The current diagnostic criteria for celiac disease require. Diagnosing Mild Enteropathy Celiac Disease: A Randomized, Controlled Clinical Study

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1 GASTROENTEROLOGY 2009;136: Diagnosing Mild Enteropathy Celiac Disease: A Randomized, Controlled Clinical Study KALLE KURPPA,* PEKKA COLLIN,, MERVI VILJAMAA,* KATRI HAIMILA, PÄIVI SAAVALAINEN, JUKKA PARTANEN, # KAIJA LAURILA,* HEINI HUHTALA,** KAIJA PAASIKIVI, MARKKU MÄKI,* and KATRI KAUKINEN, *Paediatric Research Centre, University of Tampere and Tampere University Hospital, Tampere, Finland; Department of Gastroenterology and Alimentary Tract Surgery, Department of Internal Medicine, Tampere University Hospital, Tampere, Finland; Medical School, **Tampere School of Public Health, University of Tampere, Tampere, Finland; Clinical Services, # Research and Development, Finnish Red Cross Blood Service, Helsinki, Finland; and the Department of Medical Genetics and Research Program for Molecular Medicine, University of Helsinki, Helsinki, Finland Background & Aims: The diagnostic criteria for celiac disease require small-bowel mucosal villous atrophy with crypt hyperplasia (Marsh III). However, mucosal damage develops gradually and patients may evince clinical symptoms before histologic changes appear. Endomysial antibodies are specific in predicting forthcoming villous atrophy. We hypothesized that patients with mild enteropathy but positive endomysial antibodies benefit from a gluten-free diet (GFD) similarly to patients with more severe enteropathy. Methods: Small-bowel endoscopy together with clinical evaluations was performed in all together 70 consecutive adults with positive endomysial antibodies. Of these, 23 had only mild enteropathy (Marsh I II) and they were randomized either to continue on a gluten-containing diet or start a GFD. After 1 year, clinical, serologic, and histologic evaluations were repeated. A total of 47 participants had small-bowel mucosal lesions compatible with celiac disease (Marsh III), and these served as disease controls. Results: In the gluten-containing diet group (Marsh I II) the small-bowel mucosal villous architecture deteriorated in all participants, and the symptoms and abnormal antibody titers persisted. In contrast, in the GFD group (Marsh I II) the symptoms were alleviated, antibody titers decreased, and mucosal inflammation diminished equally to celiac controls (Marsh III). When the trial was completed, all participants chose to continue on a life-long GFD. Conclusions: Patients with endomysial antibodies benefit from a GFD regardless of the degree of enteropathy. The diagnostic criteria for celiac disease need re-evaluation: endomysial antibody positivity without atrophy belongs to the spectrum of genetic gluten intolerance, and warrants dietary treatment. The current diagnostic criteria for celiac disease require small-bowel mucosal villous atrophy with crypt hyperplasia (Marsh III). 1,2 Evidently, small-bowel mucosal deterioration develops gradually 3 and mild enteropathy eventually may progress to overt villous atrophy and crypt hyperplasia, provided ingestion of gluten is continued. 4,5 Current data suggest that some patients with only mild enteropathy may even suffer from glutendependent symptoms or complications before the development of villous atrophy This raises the inevitable question of whether the diagnostic criteria for celiac disease 1 are valid anymore. There have been attempts to find reliable markers for early stage celiac disease, but no single diagnostic test has been devised to date. Small-bowel mucosal intraepithelial lymphocytosis precedes the development of morphologic changes. However, lymphocytosis is a somewhat unspecific finding, and only less than half of such cases will eventually develop celiac disease, showing that the infiltrativity of lymphocytes as such is not a sufficient criterion for celiac disease. 2 Serum endomysial (EmA) and tissue transglutaminase antibodies (ttg-ab) have been shown to afford sensitive and specific tools for celiac disease screening As a consequence of an intensified screening policy, subjects with positive antibodies but without diagnostic small-bowel mucosal villous atrophy frequently are found. 6 The condition often is considered false-positive, but there also is evidence to suggest that such a finding is indicative of early stage celiac disease. 20 Randomized clinical trials on the natural history and treatment of celiac disease patients with only mild enteropathy and positive antibodies are lacking, and there is no consensus as to whether patients should be treated at all with a gluten-free diet (GFD) before villous atrophy has developed. In this prospective, randomized, controlled clinical trial, we investigated the effect of gluten intervention in consecutive EmA-positive adults with celiac disease suspicion who were found to have small-bowel mucosal lymphocytosis but normal villous architecture. The participants were randomized either to continue on a nor- Abbreviations used in this paper: CD, celiac disease; EmA, endomysial antibodies; GFD, gluten-free diet; IEL, intraepithelial lymphocyte; ttg-ab, tissue transglutaminase antibody; Vh/CrD, villous height/crypt depth ratio by the AGA Institute /09/$36.00 doi: /j.gastro

