Limitations of faecal calprotectin in detecting histological changes and persistent villous atrophy in patients with coeliac disease on a gluten-free diet

Alimentary Pharmacology & Therapeutics, 2025; 61:1815–1819 https://doi.org/10.1111/apt.70114 1815 Alimentary Pharmacology & Therapeutics RESEARCH COMMUNICATION OPEN ACCESS Limitations of Faecal Calprotectin in Detecting Histological Changes and Persistent Villous Atrophy in Patients With Coeliac Disease on a GlutenFree Diet VerónicaSegura1 | ÁngelaRuiz-Carnicer1 | ÁngelesPizarro2 | CarmenGonzález-Naranjo2 | JacoboDíaz3 | CristóbalCoronel-Rodríguez4 | FedericoArgüelles-Arias5 | MartaGarzón-Benavides2 | CarolinaSousa1 | IsabelComino1 1Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Seville, Seville, Spain | 2Digestive Disease Clinical Unit and CIBERehd, Institute of Biomedicine of Seville (IBiS), SeLiver Group, Virgen del Rocío Hospital/CSIC/US, Seville, Spain | 3Clinical Analysis Service, Hospital Universitario INGESA, Ceuta, Spain | 4Centro de Salud Amante Laffón, Seville, Spain | 5Digestive Diseases Clinical Unit, Department of Medicine, Faculty of Medicine, Virgen Macarena Hospital, University of Seville, Seville,Spain Correspondence: Isabel Comino ([email protected]) Received: 7 February 2025 | Revised: 26 February 2025 | Accepted: 22 March 2025 Handling Editor: Jason A TyeDin Funding: This work was supported by Grant PI00532018 provided by Junta de Andalucía (Fundación Pública Andaluza Progreso y Salud, Spain). Keywords: coeliac disease| faecal calprotectin| histological changes| villous atrophy ABSTRACT Faecal calprotectin is used to assess intestinal inflammation, but its role in monitoring mucosal healing in coeliac disease is unclear. This study followed 48 adults with coeliac disease on a glutenfree diet over 12 months, evaluating faecal calprotectin levels in correlation with antitransglutaminase antibodies, glutenfree diet adherence by dietary questionnaires and histology. Although significant histological lesions (Marsh II–III) decreased from 24% to 10%, faecal calprotectin levels fluctuated without correlation to antitransglutaminase, adherence or histological remission, and did not differentiate between lesion grades. Our findings underscore faecal calprotectin's unreliability in monitoring mucosal healing in adults with coeliac disease, highlighting the urgent need for alternative biomarkers. 1 | Introduction Coeliac disease is a systemic autoimmune disorder triggered by gluten peptides, which elicit a T cell–mediated immune response, resulting in small intestinal villous atrophy and chronic inflammation [1]. A lifelong glutenfree diet is the mainstay of coeliac disease treatment, improving symptoms and intestinal lesions while preventing longterm complications. However, persistent villous atrophy may occur in some patients despite following a glutenfree diet, commonly due to poor dietary adherence, delayed mucosal response or ongoing gluten sensitivity [2]. As in other chronic intestinal disorders, effective monitoring of coeliac disease requires tools that accurately reflect mucosal status. Although intestinal biopsy is the gold standard, routine acquisition of serial intestinal biopsies is hindered by invasiveness, high cost and inherent risks. Antitissue transglutaminase IgA antibodies are sensitive for diagnosis but lose reliability after diagnosis due to poor correlation with histology in patients on a glutenfree diet [3]. Furthermore, symptoms and dietary questionnaires are unreliable [4], underscoring the need for objective, noninvasive biomarkers to assess intestinal inflammation in coeliac disease. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. © 2025 The Author(s). Alimentary Pharmacology & Therapeutics published by John Wiley & Sons Ltd. 1816 Alimentary Pharmacology & Therapeutics, 2025 Faecal calprotectin is a neutrophilderived protein released into the gut during inflammatory processes [3]. Its resistance to enzymatic degradation ensures stability and easy measurement over time. Clinically, faecal calprotectin is valuable because it reduces unnecessary endoscopic procedures, and several commercially available methods exist for its measurement [5]. Elevated faecal calprotectin levels are associated with inflammatory bowel disease, colorectal cancer, nonsteroidal antiinflammatory drug enteropathy, chronic pancreatitis and cirrhosis, although evidence outside inflammatory bowel disease remains limited [6]. In inflammatory bowel disease, faecal calprotectin is a wellestablished noninvasive biomarker in adults and children. However, its role in coeliac disease remains unclear [7]. Some studies have shown that elevated faecal calprotectin levels