Non-invasive examination methods are increasingly important in diagnosing celiac disease (CD). New options for diagnosing CD have been discovered as a result of progress in immunology (1). The detection of circulating autoantibodies is becoming more frequent clinically. As a result, many new, highly heterogeneous cases of CD have been diagnosed, and consequently, recognition of the incidence of CD in the pediatric and adult populations has increased. Detection of anti-endomysial antibodies (AEA) and anti-tissue transglutaminase antibodies (t-TG), characteristic for their high sensitivity and specificity, play important roles in diagnosing and clinically monitoring CD (2,3). Nevertheless, histopathology remains the major tool for clinicians, and particularly gastroenterologists, for definitively diagnosing CD (4,5). A variety of studies have documented that AEA, and t-TG, are highly specific in predicting CD (6,7). There are also seronegative data, which is particularly true for the adult population (8). The diagnosis in children is more successful (9), as pediatric CD has clear features in comparison to adult CD (10). It is often difficult to diagnose CD quickly, due to the fact that CD has a wide range of symptoms. However, we do not know if the same problems exist in the diagnosis of pediatric patients. We decided to test this hypothesis on a population of children in Eastern Slovakia. The objective of this study was to compare the levels of AEA and t-TG (IgA, IgG) in the plasma with that of histological abnormalities in pediatric patients with CD.

In this study, we wanted to demonstrate the lack of association between autoantibodies and lesions in certain cases of pediatric CD. This may justify the necessity of biopsies for the accurate diagnosis of CD. We show lesions, found in our samples, that are diagnostically important. The findings of Rostami et al(15) demonstrated that AEA have limited value in CD screening programs. Their sensitivity in patients with total villous atrophy (Marsh IIIC) was 100%, but with partial atrophy (Marsh IIIA) the sensitivity of AEA decreased to 3l%. Furthermore, a high IgA t-TG titer correlates with the histopathological finding of Marsh IIIC (16,17). Another study revealed that positive AEA are observed mostly in CD patients with severe tissue damage due to partial or total villous atrophy, but they have a low sensitivity in CD patients with partial villous atrophy in spite of the use of different substrates (18,19). Additionally, a study by Tărmure et al(20) demonstrated that in patients with Marsh I and Marsh II, the sensitivity of AEA was lower. These results demonstrated that the autoantibody titers may correlate with the damage to the small intestine. In our study, there have been negative antibodies with Marsh IIIC, demonstrating that serological screening is not 100% accurate for the diagnosis of CD. That serological tests would be negative was known for adult CD patients but was not previously known for pediatric CD patients. It is possible that clinical symptomatology is poor in certain children, while the disease remains active, and these diagnostic problems require a solution. Therefore, a biopsy must always be performed, so that an expert pathologist may accurately distinguish CD from other diseases (21).

The literature has provided evidence that AEA titers are the first to disappear following the introduction of a gluten-free diet, as CD is induced by gluten and related cereal proteins. Midhagen et al(22) demonstrated that antibody titers decreased sharply within the first month following the introduction of a gluten-free diet. It is possible that weak reactions of AEA or t-TG are caused by the ingestion of gluten-free food, but the degree of histological damage remains high. Fernández-Salazar et al(23) documented that AEA negativity is not rare in patients with CD. These results demonstrate that sensitivity to serology varies according to the intestinal damage and the introduction of a gluten-free diet. The results of other studies also support this hypothesis (24,25). Therefore, we recommend multiple endoscopic biopsies from the proximal and distal duodenum (26,27). Furthermore, in cases of low IgA t-TG titers or in patients with IgA deficiency, intestinal biopsies should remain mandatory (28). The results of certain studies have revealed that only patients with a IgA t-TG level ≥100 U/ml and whose symptoms improve upon the introduction of a gluten-free diet may not require a biopsy of the small intestine to confirm CD (29,30). Although these results are promising, in our opinion, biopsies should not be rejected. However, there are children who are AEA positive and exhibit a normal mucosal villous morphology of the small intestine, which indicates that the diagnostic criteria for CD should be re-evaluated (31). These disparities were described for other antibodies [e.g., AGA (IgA), ARA (IgA) and JAB (IgA)], which is also important for the pathologist to avoid rejection of biopsies based on insufficient evidence (32). Certain studies report that an accurate diagnosis is supported by the genetic analysis of HLADQ2 or HLADQ8 (33,34), but these tests are not yet standard practice in all workplaces, and HLA association alone is inadequate for providing an explanation of the inheritance pattern. For children with a biopsy consistent with CD, in addition to a positive response to a gluten-free diet, HLA testing is not required (35). Di Osdado et al(36) found variations in the expression levels of 109 genes by comparing Marsh III and Marsh 0 lesions, which have roles in the proliferation and differentiation pathways. These results are crucial and encouraging for medical practice, as they may lead to alternative future genetic analyses and the discovery of combinations with new candidate genes for CD. Recent studies have focused on ways to speed up the diagnosis of CD (37,38), but according to our study, biopsies remain necessary. Until we find a combination of diagnostic methods that are 100% reliable, we should continue to perform biopsies. We believe that currently biopsies are the optimum way for the gastroenterologist to accurately diagnose CD in at risk pediatric groups, as well as in children with unspecified long-term related digestive problems. Histological results are important for clinicians, but particularly for the gastroenterologist, in order to establish tests and interpret all available data (39,40).

It has been many years since CD was considered to be a rare disease and experts have agreed that the incidence of CD will increase, since more programs have started to investigate the ‘celiac iceburg’ (41). Autoantibodies are considered to only be helpful in clinical practice, with regards to CD screening. In accordance with this, a biopsy of the small intestine is always required for the accurate diagnosis of CD. We recommend a minimum of four endoscopic biopsies to be obtained from the bulb and third duodenal portions, as a section of the biopsy may be damaged, and the absence of certain material that should be investigated may result in incorrect histopathological interpretation. Slice descriptions should include all relevant information, and the histopathological report should be clear and conform to the recommended standards for intestinal biopsies. We consider this information to be particularly important for pathologists, which is in accordance with the views expressed in other studies (42). We recommend the use of autoantibodies only as a supportive method, as a control in gluten-free diets or as an early predictor of CD with necessary further follow-up. Recent European Society of Pediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) guidelines state that in cases where a child manifests clear symptoms of CD and demonstrates particularly high levels of t-TG, with positive HLA testing, the physician may consider omitting a biopsy. However, there are some instances where pediatric CD has remained undiagnosed, providing support for a revision to the diagnostic criteria in a clinical, endoscopic and serological context. However, since there are no reliable data on the incidence of CD in children in the East region of the Slovak Republic, our results may be considered to be valid for other countries as well, given that many countries have no screening, only local screening or use different diagnosis methods. The incidence of CD in pediatric patients is higher than previously assumed.

Recent and current literature indicates that celiac disease is underdiagnosed. From our results we conclude that the screening program should be considerably more intense, particluarly in pediatrics. In numerous cases it is difficult to diagnose only from the serum. Biopsies should be part of the investigation, regardless of the outcome of serology.