Performance assessment of a new G12/A1 antibody-based rapid ELISA using commercially available and gluten-spiked food samples.

OBJECTIVE: Food products with <20 mg/kg gluten can be labeled 'gluten-free' according to international regulations. Several antibodies-based ELISAs have been develop to track gluten traces in food products. Among them, R5 and G12 antibody-based ELISAs are the frequently used methods. However, these antibodies have certain limitations. We evaluated the accuracy of G12/A1 antibody-based 'Glutentox ELISA Rapid G12' and compared the results with the current reference method i.e., R5 antibody-based 'Ridascreen R5 ELISA'. METHODS: In the first step, the performance of Glutentox ELISA Rapid G12 kit was inspected by determination of the threshold value i.e., > or <20 mg/kg gluten in different food products. In the second step, quantification accuracy was assessed by quantification of gluten in gluten-free food products spiked with gliadin reference material. RESULTS: In total 47 food products (naturally and labeled gluten-free, and food with traces of gluten) were included. Of them, 29 products were quantified with <20 mg/kg, and 18 with a low level of gluten by both the kits. Six out of 29 gluten-free products were used for the recovery test at different spike levels. Gluten concentration and mean recovery rates of individual kits showed consistency. CONCLUSION: GlutenTox Rapid G12 ELISA could be an appropriate choice for detecting gluten in food products but needs more in-house validation and collaborative tests.

Vitamin D status in healthy Italian school-age children: a single-center cross-sectional study.

BACKGROUND: Vitamin D is involved in calcium homeostasis and bone metabolism, although its extra-skeletal actions are also well-known. Low serum 25(OH)D levels are common both in adults and children worldwide. METHODS: The purpose of this cross-sectional study was to determine the distribution of 25(OH)D levels in a cohort of healthy Italian school-age children, aged 5-10 years, in relationship to determinants of vitamin D deficiency such as season, BMI, gender, age and ethnicity. RESULTS: The mean serum 25(OH) D level was 28.2 ng/mL; the prevalence of 25(OH)D sufficiency (> 30 ng/mL), insufficiency (20-30 ng/mL), deficiency (10-20 ng/mL) and severe deficiency (< 10 ng/mL) was 36%, 37%, 21% and 6% of the study-group population, respectively. The lower serum 25(OH)D values were observed during winter (21.6 ng/mL) and spring (22.9 ng/mL), as compared to summer (46.7 ng/mL) (p < 0.001). Higher BMI z-scores were associated with lower 25(OH)D level while no statistical difference was observed as related to gender and age groups. CONCLUSIONS: Healthy Italian schoolchildren show low 25(OH)D levels, particularly during winter and spring time. Seasonality, ethnicity and overweight/obesity were confirmed to influence the vitamin D status, thus indicating the need for effective initiatives to support adequate vitamin D status in this population group.

Determination of Urinary Gluten Immunogenic Peptides to Assess Adherence to the Gluten-Free Diet: A Randomized, Double-Blind, Controlled Study.

INTRODUCTION: The adherence to a gluten-free diet (GFD) is a trending topic in the management of celiac disease. The aim of our study was to evaluate the diagnostic performance of urinary gluten immunogenic peptides (GIP) determination to detect gluten contamination of the GFD. METHODS: In study A, 25 healthy adults on a standard GFD performed 6 gluten challenges (0, 10, 50, 100, 500, and 1,000 mg) with quantification of urinary GIP before (T0) and during the following 24 hours. In study B, 12 participants on a gluten contamination elimination diet underwent urinary GIP determination at T0 and after challenge with 5 or 10 mg gluten. Urine GIP concentration was determined by an immunochromatographic assay. RESULTS: In study A, 51 of 150 baseline urine samples were GIP+ on GFD and 7 of 17 were GIP+ after the zero-gluten challenge, whereas only 55 of 81 were GIP+ after the 10-1,000 mg gluten challenges. There was no significant change in the 24-hour urinary GIP when increasing gluten from 10 to 1,000 mg. In study B, 24 of 24 baseline urine samples were GIP-, whereas 8 of 24 were GIP+ after 5 or 10 mg of gluten. DISCUSSION: Traces of gluten in the standard GFD may cause positivity of urinary GIP determination, whereas a false negativity is common after a gluten intake of 10-1,000 mg. Owing to the impossibility of standardizing the test in normal conditions, it seems unlikely that urinary GIP determination may represent a reliable tool to assess the compliance to the GFD of patients with celiac disease or other gluten-related disorders.