in paediatric patients newly diagnosed with coeliac disease normalise on a glutenfree diet; others reported no significant correlation between faecal calprotectin levels, clinical symptoms or histological findings. These discrepancies underline the need for additional research on the role of faecal calprotectin in managing coeliac disease. We aimed to evaluate faecal calprotectin levels systematically over time in adult patients with coeliac disease adhering to a glutenfree diet for at least 24 months. Specifically, we compared faecal calprotectin levels with serological markers and glutenfree diet adherence tests, while assessing their correlation with duodenal histology through biopsies at baseline and 12 months. Through this analysis, we sought to determine the potential utility of faecal calprotectin as a noninvasive biomarker for monitoring disease activity and mucosal status in coeliac disease. 2 | Methods We conducted a longitudinal prospective study on faecal calprotectin detection in patients with coeliac disease on a glutenfree diet for at least 24 months between June 2019 and March 2023. Participants had study visits at inclusion and at 3, 6 and 12 months, where clinical examinations, blood sampling for coeliac disease serology and stool collection for faecal calprotectin quantification were performed. They also completed a glutenfree diet adherence questionnaire. Duodenal biopsies were obtained at the time of inclusion and after 12 months to assess lesion progression. The study protocol was approved by the ethics committee of each institution, and written informed consent was obtained from all the participants. Faecal calprotectin levels were measured with the Bühlmann fCAL ELISA kit and categorised as > 200 μg/g (moderate/ severe inflammation), 50–200 μg/g (mild inflammation) and < 50 μg/g (no inflammation). Endoscopic biopsies were graded histologically using the Marsh–Oberhuber classification. At both baseline and 1year followup endoscopies, four duodenal biopsies were obtained from the second portion of the duodenum. Histological evaluation was performed independently by expert pathologists, based on the most severe lesion identified among the samples; atrophy was further classified as total, subtotal or focal. Histological evolution was assessed by comparing the two reports. Intraepithelial lymphocytes were quantified using the Ventana BenchMark ULTRA and CD3 antibodies (Roche Holding AG). Antitissue transglutaminase IgA levels were assessed using the EliA Celikey IgA/IgG kits, and adherence to a glutenfree diet was evaluated using the Spanish version of the Celiac Dietary Adherence Test. Sample size was calculated with a 95% confidence level (α = 0.05), 80% power (β = 0.2) and an expected proportion of 0.5, adjusted for a 10% loss rate, resulting in 40 participants (GRANMO v8.0 tool). Statistical analysis included normality tests and temporal measurement comparisons using Cochran's Q, Mann–Whitney U, Friedman, and Wilcoxon signedrank tests. Data were analysed with SPSS (v29), with p < 0.05 considered statistically significant. 3 | Results 3.1 | Patients We included 48 patients; seven were excluded due to missing stool samples or followup visits. Thus, 41 patients (33 women) with a median age of 34 years (interquartile range [IQR]: 20.5– 47.5) completed the study. 3.2 | Clinical, Analytical and Histological Evolution of Patients During FollowUp At inclusion, 24% of patients (10/41) had significant Marsh II– III histological lesions despite following a glutenfree diet for at least 24 months. This proportion decreased to 10% (4/39) after 12 months. Histology remained stable without mucosal damage in 29 patients from inclusion to the 12month followup (Marsh 0–I). Six patients showed histological improvement, with mucosal damage resolving by 12 months (Marsh II–III to Marsh 0–I). In contrast, mucosal damage persisted in three patients (Marsh II–III to Marsh II–III), while one experienced histological progression (Marsh 0–I to Marsh II–III). The proportion of nonadherent patients, as measured by the Celiac Dietary Adherence Test, remained stable (33% at inclusion, 32% at 12 months). Analysis of antitransglutaminase antibody analysis showed that 10% of patients (4/41) had elevated concentrations (> 10 U/mL) at inclusion, which decreased to 5% (2/39) at 12 months. Median antitissue transglutaminase levels remained below the 10 U/mL threshold throughout the followup period. To inclusion, 35% of patients (14/40) had faecal calprotectin > 50 μg/g, including 10% (4/40) with values > 200 μg/g. These proportions remained stable at 3 months (36% and 10%). By 6 months, faecal calprotectin > 50 μg/g increased to 51% (20/39), while faecal calprotectin > 200 μg/g remained at 13% (5/39). At 12 months, 46% (18/39) had faecal calprotectin > 50 μg/g, with 10% (4/39) exceeding 200 μg/g. Median faecal calprotectin levels increased from 30.70 μg/g at inclusion to 39.50 μg/g at 12 months, but no statistically significant differences were observed across followup visits (Figure1). 