Materials and Methods
A multicentre prospective observational study of detection of GIP in stool in a cohort of children newly diagnosed with coeliac disease was performed at seven Spanish secondary and tertiary referral hospitals: Hospital Nuestra Señora de la Candelaria (Tenerife), Hospital Regional Carlos Haya (Málaga), Hospital Universitario y Politécnico La Fe (Valencia), Hospital Universitario Virgen del Rocío (Sevilla), Hospital Universitario Virgen de Valme (Sevilla), Instituto Hispalense de Pediatría (Sevilla), Hospital Universitario Río Hortega (Valladolid) and Hospital Sant Joan de Reus (Tarragona). There were four study visits at diagnosis and, 6 (from 4–9 months), 12 (from 10–15 months) and 24 (from 16–24 months) months after diagnosis. The first visit (diagnosis) was at the time of diagnostic endoscopy when patients were untreated (diet with gluten). All participants were instructed to follow a GFD by clinical dieticians with expertise in coeliac disease. Samples of faeces and blood were collected at each study visit and participants completed a four-day food record according to the dietician instructions. The study protocol was reviewed by the ethics committee at each participating hospital and written informed consent was obtained from the parents or legal guardians.
Children (less than 18 years of age) with active coeliac disease were recruited at time of diagnostic endoscopy. Inclusion criteria were: (a) symptoms—either gastrointestinal (eg, abdominal pain, diarrhea, constipation, weightloss, flatulence, bloating, vomiting, lack of appetite) or atypical (e.g., iron deficiency anaemia, chronic fatigue, behavioural changes, poor growth); (b) elevated serum EMA IgA, tTG IgA/IgG and/or DGP IgA/IgG; (c) HLADQ2 and/or HLADQ8 genotype; and, (d) small intestinal histology consistent with coeliac disease (Marsh II-III). Exclusion criteria were: (a) history of kidney, liver or severe psychiatric disease; (b) seizure disorder and/or current use of anticonvulsants; (c) use of any antibiotics within the year prior to enrolment.
All subject data were recorded in an electronic data capture (EDC) system, including: age, weight, height, intestinal histology, symptoms, comorbiditions, clinical test results, adverse events, family history of coeliac disease, date of coeliac disease diagnosis, adherence to GFD, and details sample collection and visit attendance.
Faeces and Blood Collection
Subjects were instructed to collect 2–4 g stool in a sealed container after recording their food intake for 4 days. Specimens were dropped-off within 24 hours of collection and were kept at −20°C at all times until processing.
Blood samples were collected in two 3 mL vacutainer tubes with EDTA-K3 anticoagulant and centrifuged at 2000 g within 30 minutes of collection to obtain plasma which was stored at −80°C until analysis. Investigators performing stool and serum analysis were blinded to GFD status at the time of sample collection.
Quantification of GIP in Stool Samples
Stool GIP concentration was determined by sandwich enzyme-linked immunosorbent assay (ELISA; iVYDAL In Vitro Diagnostics iVYLISA GIP Stool kit, Biomedal S.L., Seville, Spain) according to the manufacturer's protocol. Briefly, stool samples were mixed with 9 ml Universal Gluten Extraction Solution (UGES; Biomedal S.L., Seville, Spain) per gram of stool then incubated at 50°C for 60 minutes with gentle agitation to release the GIP from the stool matrix. After extraction, samples were diluted 1:10 with dilution solution and ELISA was performed using the provided G12 coated microtiter plate, standards (50, 25, 6.25, 3.13, 1.56 ng/ml GIP) and positive and negative controls. Thus, results were expressed as μg GIP per gram faeces. Each sample was run in duplicate and at least two different aliquots of each sample were tested on different days.
The validity of this method in detecting GFD transgressions was determined by the analytical sensitivity (limits of detection and quantification 0.06 and 0.16 μg GIP per gram faeces, respectively) and the diagnostic sensitivity and specificity (98.5% and 100%, respectively).
Estimation of Gluten Consumption
A calibration factor allowed estimation of the ingestion of gluten in coeliac patients from stool measurements. Specifically, the equation for estimating daily gluten consumption in milligrams (y variable) based upon faecal GIP concentration (in micrograms per gram) (x variable) was determined from measured mean values of 6.2 and 14.9 μg GIP per gram faeces during controlled gluten challenges of 9 and 30 grams per day.[19,20,23] Fitting to a second-order polynomial going through the origin gave the relation y = 0.0649x2 + 1.0461x.
The levels of tTG IgA and DGP IgA antibodies (IgG in IgA-deficient patients) were determined by ELISA using the EliA™ Celikey® and EliA™ Gliadin kits, respectively, according to the manufacturer's protocol (Phadia, Freiburg, Germany). Measurements were performed in duplicate and the results expressed as U/ml. The manufacturer recommended cut-off of >10 U/mL was used.
To assess gluten exposure, a structured food questionnaire of 27 items was administered to record the foods consumed during the 4 days prior to stool and blood collection. The food items were classified into eight predefined groups: dairy (milk and cheese); complex carbohydrates (bread, cereals, pasta, rice, potato, legumes, and nuts); meats (red meat, fish, cold cuts, and eggs); fruits (whole or juiced); vegetables; fats (vegetable oils, butter, and cream); sweetened beverages (sodas, bottled juices, and energy drinks); and other (baked goods, candy, snacks, etc.). Images of standard portion sizes were included as a guideline for portion quantification. Subjects were asked to record the amount and type of food consumed, brand, time of meal, and if it was labelled as gluten-free. They were also asked to note if they were aware of having consumed any gluten-containing foods.
Quantitiative variables are expressed as median with interquartile range (IQR), and the categorical variables as percentages. Goodness-of-fit to normal was checked using the Shapiro-Wilk test. Pearson's chi-squared test was used for categorical variables, and the chi-squared test for ordinal variables. Statistical analysis of the degree of concordance of the dichotomously evaluated diagnostic techniques was performed using Cohen's kappa index (κ), following the criteria of Landis and Koch for the qualitative interpretation of the strength of concordance. The Mann-Whitney U test was used to compare quantitative variables in independent groups. For all cases, P < 0.05 was considered statistically significant. Data analysis was performed with SPSS 23.0 for Windows (SPSS Inc).
Aliment Pharmacol Ther. 2019;49(12):1484-1492. © 2019 Blackwell Publishing