Fecal Gluten Peptides Reveal Limitations of Serological Tests and Food Questionnaires for Monitoring Gluten-Free Diet in Celiac Disease Patients

Abstract

Objectives: Treatment for celiac disease (CD) is a lifelong strict gluten-free diet (GFD). Patients should be followed-up with dietary interviews and serology as CD markers to ensure adherence to the diet. However, none of these methods offer an accurate measure of dietary compliance. Our aim was to evaluate the measurement of gluten immunogenic peptides (GIP) in stools as a marker of GFD adherence in CD patients and compare it with traditional methods of GFD monitoring.

Methods: We performed a prospective, nonrandomized, multicenter study including 188 CD patients on GFD and 84 healthy controls. Subjects were given a dietary questionnaire and fecal GIP quantified by enzyme-linked immunosorbent assay (ELISA). Serological anti-tissue transglutaminase (anti-tTG) IgA and anti-deamidated gliadin peptide (anti-DGP) IgA antibodies were measured simultaneously.

Results: Of the 188 celiac patients, 56 (29.8%) had detectable GIP levels in stools. There was significant association between age and GIP in stools that revealed increasing dietary transgressions with advancing age (39.2% in subjects ≥13 years old) and with gender in certain age groups (60% in men ≥13 years old). No association was found between fecal GIP and dietary questionnaire or anti-tTG antibodies. However, association was detected between GIP and anti-DGP antibodies, although 46 of the 53 GIP stool-positive patients were negative for anti-DGP.

Conclusions: Detection of gluten peptides in stools reveals limitations of traditional methods for monitoring GFD in celiac patients. The GIP ELISA enables direct and quantitative assessment of gluten exposure early after ingestion and could aid in the diagnosis and clinical management of nonresponsive CD and refractory CD. Trial registration number NCT02711397.

Conflict of interest statement

Guarantor of the article: Carolina Sousa, PhD. Specific author contributions: Study concept and design: I.C., F.F.-B., M.E., L.O., G.C., B.F., C.R.K., C.S., A.R.-H., J.C.S., Á.C., J.M.M.M., J.A.G., S.V., O.L.I., A.N., L.V., A.M.V., L.C., L.F.-S., E.A., V.A.J.G., M.A.M.-C., B.E., A.G., J.V., F.J.G., M.B., A.M., C.G., J.R.A., M.R., M.R.-G., V.S., F.L., J.M., A.M.S., Á.C. and C.S.; acquisition of data: I.C., V.S., F.L., A.M.S., Á.C. and C.S.; analysis and interpretation of data: I.C., V.S., F.L., A.M.S., Á.C. and C.S.; technical and material support: I.C., V.S., F.L., A.M.S., Á.C. and C.S.; drafting of the manuscript: I.C., Á.C. and C.S.; critical revision of the manuscript and important intellectual content: I.C., F.F.-B., M.E., L.O., G.C., B.F., C.R.K., C.S., A.R.-H., J.C.S., A.C., J.M.M.M., J.A.G., S.V., O.L.I., A.N., L.V., A.M.V., L.C., L.F.-S., E.A., V.A.J.G., M.A.M.-C., B.E., A.G., J.V., F.J.G., M.B., A.M., C.G., J.R.A., M.R., M.R.-G., V.S., F.L., J.M., A.M.S., Á.C. and C.S. Financial support: This work was supported by grants from Ministerio de Ciencia e Innovación and FEDER funds (DELIAC, IPT-2011-0952-900000), and Corporación Tecnológica de Andalucía (SINGLUCHECK, 1737/0118). We also thank the generous volunteer subjects who enrolled in the study. Potential competing interests: Á.C. and F.L. own stock in Biomedal SL. Other authors declare no conflict of interest.

Figures

Figure 1

Concentration of GIP in stools of healthy controls and celiac patients on a GFD. (a) Levels of fecal GIP in positive controls, negative controls, and GFD-treated celiac patients. (b) Levels of fecal GIP for GFD-treated celiac patients and controls with weak positive and negative values (<0.3 μg per g sample) represented in a log base 10 scale. (c) Percentage distribution of controls and GFD-treated celiac patient according to GIP concentration. Each point in dot-plots represents the mean of two replicates of each sample. Celiac patients (n=188) were children and adults on GFD for ≥1 year and healthy controls (n=84) were classified into positive controls (children and adults on a gluten-containing diet, n=73); and negative controls (babies between 0 and 8 months of age who were only fed GFD formula milk, n=11). GFD, gluten-free diet; GIP, gluten immunogenic peptides.

Figure 2

GFD adherence according to patient age. (a) Percentage distribution of celiac patients according to GIP concentration and age. (b) GIP concentration of celiac patients with GIP-positive stool samples (>0.16 μg GIP per g sample) represented in a log base 10 scale. Each point represents the mean of two replicates for each sample. GFD, gluten-free diet; GIP, gluten immunogenic peptides.

Figure 3

Percentage distribution of celiac patients according to GIP content in stools and duration of GFD. GFD, gluten-free diet; GIP, gluten immunogenic peptides.

Figure 4

Evaluation of GFD adherence in celiac patients according to celiac disease serologies and association with fecal GIP content. (a) Correlation between anti-tTG-IgA and fecal GIP. (b) Correlation between anti-DGP-IgA and fecal GIP. GIP content was divided into three groups according to GIP titers by enzyme-linked immunosorbent assay (ELISA): positive (>0.30 μg GIP per g feces), weak positive (0.16 and 0.30 μg GIP per g feces), and negative (<0.16 μg GIP per g feces). Levels of anti-tTG-IgA and anti-DGP-IgA are expressed as EliA U/ml and classified as positive (>10 EliA U/ml), indeterminate (7–10 EliA U/ml), and negative (<7 EliA U/ml). Anti-DGP, anti-deamidated gliadin peptide antibody; Anti-tTG, anti-tissue transglutaminase antibody; GFD, gluten-free diet; GIP, gluten immunogenic peptides.