May Antitransglutaminase Levels Predict Severity of Duodenal Lesions in Adults with Celiac Disease?

Background and Objective: Pediatric guidelines on celiac disease (CD) state that children with anti-transglutaminase antibodies (TGAs) >×10 upper limit of normal (ULN) may avoid endoscopy and biopsy. We aimed to evaluate whether these criteria may be suitable for villous atrophy diagnosis in CD adults. Materials and Methods: We retrospectively enrolled patients with CD aged >18 years. TGAs were expressed as xULN. Duodenal lesions were classified as atrophic or non-atrophic according to Marsh-Oberhuber. Fisher's exact and t-test were used for variables comparison. Receiver operating characteristics (ROC) curve analysis was performed with estimation of area under the curve (AUC), sensitivity, specificity, and positive and negative predictive value (PPV/NPV). Results: One hundred and twenty-one patients were recruited. Sixty patients (49.6%) had TGA >×10 ULN, and 93 (76.8%) had villous atrophy. The cut-off of >×10 ULN had sensitivity = 53.7%, specificity = 64.3%, PPV = 83.3%, and NPV = 29.5% to predict atrophy. Therefore, considering pediatric criteria, in 50 (41.3%) patients, biopsy could have been avoided. Patient subgroup with atrophy had higher TGA levels despite being not significant (37.2 ± 15.3 vs. 8.0 ± 1.3 ULN, p = 0.06). In adults, a slightly better diagnostic performance was obtained using a cut-off of TGA >×6.2 ULN (sensitivity = 57.1%, specificity = 65.6%, and AUC = 0.62). Conclusions: Despite our confirmation that villous atrophy is linked to high TGA levels, CD and atrophy diagnosis based only on serology is not reliable in adults.

Human Leukocyte Antigen (HLA) Haplotype Does Not Influence the Inflammatory Pattern of Duodenal Lymphocytosis Linked to Irritable Bowel Syndrome.

Background and objectives: Duodenal lymphocytosis (DL) is a condition characterized by enhanced infiltration of intraepithelial lymphocytes (IELs) in the duodenal mucosa, and it can be linked to both gluten- and non-gluten-related diseases, such as irritable bowel syndrome (IBS). Materials and methods: We retrospectively selected patients with DL linked to IBS. Formalin-embedded biopsy samples of the duodenum were collected. CD3 lymphocyte immunohistochemistry was used for IELs. The real-time polymerase chain reaction was used to quantify the amount of mRNA coding for tissue transglutaminase 2 (tTG2), interferon-gamma (IFNγ), toll-like receptor 2 (TLR2), and myeloid differentiation primary response 88 (MyD88). All subjects underwent DQ2-8 haplotype analysis. Controls were represented by subjects with IBS without DL. Results: Thirty-two patients with IBS-DL were retrospectively recruited. Fourteen subjects (43.8%) had a DQ2-8 haplotype. DQ2-8 positive subjects had similar levels compared to negative ones for tTG2, IFNγ, TLR2, and MyD88. Cigarette smoke did not influence molecular expression in our study. Smokers had a statistically higher IELs count than non-smokers (54.2 ± 7.7 vs. 36.0 ± 8.8, p < 0.001). A significant, direct correlation between IELs and duodenal expression of IFNγ was found (r = 0.36, p = 0.04). Conclusions: IBS with DL showed higher expression of inflammatory markers than controls, but DQ2-8 haplotype did not seem to affect their expression. Smoking might increase IELs infiltration.

Predictivity of Autoimmune Stigmata for Gluten Sensitivity in Subjects with Microscopic Enteritis: A Retrospective Study.