2 March 2009 DIAGNOSING MILD ENTEROPATHY CELIAC DISEASE 817 mal, gluten-containing diet, or to start a GFD. We hypothesized that adults with positive EmA but only mild enteropathy in the small-bowel mucosa have a glutendependent disorder, as measured by clinical, serologic, and histologic indicators. If this were the case, the current diagnostic criteria for celiac disease would need revision. Materials and Methods Patients and Study Design The study was a prospective, randomized, controlled clinical trial performed between the years 2003 and 2008 at the Department of Gastroenterology and Alimentary Tract Surgery in Tampere University Hospital. The participants were recruited by general practitioners working in primary health care, who were encouraged to remit adults with any suspicion of celiac disease. Patients with immunosuppressive medication or suspicion of dermatitis herpetiformis were excluded. All together 145 adults were sent to the hospital for EmA testing, upper-gastrointestinal endoscopy, and smallbowel biopsy. Before the initial investigations 10 patients refused to participate. Small-bowel mucosal morphology was classified according to the Marsh criteria. 2 None of the EmA-positive patients had completely normal mucosa (Marsh 0). Participants with normal villi but mucosal inflammation with or without crypt hyperplasia (Marsh I and II, respectively) comprised the study group. They were randomized either to continue on a normal gluten-containing diet (gluten group) or to adhere to a strict GFD (GFD group). Randomization was performed by means of random number tables with permuted blocks, 21 and patients entered the trial before the treatment group was specified. After 1 year, GFD treatment also was offered to all participants in the gluten group, and a second follow-up evaluation was performed after 1 year. After completion of the trial, all participants in the study group could either continue on a GFD or revert to a normal glutencontaining diet. Patients with small-bowel mucosal villous atrophy and crypt hyperplasia (Marsh III) comprised the celiac disease (CD) control group (CD group), and they were placed on a GFD. For reference data on small-bowel mucosal morphology and immunohistochemistry, 34 consecutive EmA-negative adults suffering from dyspepsia (n 19), upper abdominal pain (n 11), and/or heartburn (n 14), and no suspicion of celiac disease, were used as nonceliac controls (non-cd group). Dietary Assessment A dietitian interviewed all participants at baseline and 1 year after entering the study. The subjects in the study group were advised either to continue the normal gluten-containing diet or to start a GFD, depending on the result of randomization. Subjects in the CD group started a GFD. The diet was to be as strict as possible, and in the gluten group the target was to ensure approximately the average daily gluten consumption (ie, g of gluten/day 22 ) in normal bread, cake, or pasta. Small-Bowel Mucosal Morphology At the upper-gastrointestinal endoscopy, 6 forceps biopsy specimens were taken from the distal part of the duodenum. Three biopsy specimens were processed and stained with H&E, and small-bowel mucosal morphology was determined under light microscopy from at least 3 well-oriented biopsy sections. Poorly orientated sections were dissected again until they were of good quality. All together 3 independent specialists in 2 centers evaluated the biopsy samples without prior knowledge of the clinical symptoms, EmA titers, or the randomization groups of the patients. The lesions were scored as Marsh 0 when there were normal finger-like villi of a normal crypt depth and no excess of intraepithelial lymphocytes (IELs). In Marsh I II intraepithelial lymphocytosis was seen in normal villous structure without (Marsh I) or with (Marsh II) hyperplastic crypts. Marsh III comprised villous atrophy with crypt hyperplasia, and was considered diagnostic for celiac disease. 