Evaluation of a Single Determination of Gluten Immunogenic Peptides in Urine from Unaware Celiac Patients to Monitor Gluten-Free Diet Adherence

**Background:** Celiac disease (CD) is a systemic immune-mediated disorder triggered by gluten ingestion in genetically susceptible individuals, affecting 0.5–1% of the population worldwide. The only effective therapy is a strict lifelong gluten-free diet (GFD). Monitoring GFD adherence remains challenging: serological tests (tTGA) are unreliable during follow-up, dietary questionnaires (CDAT) are subjective, and duodenal biopsy is invasive. Gluten immunogenic peptides (GIP) resistant to gastrointestinal digestion can be detected in urine (uGIP), providing a direct measure of recent gluten ingestion. This study evaluated the clinical utility of a single uGIP determination in CD outpatients who reported complete GFD adherence.

**Methods:** From April 2019 to February 2020, CD patients reporting complete GFD adherence for at least 6 months were prospectively enrolled at a tertiary referral center in Milan, Italy. Patients were unaware of the purpose of urine collection. Urinary GIP was assessed using the GlutenDetect lateral flow test (Biomedal, Seville, Spain), which detects GIP in urine from gluten ingestion in the preceding 12–24 hours. All patients completed the CDAT questionnaire (score range 7–35; <13 indicates good adherence) and visual analog scales (VAS) for abdominal symptoms and overall well-being. Serum tTGA levels were measured. Upper endoscopy with duodenal biopsies (graded by Marsh-Oberhuber scale) was performed in 113 patients, and video capsule endoscopy (VCE) in 61 patients, following international guideline indications. Statistical analysis used t-tests, chi-squared tests, and Fisher's exact tests with significance at α=0.05.

**Key Results:** Of 285 enrolled patients, 280 completed the study (232 females, 82.8%; 48 males, 17.2%; median age 31.7±16.9 years). Thirty-two patients (11.4%) had a positive uGIP test (uGIP+). No significant differences were found between uGIP+ and uGIP− patients in sex, age at diagnosis, age at enrollment, BMI, or CDAT scores. Among VAS items, only abdominal bloating severity was significantly higher in uGIP+ patients (p<0.05). Sixty-nine patients (24.6%) had elevated tTGA titers. Among uGIP+ patients, 10 (31.2%) were tTGA-positive and 22 (68.8%) were tTGA-negative, with no statistically significant association (14.4% tTGA+ vs. 10.9% tTGA− were uGIP+, p=NS). Of 113 patients who underwent duodenal biopsy, 42 (37.2%) had mucosal atrophy (Marsh 3a–c). Among these, 10 (23.1%) were uGIP+ compared to 5 of 71 (7.0%) without atrophy (p<0.05). In the uGIP+ group, 66.7% had duodenal atrophy vs. 32.7% in the uGIP− group (p=0.01). tTGA did not correlate with duodenal atrophy. Among 61 patients who underwent VCE, 29 (47.5%) showed mucosal atrophy; 5 of these (17.2%) were uGIP+ vs. 4 of 32 (12.5%) without atrophy (p=NS). Notably, 21.4% of uGIP+ patients had no detectable tTGA or mucosal atrophy.

**Clinical Implications:** A single uGIP test detected otherwise unrecognized gluten ingestion in 11% of CD patients reporting strict GFD adherence. uGIP positivity significantly correlated with duodenal mucosal atrophy on biopsy, the current reference standard for assessing CD activity, with a specificity of 93% for predicting villus atrophy. The lack of correlation with CDAT scores and tTGA serology indicates that questionnaires and serological markers alone are insufficient to identify patients with ongoing gluten exposure. uGIP testing may serve as a practical, non-invasive point-of-care tool to identify patients requiring dietary reinforcement or further investigation. Limitations include the single urine sample (which may underestimate intermittent gluten exposure), the lack of uniform testing across all patients, and the single-center design. Future studies with multiple sampling over weekdays and weekends may improve sensitivity.