Non-celiac gluten sensitivity (NCGS) is an emerging gluten-related condition. We investigated whether the presence of autoimmune stigmata in a group of patients with clinical suspicion of NCGS and a histological picture of microscopic enteritis (ME) could be a predictive factor of NCGS. Patients with ME were followed up by periodical examinations. At baseline, we collected data about previous clinical history, including autoimmune diseases. NCGS was diagnosed according to Salerno criteria; other causes of ME were diagnosed according to well-established protocols. Patients with celiac disease were excluded. Student's and chi-square tests were used in univariate analysis. Kaplan-Meier curves and Cox regression were used to estimate hazard ratios (HR). Sixty-three patients were included. Twenty-two had a final diagnosis of NCGS; the remaining 41 had non-gluten-related causes of ME. Prevalence of autoimmune thyroiditis was higher among NCGS patients (40.1%) than in other ME (14.6%; p = 0.03). NCGS showed higher positivity rate for anti-gliadin (27.3% versus 2.5%; p = 0.006) and anti-nucleus (45.4% versus 12.2%; p = 0.005). Autoimmune thyroiditis had a non-significant trend (p = 0.06) for NCGS diagnosis, (HR = 2.4). Both anti-gliadin (HR = 2.4; p = 0.04) and anti-nucleus (HR = 2.7; p = 0.04) were directly associated with NCGS diagnosis. In conclusion, NCGS may have a cohort of autoimmune stigmata that can precede its diagnosis.

Helicobacter pylori DNA isolation in the stool: an essential pre-requisite for bacterial noninvasive molecular analysis.

PURPOSE: Real-time polymerase chain reaction (RT-PCR) is a widely used technique for bacterial and viral infection diagnosis. Herein, we report our preliminary experience in retrieving H. pylori genetic sequences in stools and analyzing genotypic clarithromycin resistance by RT-PCR (noninvasive), with the aim of comparing this procedure with that performed on biopsy samples (invasive). MATERIALS AND METHODS: After 'in vitro' demonstration of H. pylori DNA detection from pure and stool-mixed bacteria, 52 consecutive patients at the first diagnosis of infection were investigated. DNA was extracted from biopsy tissue and stool samples (THD® Fecal Test, Italy). RT-PCR was performed to detect 23S rRNA encoding bacterial subunit gene and search A2143G, A2142C, A2142G point mutations for clarithromycin resistance assessment. RESULTS: RT-PCR showed H. pylori positive DNA in all infected patients with full concordance between tissue and stool detection (100%). We found A2143G mutation in 10 (19.2%), A2142G in 4 (7.7%) and A2142C in 5 (9.6%) patients; there was a full agreement between biopsy and fecal samples. A2143G was found in all the four A2142G positive cases and in three out of the five A2142C positive strains. Overall clarithromycin resistance rate in our series was 23%. CONCLUSIONS: Despite the need of confirmation on large sample, stool RT-PCR analysis could represent a feasible tool to detect H. pylori DNA sequences and antibiotic resistance point mutations. As compared to tissue molecular analysis, this technique is noninvasive, with potential advantages such as improvement of patient compliance, reduction of diagnostic procedure time/cost and improvement of therapeutic outcome.

May the assessment of baseline mucosal molecular pattern predict the development of gluten related disorders among microscopic enteritis?