3 Cases with patchy small-bowel mucosal villous atrophy were classified as Marsh III. To further evaluate precisely the mucosal morphology, villous height/crypt depth ratio (Vh/CrD) was measured as a mean of at least 5 well-orientated villouscrypt pairs as previously described 23 and a ratio of less than 2.0 was regarded as Marsh III and compatible with celiac disease. An example of the interpretation of smallbowel mucosal lesion is shown in Figure 1. Small-Bowel Mucosal Inflammation Immunohistochemical stainings were performed on 5- m thick frozen sections from 3 small-bowel mucosal biopsy specimens. CD3 IELs were stained with monoclonal antibody Leu-4 (Becton Dickinson, San Jose, CA), IELs were stained with monoclonal F1 antibody (Endogen, Woburn, MA), and IELs were stained with T-cell receptor- antibody (Endogen). Positive IELs were counted with a 100 flat field, light microscope objective throughout the surface epithelium; at least 30 fields measuring 1.6 mm in epithelial length were counted and IEL density was expressed as cells/mm of epithelium, as previously described To further show mucosal immunoactivation, mucosal HLA DR expression was detected with monoclonal HLA-DR antibody (Becton Dickinson) at a dilution of 1: ,28 HLA-DR expression was considered normal (grade 0) when there was no staining in crypts and only slight or moderate expression in the villous epithelium. When any HLA-DR expression was seen on the crypts or was strong in the villous epithelium, the expression was considered enhanced. All

3 818 KURPPA ET AL GASTROENTEROLOGY Vol. 136, No. 3 Figure 1. Small-bowel mucosal biopsy samples from 2 patients (A and B) with mild enteropathy celiac disease. The Vh/ CrD ratios were determined from 3 separate biopsy samples and at least 5 well-orientated villouscrypt units (as shown in panel A, black bars). evaluations were performed without prior knowledge of the history of the study subjects. Serology, Laboratory Parameters, and HLA Typing Serum immunoglobulin (Ig)A class EmA values were determined by an indirect immunofluorescence method using human umbilical cord as substrate; a dilution of 1:5 or more was considered positive. 29 Positive sera were diluted further: 1:50, 1:100, 1:200, 1:500, 1:1000, 1:2000, and 1:4000. We also determined serum IgA class tissue transglutaminase antibodies, which are used widely in clinical practice. 19 The antibodies were investigated by an enzyme-linked immunosorbent assay (Celikey; Phadia, Freiburg, Germany) according to the manufacturer s instructions; values of 5.0 U or greater were considered positive. The following laboratory values were measured by standard laboratory methods: blood hemoglobin level (reference values: men, g/dl; women, g/dl), serum albumin level (reference values, g/dl), ionized calcium level (reference values, mmol/l), serum iron level (reference values, g/dl), red blood cell folate level (Efolate) (reference values, nmol/l), and serum vitamin B 12 level (reference values, pmol/k). Genotyping of the participants was performed either by using the DELFIA Celiac Disease Hybridization Assay (PerkinElmer Life and Analytical Sciences, Wallac Oy, Turku, Finland) or the SSP DQB1 low-resolution kit (Olerup SSP AB, Saltsjöbaden, Sweden/Qiagen Vertriebs GmbH, Vienna, Austria) according to the manufacturer s instructions. Clinical Evaluation Past medical history, signs and symptoms leading to the suspicion of celiac disease, duration of symptoms, and family history of celiac disease were recorded. The clinical symptoms were classified semiquantitatively in 4 categories: no symptoms, slight symptoms (occasionally 1 or more of the following: abdominal pain, flatulence, diarrhea, belching, tiredness, or joint pain), moderate symptoms (symptoms more persistent, disturbing normal life), or severe symptoms (severe daily symptoms significantly restricting normal life, or excessive weight loss). The symptoms were evaluated with similar questions at each visit and the scoring was calculated blinded, without knowledge of the previous score. Statistics Quantitative data were expressed as medians and ranges or means and standard deviations. A 2-tailed Student t test was used to compare laboratory values, Vh/ CrD ratios, and IELs between the groups at baseline, and a paired t test or Wilcoxon signed-rank test was used to compare changes within the groups. The Mann Whitney U test was used to compare changes between the groups in ttg-ab values. The chi-square test in cross-tabulations was used to compare differences in clinical symptoms and HLA-DR expression between the groups at baseline and also in changes of clinical symptoms between the groups. A P value of less than.05 was considered statistically significant.