13652036, 2025, 11, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/apt.70114 by Readcube (Labtiva Inc.), Wiley Online Library on [26/05/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 1817 3.3 | Analysis of Concordance Between Different Coeliac Disease Biomarkers During FollowUp 3.3.1 | Faecal Calprotectin, Antitissue Transglutaminase and Celiac Dietary Adherence Test Scores We evaluated the relationship between faecal calprotectin levels and circulating antitissue transglutaminase levels across visits using the Spearman correlation coefficient (ρ). We found no significant correlation, suggesting that fluctuations in faecal calprotectin do not correlate with changes in antitissue transglutaminase levels, indicative of an immune response to gluten (FigureS1). Similarly, no significant association was observed between faecal calprotectin levels and the Celiac Dietary Adherence Test scores. 3.3.2 | Antitissue Transglutaminase, Celiac Dietary Adherence Test and Marsh Score No patients with Marsh II–III lesions had antitissue transglutaminase concentrations above the threshold, indicating no correlation between antitissue transglutaminase levels and mucosal damage (Mann–Whitney U test, p > 0.05). Notably, all patients with antitissue transglutaminase > 10 U/mL remained without mucosal damage (Marsh 0–I) (Figure2a–c). Additionally, Cochran's Q test (p > 0.05) showed no significant association between histological progression and glutenfree diet adherence, as assessed by the Celiac Dietary Adherence Test. 3.3.3 | Faecal Calprotectin and Marsh Score In the 29 patients without mucosal damage, median faecal calprotectin levels remained below 50 μg/g at all visits. Among the six patients achieving histological remission (Marsh II–III to Marsh 0–I), faecal calprotectin levels increased over 12 months (median: 39.55 μg/g at inclusion; 78.90 μg/g at 12 months), although this was not statistically significant (Friedman test, p > 0.05). In contrast, faecal calprotectin levels decreased in the three patients without remission (Marsh II–III to Marsh II–III) (median: 43.6–28.2 μg/g). One patient progressing from Marsh 0–I to Marsh II–III showed an increase from 40.3 to 89 μg/g (Figure2d–f). 4 | Discussion This study highlights the limitations of faecal calprotectin as a biomarker for monitoring histological recovery in adult patients with coeliac disease on a glutenfree diet. The lack of significant changes in faecal calprotectin levels, even in patients with histological improvement, underscores its limited sensitivity in reflecting mucosal regeneration. Similarly, coeliac disease serology and the Celiac Dietary Adherence Test score also failed to detect meaningful improvements in mucosal recovery. In contrast, some studies in both paediatric and adult populations have suggested that faecal calprotectin levels decrease following the initiation of a glutenfree diet and correlate with dietary adherence, particularly in patients with a gastrointestinal FIGURE 1 | Evolution of histological lesions, serological markers, Celiac Dietary Adherence Test scores and faecal calprotectin levels at inclusion and at subsequent followup visits (3, 6 and 12 months). Percentage of patients with different biomarkers assessed at inclusion and at subsequent followup visits (3, 6 and 12 months). Faecal calprotectin levels were interpreted according to the manufacturer's recommended cutoff, which > 200 μg/g suggests inflammation ranging between moderate to severe and 50–200 μg/g may indicate mild organic disorders. AntitTG, antitissue transglutaminase antibody; CDAT, coeliac dietary adherence test; FC, faecal calprotectin. 13652036, 2025, 11, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/apt.70114 by Readcube (Labtiva Inc.), Wiley Online Library on [26/05/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 1818 Alimentary Pharmacology & Therapeutics, 2025 presentation of the disease [8–10]. These studies support a potential role for faecal calprotectin in monitoring response to treatment. However, we did not observe such associations in adults, which may reflect variations in disease severity, duration or differences in study design. Conversely, numerous studies have failed to establish a significant relationship between faecal calprotectin levels and histological outcomes in coeliac disease. Specifically, several