Celiac disease: Is it common with paediatric IBD?Abstract

Background:

   Inflammatory bowel disease and celiac disease (CeD) are two immune-mediated diseases characterized by chronic intestinal inflammation. Recent findings demonstrate shared genetics and functional pathways. We reviewed the extension of these overlaps to other disease features and suggest future research approaches. In the case of IBD, there is also an overlap between symptoms of CeD and IBD that could result in misdiagnosis and delayed diagnosis of CeD.

Aim of work:

The aim of the present study is to determine how common celiac disease in children with inflammatory bowel disease is.

Methods:

We prospectively enrolled 101 IBD patients aged 1.4 - 17 (mean 8.7 ± 4) years. Screening of CeD was based on the most sensitive tests namely immunoglobulin A (IgA) human tissue transglutaminase antibody (IgA-tTGA) by enzyme-linked immunosorbent assay and confirmed the diagnosis by upper gastrointestinal endoscopy and histo-pathological findings in small bowel biopsy.

Results:

Out of 101 IBD patients, Seventeen patients (16.8%) had Crohn’s disease while 84 patients (83.2%) had ulcerative colitis, IgA-tTGA was positive in ten of them (9.9%) and they had grade III marsh classification in small bowel biopsy. The range of IgA-tTGA in this group of patients was (11.5 – 310). IgA-tTGA showed significant positive (proportionate) correlation with severity of both Ulcerative Colitis & Crohn's disease.Conclusion:

There is a significant overlap/association between celiac disease and inflammatory bowel disease. Anti-IgA-tTGA antibody titer levels could be used to monitor inflammatory bowel disease activity in children with IBD

Key words: Inflammatory Bowel Disease, Celiac disease, Tissue transglutaminase antibody

Introduction:

Recent findings demonstrate the common genetic basis for many immune-mediated diseases, and consequently, the partially shared pathogenesis. Two autoimmune diseases were selected that also share the specific target organ, the bowel. The etiology and immunopathogenesis of both conditions characterized by chronic intestinal inflammation, inflammatory bowel disease (IBD) and celiac disease (CeD), are not completely understood (1). According to the ESPGHAN definition, “Coeliac disease is an immune-mediated systemic disorder elicited by gluten and related prolamines in genetically susceptible individuals, characterised by the presence of a variable combination of gluten dependent clinical manifestations, coeliac disease specific antibodies, HLA-DQ2 and DQ8 haplotypes and enteropathy” (2,3,4). CeD is a lifelong disorder that affects both genders of all ages and is one of a few immune disorders that can be controlled when diagnosed and treated early (5). The classic clinical manifestations for CD are mostly gastrointestinal in nature such as diarrhoea, malnutrition, weight loss, steatorrhea and oedema secondary to hypoalbuminemia (6). Subsequently, the disease has also now been diagnosed in patients suffering from a variety of atypical symptoms such as anemia or osteoporosis and even in asymptomatic subjects (also formerly known as silent celiac disease or latent celiac disease) (7). IBD manifests during childhood or adolescence in at least 20% of patients. This presentation is commonly more severe and extensive than that observed in adult-onset disease (8). A single reference standard for the diagnosis of Crohn’s disease (CD) or ulcerative colitis (UC) does not exist. The diagnosis of CD or UC is based on a combination of clinical, biochemical, stool, endoscopic, cross-sectional imaging, and histological investigations (9). Extra intestinal manifestations may be present in IBD and CeD. In IBD, most of the extra intestinal manifestations are shared by CD and UC. They may accompany intestinal symptoms or, less commonly, precede or overshadow them. These manifestations seem to be related to activity (relapse/remission) and location of the disease (10). The prevalence of CeD in patients with IBD is not clear. There are several cases in the literature describing the coexistence of both diseases in the same family or even in the same patient (11). However, some authors consider that this is an incidental association and the prevalence of CeD is similar between IBD patients and the general population (12). In fact, CD and UC patients are not considered a risk group for routine CeD screening. As frequently observed in autoimmune diseases, CeD is characterized by the presence of autoantibodies, which are currently included in the definition and diagnostic guidelines for CeD (13; 14). Transglutaminase type 2 (TG2) is the major autoantigen in CeD and the target antigen for endomysial antibodies (EMA) and anti-TG2 antibodies. Therefore, those two antibodies are the most specific for CeD diagnosis (2).

Methodology:

Study design: The present study is cross sectional study that included 101 children with an established diagnosis of IBD based on clinical, endoscopic, histopathological. The study was conducted at the Paediatric gastroenterology Unit, Cairo University Paediatric Hospital, from April 2016 to April 2018.

Ethical considerations: The study protocol was approved by the Research Ethics Committee of the Paediatrics Department, Faculty of Medicine, Cairo University, Egypt. The research was conducted in accordance with the Helsinki Declaration. All

patients were enrolled in the study after informed consent was obtained from their parent/guardian.