Quantification of Accidental Gluten Contamination in the Diet of Children with Treated Celiac Disease.

A strict gluten-free diet is extremely difficult to maintain. Protracted ingestion of gluten traces (>10 mg/day) is sufficient to cause significant damage in the architecture of the small intestinal mucosa in patients on treatment for celiac disease. The aim of this study was to directly measure the level of contaminating gluten in the daily diet of celiac children following a gluten-free diet. From April 2019 to December 2019, celiac disease children (2-18 years old) on a gluten-free diet for ≥6 months were offered to participate in this prospective-observational study. Patients and their caregivers were invited to provide a representative portion (about 10 g) of all meals consumed during a 24-h period. Participants were requested to weigh all ingested food and report items in a 24-h food diary. The gluten content was quantified by the R5 sandwich enzyme-linked immunosorbent assay method. Sixty-nine children completed the protocol. Overall, 12/448 (2.7%) food samples contained detectable amounts of gluten; of them, 11 contained 5-20 ppm and 1 >20 ppm. The 12 contaminated food samples belonged to 5/69 enrolled patients. In these 5 children, the daily gluten intake was well below the safety threshold of 10 mg/day. The present findings suggest that in a country characterized by high celiac disease awareness, the daily unintended exposure to gluten of treated celiac children on regular follow-up is very low; reassuringly, the presence of gluten traces did not lead to exceed the tolerable threshold of 10 mg/day of gluten intake in the gluten-free diet.

Nutritional Status, Dietary Intake, and Adherence to the Mediterranean Diet of Children with Celiac Disease on a Gluten-Free Diet: A Case-Control Prospective Study.

The only effective treatment for celiac disease (CD) is a life-long strict gluten-free diet (GFD). Nutritional adequacy of the GFD has remained controversial and a matter of debate for a long time. No large case-control studies on children regarding the nutritional adequacy of the GFD has been performed. In this study, children diagnosed with CD on a GFD for ≥ 2 years were recruited. Controls were age and gender-matched healthy children not affected with CD. In both groups, anthropometric measurements and energy expenditure information were collected. Dietary assessment was performed by a 3-day food diary. Adherence to the Mediterranean diet was estimated by the KIDMED index. Overall, 120 children with CD and 100 healthy children were enrolled. No differences were found between CD children and controls in anthropometric measurements and energy expenditure. In the CD group, the daily intake of fats was significantly higher while the consumption of fiber was lower in comparison with the control group. The median KIDMED index was 6.5 in CD children and 6.8 in healthy controls. The diet of children with CD in this study was nutritionally less balanced than controls, with a higher intake of fat and a lower intake of fiber, highlighting the need for dietary counseling.

Misuse of serological screening tests for celiac disease in children: A prospective study in Italy.

BACKGROUND: Despite a well-established diagnostic algorithm for celiac disease, it remains unclear whether prescriptions for celiac serological tests comply with the current pediatric guidelines. AIM: To analyze the appropriateness of test prescription in children investigated for celiac disease in Italy, compared to the current European pediatric guidelines. METHODS: All children who had performed a first evaluation for celiac disease were prospectively enrolled. Prescribed tests and related indications for testing were recorded, and compared to the European pediatric guidelines. RESULTS: Overall, 202 children were enrolled (females 59%, mean age 7.1 years ±4.1) in two centers. The reasons for celiac disease testing were typical, atypical symptoms or celiac disease-associated conditions in 46.5%, 49%, and 4.5% of cases, respectively. First-line tests were IgA and IgG anti-transglutaminase antibodies in 88.1% and 29.7% of children, IgA and IgG anti-deamidated gliadin peptide antibodies in 43% and 47%, IgA and IgG anti native gliadin in 15.8%, IgA anti-endomysium antibodies in 44.5%, HLA predisposing genes in 10% of patients. Test redundancy was very common, and the current diagnostic guidelines were correctly followed only in 23/202 patients (11.4%). CONCLUSIONS: Diagnostic European guidelines for celiac disease screening are often disregarded in Italy. Intervention to implement adherence to these guidelines is needed, with the aim of improving resource utilization, and quality of patient care.

Contribution of Oral Hygiene and Cosmetics on Contamination of Gluten-free Diet: Do Celiac Customers Need to Worry About?