4 March 2009 DIAGNOSING MILD ENTEROPATHY CELIAC DISEASE 819 None of the participants had reduced gluten intake before the first endoscopy. Figure 2. Flow chart of the study. Ethical Considerations The study protocol was approved by the Ethical Committee of Tampere University Hospital. All subjects gave written informed consent. Results Patients and Dietary Intervention Of the total 145 patients admitted, 70 were EmApositive and all had at least Marsh I type, small-intestinal mucosal lesions; none had patchy villous damage. Of the 70 patients, 23 had normal villi and small-bowel mucosal intraepithelial lymphocytosis with (Marsh II) or without (Marsh I) crypt hyperplasia (Figure 2); this means that they did not fulfill the current diagnostic criteria for celiac disease 1,2 (study group). Ten of these 23 subjects were randomized to a gluten group and 13 to a GFD group (Figure 1). A total of 47 participants already had Marsh III lesion in the mucosa (CD group). At baseline, all 3 groups were comparable with respect to age, sex, primary reason for celiac disease suspicion, duration of symptoms, and family history of celiac disease (Table 1). Small-Bowel Mucosal Morphology At the beginning of the trial the Vh/CrD was 2.0 or greater in all participants in the gluten and GFD groups, and less than 2.0 in all subjects in the CD group. As shown in Figure 3A, in the gluten group the Vh/CrD decreased in all subjects on a gluten-containing diet. In contrast, in the GFD group the Vh/CrD increased in all, although the initial values were within the normal range. When the participants in the gluten group adopted a GFD after the first year, the mucosal architecture recovered, which also was observed in the CD group. Small-Bowel Mucosal Inflammation At baseline, there were no significant differences between the densities of CD3,, and IELs in the trial groups (gluten, GFD, and CD groups), but the densities were increased in all 3 groups when compared with the non-cd group (Table 2). In the gluten group, IELs remained increased after 1 year on a gluten-containing diet, whereas in the GFD and CD groups the IELs decreased significantly on a GFD (Table 2). While on a GFD, the IELs also decreased in the gluten group, albeit not significantly (P.104). At baseline, HLA-DR expression in the mucosa clearly was aberrant in all trial groups compared with the non-cd group. Serology and HLA Typing By definition, all the EmA titers were increased in the gluten and GFD groups before the intervention, but were significantly lower than those in the CD group (P.001) (Figure 3B). In the gluten group, the EmA titers remained basically at the same level on continued gluten consumption. In contrast, in the GFD group the titers normalized in all but 1 subject. When the GFD was prescribed to subjects in the gluten group, EmA disap- Table 1. Demographic Data and Primary Reason for Celiac Disease Suspicion Study groups Control groups Gluten group (n 10) GFD group (n 13) CD group (n 47) Non-CD group (n 34) a Female, n (%) 6 (60) 9 (75) 41 (87) 21 (62) Median age, y (range) 53 (16 69) 49 (16 70) 43 (16 71) 53 (22 72) Primary reason for endoscopy, n (%) Abdominal symptoms 8 (80) 10 (77) 25 (53) 34 (100) Malabsorption or anemia 1 (8) 11 (23) Extraintestinal symptoms b 1 (8) 5 (10) Risk group of celiac disease c 2 (10) 1 (8) 6 (13) Median duration of symptoms, y (range) 5 (0 50) 5 (0.5 40) 4 (0 50) Not defined Family history of celiac disease, n (%) 5 (50) 5 (45) 16 (34) 0 (0) a For histologic comparisons only. b Arthritis, dental enamel defects, neurologic symptoms, increased liver enzyme levels, alopecia, aphthous stomatitis, gynecologic disorders, and osteoporosis. c Type I diabetes mellitus, autoimmune thyroid disease, Sjögren s syndrome, and family history of celiac disease.