Participants: The age of the enrolled IBD patients of both sexes was from 2-18 years of age on gluten –containing diet. Patients were recruited regardless of the disease activity. We excluded infants younger than 2 years of age, adult more than 18 years and those with other causes of colitis, on gluten-free diet, having total IgA deficiency.

Assessment and evaluation: All patients were subjected to a full clinical assessment focusing on the age of onset of IBD, symptoms and signs of active disease, e.g., abdominal pain (site and severity), nausea and vomiting, diarrhea (amount, frequency, consistency, presence of blood or mucus in stool), tenesmus, bleeding per rectum (amount, color of blood), clubbing, weight loss, constitutional manifestations, general well being and activity as well as per rectal examination was performed searching for tags, fistula, fissures, abscess or drainage. Assessment of IBD activity were calculated by inflammatory bowel disease activity indices; pediatric ulcerative colitis activity index (PUCAI) and pediatric Crohn’s disease activity index (PCDAI). The criteria of ulcerative colitis activity index (PUCAI) were used in our study to monitor the severity of disease in ulcerative colitis patients whereas Crohn's disease activity index (PCDI) was applied in Crohn's disease patients. Crohn's disease activity index was defined as following score : <10 remission; 10-27.5, mild disease activity; 30-37.5 moderate disease activity and >40 severe disease activity. While UC disease activity index was defined as following score: <10 remission; 10-34 mild disease activity; 35-64 moderate disease activity and >65, severe disease activity. The calculation was performed by experienced gastroenterologists. The patients’ anthropometric measurements (weight, height), which were plotted on Egyptian growth curves (Standard Egyptian Growth, 2008). The patients were clinically examined searching for extra-intestinal manifestations e.g., Fever, definite arthritis, uveitis, erythema nodosum, pyoderma gangrenosum, jaundice or organomegaly. All patients were evaluated by laboratory tests including; complete blood count (CBC), erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), and serum albumin. Upper and lower gastrointestinal endoscopic examination was performed using a Silver Karl Storz Endoskope 13821 PKS with Storz Professional Image Enhancement System (SPIES) technology with an HD system and 100-watt xenon light source. Endoscopy is fundamental in diagnosing IBD, obtaining biopsies, distinguishing UC from Crohn’s disease and to assess extent and severity of the disease during flare ups and observe effectiveness of treatment during follow up. Endoscopic parameters of assessment include mucosal vascular pattern, friability, mucosal damage up to ulceration, pseudo polyp, cobblestone appearance and stricture. Multiple biopsies were obtained from detected lesions even also normal appearing colonic and ileal mucosa by using biopsy forceps, All specimens were processed as formalin-fixed paraffin-embedded tissue blocks. Each block was cut into 5-μm-thick serial sections, which were mounted on glass slides. Sections were stained with haematoxylin and eosin. Stained sections were then examined using light microscopy. The histological diagnosis of IBD is based upon these features; mucosal architecture, lamina propria cellularity, neutrophil granulocyte infiltration, and epithelial abnormality. Neutrophils, which reflect disease activity, are present in the lamina propria and/or invade the surface or crypt epithelium, resulting in cryptitis (presence of neutrophils within crypt epithelium) or crypt abscesses (presence of neutrophils within crypt

lumina). Granulomas are not observed, except for those in response to foreign bodies or to mucin from ruptured crypts (cryptolytic granulomas). Histopathological examination of duodenal biopsies was examined for presence of villous atrophy, crypt hyperplasia, intraepithelial lymphocyte more than 30 and lamina propria inflammation compatible with grade III Marsh classification which was significant for diagnosis of celiac disease. Screening for celiac disease was performed through measuring anti-tissue transglutaminase IgA antibodies and total IgA levels. Dietary history was carefully assessed to confirm the presence of gluten in patients’ diet. Serum samples were collected from the patients through clean venipuncture under sterile conditions. Samples were then left at room temperature to allow samples to clot. Serum was then separated by centrifugation. Hemolysed and lipemic samples were excluded from the analysis and sampling was repeated. Serum was then stored at -20 °C. Total serum IgA was measured using nephlometry (Nephstar, China). Serum IgA-tTGA level was determined by ELISA using DRG®, Anti-Tissue Transglutaminase (EIA-3611) (Celikey; Pharmacia Diagnostics, Freiburg, Germany). The coefficient of variation (CV) of the assay is 7%. Principle of the test: Human recombinant tissue transglutaminase is bound to microwells. Antibodies against this antigen, if present in diluted serum or plasma, bind to the respective antigen. Washing of the microwells removes unspecific serum and plasma components serum antibodies. Horseradish peroxidase (HRP) conjugated anti-human IgA immunologically detects the bound patient antibodies forming a conjugate/anti-body/ antigen complex. Washing of the microwells removes unbound conjugate. An enzyme substrate in the presence of bound conjugate hydrolyses to form a blue color. The addition of an acid stops the reaction forming a yellow end-product. The intensity of this yellow color is measured photometrically at 450 nm. The amount of colour is directly proportional to the concentration of IgA antibodies present in the original sample. Reference range: <4.0 U/mL (negative); 4.0-10.0  U/mL (weak positive); >10.0 U/mL (positive). Patients with positive anti-IgA-tTGA antibodies were correlated to histo-pathological changes to duodenal biopsies and degree of IBD activity.