studies have shown no correlation between faecal calprotectin levels and the severity of intestinal lesions or clinical presentation [11]. Additionally, other research has found no significant differences in faecal calprotectin levels between adult coeliac disease patients and healthy controls, further limiting its utility in monitoring disease activity[12]. Consistent with these findings, our results demonstrated that faecal calprotectin levels did not correlate with Marsh scores and remained stable over time, even in patients achieving histological remission. Notably, some patients who improved histologically showed an increase in faecal calprotectin levels, while others with persistent mucosal damage experienced a decline, reinforcing the variability and lack of predictive value of faecal calprotectin in this context. This suggests a disconnect between faecal calprotectin and mucosal recovery. Supporting this view, the ESPGHAN expert group recommended against using faecal calprotectin for coeliac disease diagnosis or monitoring in paediatric patients [13]. The observed increase in faecal calprotectin levels at 12 months in patients with histological remission may reflect residual or lowgrade inflammation that histology does not fully capture, supporting the observation that faecal calprotectin can detect subclinical inflammation [14]. However, the lack of statistical significance in our Friedman's test highlights the need for larger studies to confirm these findings. Elevated faecal calprotectin levels in histological remission may also suggest other underlying gastrointestinal conditions or factors influencing intestinal inflammation, further complicating the interpretation of faecal calprotectin in coeliac disease. In conclusion, our study highlights the limitations of faecal calprotectin as a reliable biomarker for monitoring histological changes in coeliac disease. The interpretation of faecal calprotectin levels is complicated by variability in inflammatory responses and the presence of subclinical conditions, emphasising the need to consider additional factors. These findings reinforce the challenges of using faecal calprotectin as a standalone biomarker and underscore the need for more accurate noninvasive markers for assessing mucosal status in coeliac disease management. Author Contributions Verónica Segura: methodology, validation, formal analysis, data curation, writing – original draft, writing – review and editing. Ángela RuizCarnicer: methodology, validation, writing – review and editing, writing – original draft, formal analysis, data curation. Ángeles Pizarro: writing – review and editing, conceptualization, methodology. Carmen GonzálezNaranjo: writing – review and editing, FIGURE 2 | Variation of antitransglutaminase and faecal calprotectin concentration levels in patients without villous atrophy during followup and in patients who achieved histological remission at the end of the study. (a) Antitissue transglutaminase concentration. (b) Evolution of antitransglutaminase levels in patients without villous atrophy during followup. (c) Evolution of antitissue transglutaminase levels in patients who achieved histological remission at the end of the study. (d) Faecal calprotectin concentration. (e) Evolution of faecal calprotectin levels in patients without villous atrophy during followup. (f) Evolution of faecal calprotectin levels in patients who achieved histological remission at the end of the study. The differences between paired groups were tested with the Friedman and Wilcoxon tests. AntitTG, antitissue transglutaminase antibody; FC, faecal calprotectin. 13652036, 2025, 11, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/apt.70114 by Readcube (Labtiva Inc.), Wiley Online Library on [26/05/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 1819 methodology. Jacobo Díaz: formal analysis, software, writing – review and editing. Cristóbal CoronelRodríguez: writing – review and editing, methodology, conceptualization. Federico ArgüellesArias: conceptualization, methodology, writing – review and editing. Marta GarzónBenavides: conceptualization, methodology, writing – review and editing. Carolina Sousa: project administration, conceptualization, investigation, writing – original draft, writing – review and editing, visualization, supervision. 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Supporting Information Additional supporting information can be found online in the Supporting Information section. 13652036, 2025, 11, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/apt.70114 by Readcube (Labtiva Inc.), Wiley Online Library on [26/05/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License