacturer was >5 U/mL (30). Studies have indicated that a cutoff of 3 U/mL would be more correct, and therefore the results were additionally analyzed at a cutoff of 3 U/mL (31–33). Four children with tTG-IgA >30 U/mL in combination with Marsh 1 lesions and 1 child with tTG-IgA >50 U/mL who had Marsh 0 lesions were additionally tested for EMA.
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tTG-IgA (U/mL)
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FIGURE 2. Degree of mucosal damage in relation to tTG-IgA. NB: In the 1993 cohort, 2 patients with Marsh 2 lesions have been included in the Marsh 2 to 3a group. tTG-IgA ¼ anti-tissue transglutaminase antibodies of type immunoglobulin A.
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Webb et al
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Volume 60, Number 6, June 2015
TABLE 1. Serological levels in correspondence with CD diagnosis and biopsy evaluation Marsh classification Serology groups, U/mL 50 U/mL and Marsh 1 lesions and 4 patients with tTG-IgA >30 U/mL and Marsh 1 lesions, were EMA positive. Although there are many pitfalls in the diagnosis of CD, there are some plausible explanations for the inconsistent association between the level of serological markers and intestinal mucosal damage. Every step in the diagnostic process has an impact on the diagnostic outcome. The histopathological evaluation is dependent on different variables, for example, biopsies from several different locations, biopsy method, and the evaluation by an experienced pathologist (29). The biopsy procedure in the present study has been thoroughly validated (29), and we found that multiple biopsies taken from both the proximal and the distal duodenum sharpened the diagnosis; all of the biopsies in the present study were reevaluated by the same experienced pathologist. An interesting result was the subgroup with severe enteropathy in combination with low levels of tTG-IgA, which supports the results of other studies and underlines the necessity of performing a biopsy to obtain a correct diagnosis (16). It is somewhat easier to explain why some children with high levels of tTG-IgA had only mild enteropathy because it is known that the lesions can be patchily distributed in children that can lead to severe lesions being missed by the biopsy (37). Another interesting finding was that the correlation followed the same pattern regardless of sex, suggesting that boys and girls have a similar immunological response in the intestinal mucosa even though CD is more common in girls than in boys.
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One child had a normal biopsy (Marsh 0) in combination with a relatively high tTG-IgA value of 44 U/mL at the time of biopsy. In the blood sample taken at the school, the tTG-IgA value was 33 U/mL. The mucosal specimen was evaluated to be of good quality and classified as Marsh 0, by both the local and the expert pathologist. The child did not obtain a CD diagnosis and therefore continued on gluten-containing food. The child, however, had a genetic susceptibility for CD and family members with CD. The serum samples were studied annually and showed a gradual decline. After 3 years, the tTG-IgA levels were normalized. A possible explanation to the initial high levels may be that the serological markers were transient or that the gluten load unintentionally decreased over time. The child may have potential CD. Applying the 2012 ESPGHAN guidelines, when tTG-IgA levels exceed 10 times the upper limit of normal and biopsy can be omitted, seems reasonable even in screening-detected patients without symptoms suggestive of CD. The guidelines, which suggest that all of the screening-detected patients should be biopsied, may, therefore, be questioned. Notably, for the child with tTG-IgA 83 U/ mL and Marsh 1 lesions, the guidelines would not have been applicable. The local pathologist, however, reported that the mucosal specimen was of poor quality and therefore the preparation and histopathological evaluation was suboptimal in this patient. This child also had positive EMA. The patient was subjected to a 3month gluten challenge that resulted in increased levels of tTG-IgA (120 U/mL), followed by normalization after treatment with GFD. A possible explanation in this case may be a primary false-negative biopsy because of a patchy distribution and the specimen not representative of the true mucosal damage. Approximately 90% of all of the children with type 1 diabetes mellitus have the same high-risk HLA haplotype DQ2 and/or DQ8, but only 10% of these children develop CD (38–40). Thus, this group of children is subjected to frequent CD screening, and omitting the small intestinal biopsy could be beneficial for the quality of life of these children. Popp et al showed that high tTGIgA levels correlated with Marsh 3 lesions in nonsymptomatic children with type 1 diabetes mellitus (41). This observation of screening high-risk individuals is in line with our findings in the general population and suggests that the revised criteria for CD diagnosis not only are applicable in symptomatic patients but may also be of use in screening-detected patients. Also, CD has many different clinical appearances, and in another substudy of this particular screened population we found that symptoms associated with CD did not predict actual CD in a screening situation in the general population (42). To conclude, our results indicate that high levels of tTG-IgA (>50 U/mL) are related to enteropathy in screening-detected patients and that biopsies could be omitted even in this group. Further studies are needed to investigate the need for biopsy confirmation and whether the ESPGHAN guidelines concerning CD diagnosis in screening-detected patients need to be revised. www.jpgn.org
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Volume 60, Number 6, June 2015
Acknowledgments: We thank all of the participating children and their families and Phadia GmbH, Germany, for analyzing all of the blood samples.
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