5 820 KURPPA ET AL GASTROENTEROLOGY Vol. 136, No. 3 Figure 3. Small-bowel mucosal (A) Vh/CrD ratio, (B) serum IgA class EmA and (C) ttg-ab, and (D) clinical symptoms of the participants before and after 1 year with gluten-containing or GFD. The second year with a GFD in the gluten group also is shown. *Change within the group was significant compared with the change within the gluten group after 1 year; P.05. **Change within the group was significant compared with the change within the gluten group after 1 year; P.001. peared or titers decreased in all, as was the case in the CD group (Figure 3B). As shown in Figure 3C, the alterations of ttg-ab levels in the groups were parallel with EmA. All EmA-positive participants in the study carried the celiac disease associated HLA alleles DQB1*02 (DQ2) or *0302 (DQ8), or both. Clinical Evaluation At the beginning of the study, 62 of 70 (89%) participants in the trial groups evinced clinical symptoms (Table 1) and the symptoms did not differ significantly between the groups (Figure 3D). After 1 year on a glutencontaining diet, the symptoms basically were unaltered in the gluten group, whereas in the GFD group the symptoms were alleviated in all subjects while on a GFD. The beneficial effect of the GFD on symptoms also was observed in the CD group as well as in most participants in the gluten group after adoption of the GFD (Figure 3D). Laboratory Values The results of the laboratory tests are shown in Table 3. At baseline, hemoglobin and iron values were significantly higher in the GFD group compared with the CD group. During the study there were no statistically significant changes in any of the laboratory values within the gluten or GFD groups, whereas within the CD group the hemoglobin, E-folate level, and vitamin B 12 values increased significantly on a GFD. Diet Choices After the Trial Because the participants in the trial groups were shown to be gluten-sensitive when the trial was completed, a GFD was offered to all patients. All patients chose to continue with a GFD and not to return to a normal diet.