Statistical analysis

Data was entered on the computer using "Microsoft Office Excel Software" program (2010) for windows. Data was then transferred to the Statistical Package of Social Science Software program, version 23 (IBM SPSS Statistics for Windows, Version 23.0. Armonk, NY: IBM Corp.) to be statistically analysed. Data was presented using range, mean, standard deviation median and interquartile range for quantitative variables and frequency and percentage for qualitative ones. Comparison between groups was performed Mann Whitney test for quantitative variables and Chi square or Fisher's exact test for qualitative ones. P values less than 0.05 were considered statistically significant.

Results:

The present study included 101 patients with established diagnosis of IBD based on classic clinical, endoscopic, pathological features. The demographic and anthropometric data of IBD patients are summarized in Table 1. The common presenting symptoms and clinical signs were elicited in table 2. As regard severity of ulcerative colitis, fifty- five patients (65%) had mild activity while twenty-nine patients (34.5%) had moderate activity of disease and none had severe activity. While severity of Crohn’s, seven patients (41.2%) had mild activity, seven patients (41.2%) had moderate activity and only three patients (17.6%) had severe activity. Regarding the extent of inflammation in the colon by lower endoscopy (figure 1,2), 76 (75.2%) patients had proctitis, 49(48.5%) patients had distal or left sided colitis, 12 (11.9%) patients had pancolitis , and 61(60.4%) patients had ileitis. Histopathological findings of specimens from lower GI endoscopy which revealed that seventeen patients (16.8%) had crohn's disease in the form of areas of active disease interspersed with normal mucosa with rectal sparing and cobble stoning (nodular mucosa often intersected by crossing linear ulcerations). Eighty-four patients (83.2%) had picture of ulcerative colitis in the form of diffuse continuous pattern of superficial inflammation with cellular infiltration and disruption of glandular architecture, cryptitis, crypt abscesses, and goblet cell depletion figure 3. Total serum IgA and IgA-tTGA was done for all studied patients as screening for celiac disease in pediatric IBD. Ten patients (9.9%) had positive IgA-tTGA (>10mg/dl) with statistically significant difference (p value<0.001). Eight patients (80%) of them had UC and two (20%) had CD. the diagnosis was confirmed by upper gastrointestinal endoscopy and histo-pathological findings in small bowel biopsy. Histopathology of upper endoscopic duodenal biopsy shows that 10 (100%) of seropositive IBD patients had fulfilled the histological grade III Marsh classification criteria for diagnosis of CeD, including villous atrophy, crypt hyperplasia, intraepithelial lymphocyte more than 30/ high power fluid and lamina propria inflammation. While none of sero-negative IBD patients had grade III Marsh classification in their duodenal biopsies with statistically significant difference (p value< 0.001) figure 4.

We found that sero-positive IBD patients were under weight and stunted in comparison to sero-negative IBD patients but no statistical significant difference.  All patients were evaluated by complete blood count and serum albumin level and calcium level showed that the mean of haemoglobin level, calcium level and albumin level in sero-positive IBD patients was 8.2 ± 1.1; 8 ± 0.8; 3 ± 0.6 that was lower than the mean levels in sero-negative IBD patients with statistically significant difference (p-value=0.007; <0.001; <0.001) respectively. Titer of IgA-tTGA showed significant proportionate correlation with severity of both UC and CD. IgA-tTGA level was significantly higher among UC patients with moderate activity than those with mild one with statistically significant difference (p-value <0.001). Within patients with Crohn's disease, IgA-tTGA level was significantly higher among patients with severe disease than those with moderate and mild ones.

