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- Materials and methods
The diagnosis of coeliac disease requires the presence of small intestinal mucosal villous atrophy and crypt hyperplasia (Marsh III).1, 2 However, mucosal intraepithelial lymphocytosis evincing normal villous architecture (Marsh I) precedes this lesion. Marsh I lesion may thus indicate early developing coeliac disease, but this finding is also associated with other disorders. Only 2–10% of patients are eventually shown to suffer from genetic gluten intolerance.3, 4 Increased densities of γδ+5 or villous tip intraepithelial lymphocytes (IELs)6 have been of better positive predictive value than IELs in general in the diagnosis of early developing coeliac disease, but so far no single marker is considered a reliable indicator of this condition.
Serum immunoglobulin (Ig)A-class endomysial (EmA)7 and transglutaminase 2 (TG2)8, 9 antibodies are powerful tools in disclosing coeliac disease with overt villous atrophy. Some studies have suggested, however, that these autoantibodies might be less useful in the case of early developing coeliac disease,10–12 as the antibodies are thought to enter the circulation only after severe villous atrophy has developed. In contrast, there are also cases where coeliac autoantibodies have appeared in the serum before the development of villous atrophy, implying that they might be valuable in identifying patients with early developing coeliac disease.5, 13, 14
Previous studies have demonstrated that TG2 is the antigen for in vitro binding of coeliac IgA-class antibodies to intestinal and extraintestinal tissues.15, 16 Coeliac autoantibodies are produced in the small bowel mucosa,17–19 and it has been recognized that the small intestinal epithelial basement membrane region contains deposited IgA in untreated coeliac disease, though the target of this deposition remained unknown for decades.20, 21 We recently demonstrated in vivo that deposited extracellular IgA in the small intestinal mucosa targets TG2 in untreated coeliac disease patients.22 Further, our previous findings have suggested that TG2-targeted intestinal autoantibody deposits might be detectable before the development of overt villous atrophy and precede the appearance of serum coeliac autoantibodies.22, 23 These preliminary results raised the intriguing hypothesis that intestinal autoantibody deposits targeted against TG2 would precede manifest coeliac mucosal lesion, and further that this might furnish a method applicable in the diagnosis of early developing coeliac disease.
Recent evidence shows that coeliac disease is no longer restricted to severe enteropathy,24 and that patients can suffer from gluten-dependent clinical symptoms and complications of coeliac disease even before small bowel mucosal villous atrophy has developed.25, 26 Such patients benefit of gluten-free diet, and therefore, reliable markers of early developing coeliac disease are needed. However, previously published evidence of this condition has mainly consisted of case reports. This long-term follow-up study focused for the first time on early developing coeliac disease in a large, well-defined patient material. The aim was first to establish whether patients with normal villous architecture but positive serum coeliac autoantibodies, or Marsh I lesion in the small bowel mucosa, suffer from early developing coeliac disease. Secondly, we investigated whether intestinal TG2-targeted autoantibody deposits are superior in detecting early developing coeliac disease than the CD3+, γδ+ and villous tip IELs currently employed.
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- Materials and methods
This study elucidated the natural history of coeliac disease. Altogether 17 patients with normal villous architecture at baseline, while consuming a gluten-containing diet, subsequently developed mucosal damage diagnostic for coeliac disease. Majority of cases had initially positive coeliac autoantibodies in the serum. When these TG2-targeted autoantibodies were examined where they are produced, in the small bowel mucosa, 93% of all patients with early developing coeliac disease were identified in the absence of villous atrophy.
Occasionally coeliac disease diagnosis can be problematic; small bowel biopsy is considered the gold standard in coeliac disease diagnosis despite involving some obvious difficulties in interpretation. Even in the diagnostics of overt coeliac disease, a false diagnosis can be made, because at least 10% of biopsy specimens are of poor quality36 and further, villous atrophy may occasionally be patchy.37 In this study multiple small bowel biopsies were taken from several sites in order to detect also patients with patchy mucosal lesion. There is still always a possibility that a patchy form of villous atrophy is missed, and hence new methods are needed.