6 March 2009 DIAGNOSING MILD ENTEROPATHY CELIAC DISEASE 821 Table 2. Small-Bowel Mucosal IELs and Enhanced HLA-DR Expression Before and After 1 Year With a Gluten-Containing Diet or a GFD Study group Control groups Gluten group (n 10) GFD group (n 13) CD group (n 47) Non-CD group (n 34) Baseline After gluten After GFD Baseline After GFD Baseline After GFD Baseline CD3 IELs, cell/mm Median 61 a a 41 b 63 a 50 c 32 Range IELs, cell/mm Median 32 d a 21 c 45 a 30 b 24 Range IELs, cell/mm Median 19.1 a a 21.7 b 19.5 a 17.1 c 2.1 Range HLA-DR expression, n (%) Enhanced 8/9 (89) d 7/9 (78) 5/6 (83) 13/13 (100) a 7/12 (58) 42/42 (100) a 29/41 (71) 20/38 (53) Normal 1/9 (11) 2/9 (22) 1/6 (17) 0/13 (0) 5/12 (42) 0/42 (0) 12/41 (29) 18/38 (47) a P.001 compared with non-cd group. b Change within the group significant compared with baseline; P.001. c Change within the group significant compared with baseline; P.05. d P.05 compared with non-cd group. Discussion This was a randomized, controlled, clinical trial to show that individuals with mild enteropathy celiac disease and positive serum EmA suffer from a gluten-dependent condition. In the course of the study, this gluten dependency was confirmed in several ways. Small-bowel mucosal structure and inflammation, serology, and clinical symptoms were investigated both by ongoing gluten intake and gluten withdrawal. When gluten consumption was continued, progression of the mucosal damage was evident, and the abnormal serology and clinical symptoms persisted. In contrast, the beneficial effect of a GFD was indisputable and in fact similar both in mild enteropathy and in overt celiac disease. In other words, there was a definite gluten dependency in all our main variables and, regardless of the degree of enteropathy, the recovery of the subjects was comparable. Moreover, all participants in the study had the HLA-type requisite for celiac disease, further strengthening the conclusion that they were suffering from genetic gluten intolerance. 30,31 The development of celiac disease is a continuum from early small-bowel mucosal changes to overt disease. 3 The well-established concept of latent celiac disease is a retrospective diagnosis for those who have normal small- Table 3. Mean Laboratory Values With Standard Deviations of the Study Participants Before and After 1 Year With a Gluten-Containing Diet or a GFD Study groups Control group Gluten group (n 10) GFD group (n 13) CD group (n 47) Baseline After gluten After GFD Baseline After GFD Baseline After GFD Blood-Hemoglobin, g/dl mean (SD) 14.0 (0.96) 14.3 (1.11) 14.1 (1.07) 14.2 (0.89) a 14.3 (1.03) 13.3 (1.06) 13.6 (0.81) b Serum-Albumin, g/dl mean (SD) 4.4 (0.44) 4.3 (0.56) 4.3 (0.40) 4.4 (0.37) 4.1 (0.45) 4.2 (0.42) 4.1 (0.42) Serum-Ionized calcium, mmol/l mean (SD) 1.26 (0.037) 1.25 (0.042) 1.25 (0.047) 1.24 (0.032) 1.25 (0.034) 1.25 (0.041) 1.24 (0.042) Serum-Iron, mol/l mean (SD) 19.8 (7.4) 19.3 (7.1) 17.4 (7.7) 22.8 (7.5) a 23.1 (6.2) 15.6 (7.2) 17.3 (7.1) E-Folate, nmol/l mean (SD) 758 (528) 725 (366) 788 (346) 466 (135) 415 (136) 516 (391) 652 (432) b Serum-Vitamin B 12, pmol/l mean (SD) 351 (116) 373 (92) 383 (137) 384 (138) 398 (115) 341 (177) 398 (173) b a P.05 compared with CD group. b Change within the group significant compared with baseline; P.05.