OBJECTIVES: The only available treatment for celiac disease (CD) is the gluten-free diet. It is unclear whether the presence of gluten in oral hygiene products and cosmetics that are applied on the mouth is a reason of concern for CD patients. The aim of this study was to test the level of gluten contamination in oral hygiene and cosmetic products available in the Italian market. METHODS: A total of 66 products (toothpastes = 37; dental tablets = 2; mouthwashes = 5; lip-balms = 10; lipsticks = 12) labelled gluten-free or with unknown gluten content were randomly collected from different supermarkets and pharmacies. The gluten quantification was determined by the R5 ELISA method approved by EU regulations. RESULTS: Out of 66 oral hygiene and cosmetics, 62 products (94%) were found to be gluten-free (gluten level <20 ppm), while 4 (6%) (toothpastes = 3; lipsticks = 1) showed a gluten level >20 ppm (toothpastes: 20.7, 31.4, and 35 ppm; lipstick: 27.4 ppm). None of the selected products had ingredient derived from wheat, barley, or rye. CONCLUSIONS: Gluten contamination is currently not an issue in a wide array of cosmetic and oral hygiene products that are commonly in the market.

Validation of a novel single-drop rapid human leukocyte antigen-DQ2/-DQ8 typing method to identify subjects susceptible to celiac disease.

BACKGROUND AND AIM: Human leukocyte antigen (HLA)-DQ2 and/or -DQ8 is an essential risk factor for celiac disease (CD). About 90-95% of patients with CD carry HLA-DQ2/-DQ8 alleles, and HLA-DQ typing is considered an additional diagnostic test. Conventional polymerase chain reaction (PCR)-based HLA-DQ typing methods are expensive, complex, and a time-consuming process. We assessed the efficacy of a novel HLA-DQ typing method, "Celiac Gene Screen," for the detection of CD-associated HLA haplotypes. METHODS: To assess the diagnostic performance of the Celiac Gene Screen test, 100 ethylenediaminetetraacetic acid (EDTA) blood samples, already characterized by the conventional HLA-DQ typing method, that is, PCR sequence-specific oligonucleotide probes (PCR-SSOP), a concordance between both the methods were explored. For validity, a further 300 EDTA blood samples with unknown HLA-DQ status were genotyped using the Celiac Gene Screen test, including 141 samples from CD, 56 first-degree relatives (FDRs) of CD and 103 samples from controls. RESULTS: Of the 100 samples with known status of HLA-DQ alleles, 79 samples were HLA-DQ2 and/or -DQ8 positive, and 21 samples were HLA-DQ2 and/or -DQ8 negative by conventional PCR. These 100 samples were re-typed using the Celiac Gene screen kit; all 79 positives were typed positive, and 21 negatives were typed negative for HLA-DQ alleles. Among 300 samples with unknown HLA-DQ status, 118 of 141 (84%) patients with CD, 48 of 56 (86%) FDRs of CD, and 52 of 103 (50%) controls typed positive for HLA-DQ alleles. CONCLUSIONS: The Celiac Gene Screen HLA-DQ typing method showed excellent concordance with the conventional HLA-DQ typing method and could be a cost-reducing and effective method for CD-associated HLA screening.

Effects of the Exclusive Enteral Nutrition on the Microbiota Profile of Patients with Crohn's Disease: A Systematic Review.

The mechanisms behind the efficacy of exclusive enteral nutrition (EEN) in Crohn's disease (CD) remain poorly understood, despite the high rate of treatment response. Evidence accumulated in the last 20 years suggests that a positive shift of the disrupted microbiota is one of the treatment effects. The purpose of this study was to critically review and summarize data reporting the microbiological effects of EEN in patients with CD. Fourteen studies were considered in the review, overall involving 216 CD patients on EEN. The studies were heterogeneous in methods of microbiota analysis and exclusion criteria. The most frequently reported effect of EEN was a reduction in microbiota diversity, reversible when patients returned to a normal diet. The effect of EEN on specific bacteria was very variable in the different studies, partially due to methodological limitations of the mentioned studies. The EEN seem to induce some metabolomic changes, which are different in long-term responder patients compared to patients that relapse earlier. Bacterial changes can be relevant to explaining the efficacy of EEN; however, microbiological data obtained from rigorously performed studies and derived from last generation techniques are largely inconsistent.