AIM: To evaluate mucosal baseline mRNA expression of tissue transglutaminase 2 (tTG2), interferon gamma (IFNγ), toll-like receptor 2 (TLR2) and Myeloid Differentiation factor 88 (MyD88) in patients with microscopic enteritis (ME). METHODS: We retrospectively enrolled 89 patients with ME of different etiology, which was defined within a 2-year mean period of follow-up. Baseline histological examination was performed on Hematoxylin-Eosin stained sections and CD3 lymphocyte immunohistochemistry was used for intraepithelial lymphocyte count (IELs). ME was defined according to the criteria of Bucharest Consensus Conference. For each patient, formalin embedded biopsy samples of the duodenum referred to the period of ME diagnosis were retrieved. Real-time polymerase chain reaction (RT-PCR) was used to detect the amount of mRNA coding for tTG2, IFNγ, TLR2 and MyD88, and the quantity was expressed as fold change compared to controls. Control group was represented by duodenal normal specimens from 15 healthy subjects undergoing endoscopy for functional symptoms. Comparisons among continuous variables were performed by One way analysis of variance (ANOVA) and Bonferroni's test. The χ(2) test was used for categorical variables. Pearson's test was used to evaluate correlations. Receiver operating curves were drawn for all four markers to estimate sensitivity and specificity in discriminating the development of CD and GS. RESULTS: After a period of follow up of 21.7 ± 11.7 mo, the following diagnoses were achieved: gluten related disorders in 48 subjects (31 CD; 17 GS) and non-gluten related ones in 41 (29 Irritable Bowel Syndrome - IBS; 12 Others). CD patients had the highest tTG2 levels (8.3 ± 4.5). The ANOVA plus Bonferroni analysis showed that CD > Other ME > GS = IBS > negative controls. A cut off value of 2.258 was able to discriminate between CD and GS with a sensitivity of 52.94% and a specificity of 87.1%. Additionally, CD patients had the highest IFNγ levels (8.5 ± 4.1). ANOVA plus Bonferroni demonstrated CD > Other ME > GS = IBS > negative controls. A cut off of 1.853 was able to differentiate CD and GS with a sensitivity of 47.06% and a specificity of 96.77%. Patients with non gluten-related causes of ME exhibited the highest TLR2 levels (6.1 ± 1.9) as follows: Other ME > CD = GS = IBS > negative controls. TLR2 was unable to discriminate CD from GS. Patients with CD overexpressed MyD88 levels similarly to non gluten-related causes of DL (7.8 ± 4.9 and 6.7 ± 2.9), thus CD = Other ME > GS = IBS > negative controls. A cut off of 3.722 was able to differentiate CD from GS with a sensitivity of 52.94% and a specificity of 74.19%. IELs count (15-25 and more than 25/100 enterocytes) strongly correlated with mRNA levels of all tested molecules (P < 0.0001). CONCLUSION: Our results confirm that a single marker is unable to predict a discrimination among ME underlying conditions as well as between CD and GS. Mucosal high levels of tTG and IFNγ mRNA may predict the development of CD more than GS with high specificity, despite an expected low sensitivity. TLR2 does not discriminate the development of CD from GS. MyD88 levels indicate that intestinal permeability is more increased when a severe intestinal damage underlies ME in both gluten related and unrelated conditions. Therefore, the results of the present paper do not seem to show a clear translational value.

Mucosal molecular pattern of tissue transglutaminase and interferon gamma in suspected seronegative celiac disease at marsh 1 and 0 stages.

BACKGROUND/AIM: In celiac disease (CD), there is increased mRNA coding for tissue transglutaminase (tTG) and interferon gamma (IFNα). In seronegative celiac patients, the mucosal immune complexes anti-tTG IgA/tTG are found. We assayed tTG- and IFNα-mRNA in the mucosa of patients with a clinical suspicion of seronegative CD and correlated the values with intraepithelial CD3 lymphocytes (IELs). MATERIALS AND METHODS: Distal duodenum specimens from 67 patients were retrieved and re-evaluated for immunohistochemically proven CD3 IELs. Five 10 µm sections were used for the extraction and assay of tTG and IFNα coding mRNA levels using reverse transcriptase real-time polymerase chain reaction (RT-PCR). Samples from 15 seropositive CD patients and 15 healthy subjects were used as positive and negative controls. Results were expressed as fold-change. RESULTS: Our series was divided into three groups based on IEL count: >25 (14 patients: group A), 15-25 (26 patients: group B), and 0-15 (27 patients: Group C). tTG-mRNA levels were (mean ± SD): CD = 9.8 ± 2.6; group A = 10.04 ± 4.7; group B = 4.99 ± 2.3; group C = 2.26 ± 0.8, controls = 1.04 ± 0.2 (CD = group A > group B > group C = controls). IFNα-mRNA levels were: CD = 13.4 ± 3.6; group A = 7.28 ± 3.6; group B = 4.45 ± 2.9; group C = 2.06 ± 1.21, controls = 1.04 ± 0.4. CONCLUSIONS: Our results suggest that tTG- and IFNγmRNA levels are increased in both seropositive and potential seronegative patients with CD, showing a strong correlation with the CD3 IEL count at stage Marsh 1. An increase in both molecules is found even when IELs are in the range 15-25 (Marsh 0), suggesting the possibility of a "gray zone" inhabited by patients which should be closely followed up in gluten-related disorders.

Evolution of nonspecific duodenal lymphocytosis over 2 years of follow-up.