The diagnosis of early developing coeliac disease is even more challenging; minor histological abnormalities are unspecific, subjective and difficult to interpret. According to the recent studies focusing on early developing coeliac disease it has become evident that the recognition of coeliac disease without villous atrophy is important,24–26 especially because the patients benefit of dietary treatment. In agreement with this also our patients with early developing coeliac disease suffered from various symptoms typical of coeliac disease before the development of overt villous atrophy (Table 2). However, we demonstrated that Marsh classification could not be utilized reliably in the detection of early developing coeliac disease. Therefore, patients with Marsh I lesion in the small bowel mucosa should not be advised to adhere to a gluten-free diet in the absence of additional evidence.
The value of villous tip IELs in discovering patients with early developing coeliac disease was superior to Marsh I lesion, as has also previously been shown in smaller series.6, 31, 38 The clear advantage of villous tip IELs is that they can be studied in routine H & E-stained sections, whereas other histological methods applied here required frozen samples. In this study increased densities of γδ+ IELs were not only indicative of early developing coeliac disease, but also 40% of patients excluded for coeliac disease also on re-evaluation had increased densities of these cells at baseline when coeliac disease was first suspected. False-positive γδ+ cells have similarly been reported elsewhere.13, 23
The diagnosis of both overt and early developing coeliac disease should be based on reliable evidence. Histology has its limitations, as stated above. Nor are coeliac autoantibodies 100% specific. Five autoantibody-positive patients in this study did not proceed to villous atrophy during the follow-up, though it is possible that a longer follow-up might reveal progression of the disease in these individuals. Antibody testing was changing during the study period, and TG2 antibody test was not available at the time of baseline investigations in most patients. However, EmA and TG2 antibody tests correlate closely,8, 29 and the sensitivity and specificity values of these tests have been equal,39 even though some occasional patients remain negative for EmA despite positive for TG2 antibodies, and vice versa. DQ is mandatory in the identification of patients with genetic gluten intolerance, and patients with HLA DQ2 or DQ8 having positive coeliac autoantibodies in the serum are highly likely to suffer from coeliac disease. However, HLA DQ2 and DQ8 alone can only be used when patients are excluded for coeliac disease, because they are common in the population in general.40
We consider that intestinal IgA deposits targeted against TG2 are currently the best method in revealing early developing coeliac disease. Although serological tests predicted the development of villous atrophy relatively well, four (24%) of 17 patients with histologically confirmed coeliac disease had negative serology at baseline. Furthermore, there were five patients who underwent spontaneous negative seroconversion. Only one of them had intestinal IgA deposits at baseline, again showing that the method is more specific than serology. Investigation of intestinal IgA deposits is a special method requiring frozen small bowel biopsy specimens, which limits its utility. This method should be available at least in special centres, because it is clearly beneficial in cases where the conventional histology is ambiguous, especially in cases where a second investigation and biopsy is considered to confirm the diagnosis of coeliac disease. By investigating these deposits in small bowel biopsy specimens, when coeliac disease was first suspected, we were able in the majority of cases to diagnose early developing coeliac disease before the development of forthcoming villous atrophy. However, patients with early developing coeliac disease do not yet fulfil the traditional European Society for Pediatric Gastroenterology and Nutrition diagnostic criteria for coeliac disease. We have now shown that the criteria are no longer valid; many patients suffer from gluten-dependent symptoms before the development of villous atrophy, and the criteria should thus be revised.
In conclusion, the detection of intestinal TG2-specific IgA deposits proved a powerful diagnostic tool in coeliac disease without villous atrophy. We recommend that these deposits be invariably investigated when early developing coeliac disease is suspected. The presence of these intestinal TG2-targeted deposits strengthens the diagnosis of coeliac disease and dietary treatment should be considered at least in symptomatic patients. Further, patients with coeliac disease suspicion but normal villous architecture having positive coeliac autoantibodies in the serum or increased density of γδ+ or villous tip IELs in the small bowel mucosa should be followed up, whereas patients with Marsh I alone do not require routine surveillance. The detection of intestinal TG2-specific IgA deposits provides a reliable means to discover patients with early developing coeliac disease, and thus the results of this study should be taken into consideration when the diagnostic criteria for coeliac disease are revised.