Discussion

Both CeD and IBD are immune-related disorders (14). These two disorders may be linked together. The clinical manifestations of celiac disease and inflammatory bowel disease (IBD) can be similar and often patients with celiac disease, particularly those

that have no response to a gluten-free diet (GFD), are investigated for possible IBD as the 2 conditions may coexist (1).To the best of our knowledge, our Center Experience represents the first report from this region on the association between IBD and CeD. In this study the occurrence of CeD was observed in 9.9% (10/101) of the IBD cases. This incidence is slightly higher than that encountered in the study conducted by Casella et al., 2010 showed that nine of the 1711 patients (0.5%) had serological and histological findings (Marsh III villous atrophy) compatible with the diagnosis of celiac disease; six of them had UC and three had CD(15). While Tavakkoli and colleagues, they found out of 100 patients examined, 17 (17%) had IgA antitTG antibody levels above the cutoff point that consisted of 12 UC and 5 CD patients. Thirty- two patients were positive for IgA EMA that consisted of 22 UC and 10 Crohn's disease patients (16). Another Japanese study was showed that the positivity of both serum antibodies was significantly higher in IBD and correlated with disease activity. However, no biopsy-defined or HLA-defined true celiac disease was found (17). However a major limitation of their study was lack of confirmation of CeD by intestinal biopsy. The diagnosis of coeliac disease requires a specific histopathological assessment (18). In our study the diagnosis of celiac was confirmed by small bowel biopsies.

Manceñido Marcos et al., 2017 performed Celiac screening in 163 patients with de novo IBD: 65 with CD, 92 with UC and Six patients have positive anti-tTG (3.7%), 2 patients with CD (1 with colonic CD and 1 with ileocolonic CD) and 4 patients with UC (2 with ulcerative proctitis and 2 with extensive colitis), with no statistically significant differences between groups (19). While we found eight (80%) of sero-positive IBD patients had UC and two (20%) had CD. While three of sero-positive UC patients had distal or left sided colitis, one had pancolitis and the last four had proctitis. Two sero-positive Crohn's patients had ileo-colonic distribution with skip lesions.

Regarding the anthropometry; eight (80%) of sero-positive IBD patients were under weight. Seven (70%) of sero-positive IBD patients were stunted. This might be attributed to co-morbidity of IBD and CeD. Boguszewski et al., 2015 reported that group of 40 children with the diagnosis of CeD, 15% with short stature (20).

Concerning basic laboratory investigations in our study showed that the mean of Hb level in sero-positive IBD patients was 8.2 ± 1.1 that was lower than the mean of Hb level in sero-negative IBD patients with (p value=0.007). This was in concordance with Halfdanarson et al., 2007 that showed that the prevalence of anemia varies greatly according to different reports and has been found in 12% to 69% of newly diagnosed patients with CeD. 46% of cases of subclinical CeD, with a higher of iron deficiency (21).

Seven Crohn' patients had moderate disease activity, seven had mild disease activity, and 3 patients had severe activity. 29 patients had moderate disease activity, 55 patients had mild disease activity. IgA-tTGA showed significant positive (proportionate) correlation with severity of both UC and CD. IgA-tTGA level was significantly higher among patients with moderate UC than those with mild one. Within patients with CD, IgA-tTGA level was significantly higher among patients with severe disease than those with mild and moderate ones while the last two categories didn’t show significant differences between them.

This was in agreement with other study which showed that, the relationship between the positivity of celiac disease-specific antibodies and the clinical presentation of Crohn’s disease and ulcerative colitis. A highly significant correlation was found between the positivity of celiac-specific antibodies and disease activity in IBD patients (17). In comparison to Tavakkoli et al., 2012 who reported that Two of CD patients had moderate disease activity, seven had mild disease activity, and 14 patients were in remission. Spearman correlation showed no significant relationship between the severity of disease in ulcerative colitis and Crohn's disease with IgA anti-tTG antibody levels and IgA EMA (P>0.05) (16).

In conclusion, we demonstrated the high incidence of CeD in children with IBD (9.9%). Therefore, we recommend that human-tissue Transglutaminase and total serum IgA measurement should be integrated into the diagnostic routine of all patients with IBD for the early identification of CeD in this high risk group. There is a significant overlap/association between celiac disease and inflammatory bowel disease. There is significant positive correlation between changes in inflammatory bowel disease activity and changes in anti-IgA-tTGA antibody titer levels. Consequently, anti-IgA-tTGA antibody titer levels could be used to monitor inflammatory bowel disease activity in children with IBD. Using a Gluten-restricted diet will have a good clinical response on IBD patients.

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Figure (2): Lower endoscopic findings of one of IBD cases, shows edematous friable mucosa which bleeds easily on touch with loss of colonic haustrations

Figure 3: Photomicrograph of colonic mucosa showing surface ulceration (arrow), goblet cell depletion, inflammatory infiltrate, cryptitis and crypt abscess (arrow head)

Figure 4: Duodenal biopsy, A and B showed Mild – moderate villous atrophy & crypt hyperplasia

H&E, Original magnification x40(right) & x100(left)

A B

Figure 5: Duodenal biopsy, C and D showed Dense lamina propria inflammation dominated by plasma cells and intraepithelial lymphoctes >30/ 100 enterocytes (arrows)

H&E, Original magnification x100

C D