7 822 KURPPA ET AL GASTROENTEROLOGY Vol. 136, No. 3 intestinal mucosal structure but eventually develop villous atrophy while on a gluten-containing diet. 32 Case reports and small follow-up studies have indicated that before the development of villous atrophy, patients with latent celiac disease may suffer from abdominal pain, 4,9,12 diarrhea, 4,9,10,12 growth retardation or weight loss, 4,9 anemia, 4,11,12 fatigue, 10,12 amenorrhea, 11 and osteopenia. 9,13 Because there also is evidence showing that the symptoms and antibodies may disappear on a GFD, 4,6,9,11,13 it would seem unwise in these cases to delay treatment until the development of overt villous atrophy. Moreover, certain complications of celiac disease, for example, retardation of growth, osteoporosis, and dental enamel defects, 33,34 may remain permanent if left untreated, which further supports the need for early diagnosis and treatment. On the other hand, because a life-long GFD may be difficult to maintain, expensive, and socially restrictive, there should be strong evidence for the diagnosis. An intensified gluten challenge in the early developing phase has been proposed to uncover gluten-sensitive patients. 9 However, it is important to realize that even with continuous gluten consumption, the development of mucosal villous atrophy may take a number of years, 6 leading to the burdensome and expensive follow-up evaluation of symptomatic patients with repeated endoscopies. Obviously, new diagnostic criteria for celiac disease and better methods for early diagnosis are needed. It is interesting to note that there is already a wellcharacterized model for mild enteropathy celiac disease. In dermatitis herpetiformis, about 20% of patients may have normal small-bowel mucosal morphology, but there are often celiac-type inflammatory changes in the mucosa 35 and positive antibodies in the sera, 36 and it is well known that these changes improve upon adoption of a subsequent GFD. 36 Because of a lack of specificity the mild enteropathy as such is not a sufficient diagnostic criterion for celiac disease, 15,16 but combined with endomysial antibodies it seems to be possible to reliably find truly gluten-sensitive subjects. In this study we showed in an evidence-based manner that celiac patients with mild enteropathy, positive antibodies, and clinical symptoms benefit from early treatment with a GFD. Furthermore, because the benefits of the treatment should outweigh the burden, it is of note that when the trial was completed all participants were willing to continue with a life-long GFD. We propose that the criteria for celiac disease be evaluated promptly because patients such as ours would appear far from rare when an augmented screening policy is applied. In our selected series of 145 consecutive patients with celiac disease suspicion, 70 eventually were proved to be suffering from a gluten-dependent condition; of these 70 patients a total of 23 (33%) had only mild enteropathy. Moreover, awareness of the high prevalence, 37,38 diverse clinical picture, and complications of celiac disease is expanding, and the threshold for initiating early treatment of a symptomatic patient is lowered. When a patient already suffers from clinical symptoms before the development of small-bowel mucosal villous atrophy, it seems unethical to delay treatment, and it would be rational to change the criteria together with clinical practice. The present results also lead to the inevitable question of whether histologic evaluation is discriminative for the diagnosis of celiac disease, and whether the biopsy thus would not be needed for a subset of patients with associated symptoms, positive EmA, and compatible HLA. Of note, in this trial there were no EmA-positive patients with completely normal small-bowel mucosa (Marsh 0), and thus it remains obscure whether they would react in dietary interventions similarly to subjects with mild enteropathy. Nevertheless, the results of this study offer a basis for further studies assessing the role of intestinal biopsy in the diagnosis of celiac disease. The gluten dependency of EmA-positive participants also was shown by measuring ttg-ab. Although EmA and ttg-ab measure practically the same antigen, 39 the results of ttg-ab may vary between different laboratories or methods used, and the positive predictive value of ttg-ab for celiac disease may be markedly low. 40 As a consequence, there might be EmA-negative ttg-ab positive individuals. Because no such patients were detected in this study, the results of the trial should not be extended to EmA-negative subjects. In conclusion, the findings here confirm that patients with mild enteropathy and EmAs may experience clear gluten-induced symptoms, and benefit from a GFD. Again, this study proved that the spectrum of celiac disease is wider than previously thought, and it is time to extend the current diagnostic criteria. Instead of using the definition of celiac disease or gluten-sensitive enteropathy, the term genetic gluten intolerance would be more descriptive. 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BMJ 2007;334: Received June 2, Accepted November 13, Reprint requests Address requests for reprints to: Katri Kaukinen, MD, PhD, University of Tampere, Medical School, FIN-33014, Tampere, Finland. katri.kaukinen@uta.fi; fax: (358) Conflicts of interest The authors disclose no conflicts. Funding This study and the Coeliac Disease Study Group were supported by the Academy of Finland Research Council for Health, the Competitive Research Funding of the Pirkanmaa Hospital District, the Yrjö Jahnsson Foundation, the Foundation for Paediatric Research, the Finnish Coeliac Society, an EU Commission Marie Curie Excellence grant (FP6 contract MEXT-CT ), and a Marie Curie mobility grant (MRTNCT ; TRACKS).