AIM: To assess the evolution of duodenal lymphocytosis (DL), a condition characterized by increased intraepithelial lymphocytes (IELs), over 2 years of follow-up. METHODS: Consecutive patients undergoing upper endoscopy/histology for abdominal pain, diarrhea, weight loss, weakness or other extraintestinal features compatible with celiac disease (CD) were included. Evaluation of IELs infiltrate in duodenal biopsy samples was carried out by CD3-immunohistochemistry and expressed as number of positive cells/100 enterocytes. Diagnostic agreement on the IELs count was tested by calculating the weighted k coefficient. All patients underwent serological detection of autoantibodies associated with CD: IgG and IgA anti-tissue transglutaminase and endomysium. Each patient underwent further investigations to clarify the origin of DL at baseline and/or in the course of 2 years of follow-up every six months. Autoimmune thyroiditis, intestinal infections, parasitic diseases, bacterial intestinal overgrowth, hypolactasia and wheat allergy were detected. Colonoscopy and enteric magnetic resonance imaging were performed when necessary. Risk factors affecting the final diagnosis were detected by multinomial logistic regression and expressed as OR. RESULTS: Eighty-five patients (16 males, 69 females, aged 34.1 ± 12.5 years) were followed up for a mean period of 21.7 ± 11.7 mo. At baseline, endoscopy/duodenal biopsy, CD3 immunohistochemistry revealed: > 25 IELs/100 enterocytes in 22 subjects, 15-25 IELs in 37 and < 15 IELs in 26. They all had negative serum anti-transglutaminase and anti-endomysium, whilst 5 showed IgG anti-gliadin positivity. In the course of follow-up, 23 developed CD seropositivity and gluten sensitivity (GS) was identified in 19. Other diagnoses were: 5 Helicobacter pylori infections, 4 jejunal Crohn's disease, 1 lymphocytic colitis and 1 systemic sclerosis. The disease in the remaining 32 patients was classified as irritable bowel syndrome because of the lack of diagnostic evidence. At multivariate analysis, the evolution towards CD was associated with an IELs infiltrate > 25 (OR = 1640.4) or 15-25 (OR = 16.95), human leukocyte antigen (HLA) DQ2/8 (OR = 140.85) or DQA1*0501 (OR = 15.36), diarrhea (OR = 5.56) and weakness (OR = 11.57). GS was associated with IELs 15-25 (OR = 28.59), autoimmune thyroiditis (OR = 87.63), folate deficiency (OR = 48.53) and diarrhea (OR = 54.87). CONCLUSION: DL may have a multifactorial origin but the IELs infiltrate and HLA are strong predictive factors for CD development and a clinical diagnosis of GS.

Quadruple rescue therapy after first and second line failure for Helicobacter pylori treatment: comparison between two tetracycline-based regimens.

BACKGROUND AND AIMS: Antibiotic resistance is the main reason for failure of Helicobacter pylori (H. pylori) treatment. Currently, guidelines recommend a treatment guided by antimicrobial susceptibility testing after two failures. However, microbial culture is not feasible everywhere, and the limited number of effective antibiotics against the bacterium narrows the options; thus a rescue therapy combining antibiotics with a low resistance may be fitting. METHODS: Patients who have failed a first-line treatment (either prolonged triple or sequential regimens) and, successively, a levofloxacin-based triple therapy were considered for the study. Subjects underwent urea breath test (UBT), stool antigen test (ST) and endoscopy/histology to confirm the diagnosis. Cytopenia and impaired liver and kidney function were exclusion criteria. Fifty-four subjects were randomized 1:1 to two regimens: RMB Rabeprazole/Rifabutin/Minocycline/Bismuth sub-citrate or MTB Rabeprazole/Tinidazole/Minocycline/Bismuth sub-citrate both for 10 days. The results were checked 6 weeks after the end of therapy with ST/UBT plus endoscopy when indicated. RESULTS: RMB eradicated the bacterium in 21 patients. Two subjects dropped out. The eradication rate was 77.7% (CI 62.0-93.4%) at intention-to-treat and 84.0% (CI 69.6-98.4%) at per-protocol analysis. MTB was successful in 14 patients (51.9%, CI 33.1-70.7%). No patient withdrew from the treatment for adverse events. Drug-related side effects were reported only in 3 subjects, but in all cases the treatment was carried on. CONCLUSIONS: The association minocycline/rifabutin seems to have a synergic effect and a good therapeutic outcome in patients who have failed at least two previous regimens, although a trial on a large population is needed.