Fecal calprotectin (FCP) has limited specificity as diagnostic biomarker of pediatric inflammatory bowel disease (IBD), leading to unnecessary invasive endoscopies. This study aimed to develop and validate a fecal microbiota and amino acid (AA)-based diagnostic model. Fecal samples from a discovery cohort (de novo IBD and healthy controls [HC]) were used to develop the diagnostic model. This model was applied in a validation cohort (de novo IBD and controls with gastrointestinal symptoms [CGI]). Microbiota and AAs were analyzed using interspace profiling and liquid chromatography-mass spectrometry techniques, respectively. Machine learning techniques were used to build the diagnostic model. In the discovery cohort (58 IBD, 59 hC), two microbial species (Escherichia coli and Alistipes finegoldii) and four AAs (leucine, ornithine, taurine, and alpha-aminoadipic acid [AAD]) combined allowed for discrimination between both subgroups (AUC 0.94, 95% CI [0.89, 0.98]). In the validation cohort (43 IBD, 38 CGI), this panel of six markers could differentiate patients with IBD from CGI with an AUC of 0.84, 95% CI [0.67, 0.95]). Leucine showed the best diagnostic performance (AUC 0.89, 95% CI [0.81, 0.95]). Leucine might serve as adjuvant noninvasive biomarker in the diagnostic work-up of pediatric IBD. Future research should investigate whether the combination of leucine with FCP could improve specificity and may help tailor the course of diagnostics.

Purpose: This review aims to critically evaluate the potential benefit of either oral or subcutaneous administration of methotrexate (MTX) in various immune-mediated inflammatory disorders (IMIDs) through analysis of efficacy, toxicity, pharmacokinetics and pharmacodynamics of both administration routes. Recent Findings: Recent studies comparing the efficacy of oral versus subcutaneous MTX administration in IMIDs have revealed contradicting results. Some reported higher efficacy with subcutaneous administration, while others found no significant difference. Regarding toxicity, some studies have challenged the notion that subcutaneous administration is better tolerated than oral administration, while others have supported this. Pharmacokinetic studies suggest higher plasma bioavailability and increased accumulation of MTX-polyglutamates (MTX-PGs) in red blood cells (RBCs) with subcutaneous administration during the initial treatment phase. However, after several months, similar intracellular drug levels are observed with both administration routes. Summary: There is no conclusive evidence supporting the superiority of either oral or subcutaneous MTX administration in terms of efficacy and adverse events in IMIDs. Subcutaneous administration leads to higher plasma bioavailability and initial accumulation of MTX-PGs in RBCs, but the difference seems to disappear over time. Given the variable findings, the choice of administration route may be based on shared decision-making, offering patients the option of either oral or subcutaneous administration of MTX based on individual preferences and tolerability. Further research is needed to better understand the impact of MTX-PGs in various blood cells and TDM on treatment response and adherence to MTX therapy.

Background and Aims: Microbiome studies reveal distinct microbial differences in inflammatory bowel disease (IBD), indicating its potential role in pathophysiology and as a noninvasive diagnostic biomarker. This study aims to profile the gut microbiome in children with IBD, compared to both healthy controls (HC) and controls with gastrointestinal symptoms (CGI), and to assess the potential of microbiome profiles as noninvasive diagnostic markers for de novo treatment-naïve pediatric IBD, and as early predictive markers for therapy response. Methods: We analyzed baseline fecal samples and clinical data from 103 therapy-naïve children with IBD, 75 CGI, and 356 age and sex matched HC. Metagenomic sequencing was performed, and prediction models assessed diagnostic potential and prediction of induction therapy response at 3 months. Results: Alpha diversity progressively decreased from HC to CGI (P < .001) and decreased even further in IBD patients (P = .0056). Beta diversity analysis showed significant clustering differences (P < .001, R2 = 0.045). Differential abundance analysis revealed 116 species differing between HC and IBD, and 30 species between CGI and IBD. Prediction models based on microbiome features accurately distinguished IBD from HC (area under the curve [AUC] = 0.96) and from CGI (AUC = 0.71). However, these models were outperformed by clinical features, such as fecal calprotectin. Microbiome-based prediction of response to induction therapy in general showed limited accuracy (AUC = 0.63), as well as for response to nutritional induction therapy (AUC = 0.67). Conclusions: We observed profound gut microbiome differences between de novo, therapy-naïve pediatric IBD patients and controls. While microbiome profiles hold promise for improving diagnostic precision, their predictive value for therapy response seems limited.

Introduction: Faecal amino acids are promising non-invasive diagnostic biomarkers, but stability remains unclear. Objectives: This study examined whether storage conditions, sampling site within the sample, freeze-thaw cycles, and the OMNImet^®·GUT device affect faecal amino acid concentrations. Methods: Faecal samples from three donors underwent various pre-analytical conditions. Amino acids were analysed through targeted liquid chromatography-tandem mass spectrometry. Results: Most amino acids remained stable across sampling sites and freeze-thaw cycles. Storage at -20 °C preserved integrity, whereas 4 °C and 20 °C storage led to variations. The OMNImet^®·GUT device stabilised some amino acids but showed inconsistencies. Conclusion: Pre-analytical conditions influence faecal amino acid concentrations. Standardisation is essential for biomarker reliability.

The gut microbiota and its related metabolites differ between inflammatory bowel disease (IBD) patients and healthy controls. In this study, we compared faecal volatile organic compound (VOC) patterns of paediatric IBD patients and controls with gastrointestinal symptoms (CGIs). Additionally, we aimed to assess if baseline VOC profiles could predict treatment response in paediatric IBD patients. We collected faecal samples from a cohort of de novo therapy-naïve paediatric IBD patients and CGIs. VOCs were analysed using gas chromatography–ion mobility spectrometry (GC-IMS). Response was defined as a combination of clinical response based on disease activity scores, without requiring treatment escalation. We included 109 paediatric IBD patients and 75 CGIs, aged 4 to 17 years. Faecal VOC profiles of paediatric IBD patients were distinguishable from those of CGIs (AUC ± 95% CI, p-values: 0.71 (0.64–0.79), <0.001). This discrimination was observed in both Crohn’s disease (CD) (0.75 (0.67–0.84), <0.001) and ulcerative colitis (UC) (0.67 (0.56–0.78), 0.01) patients. VOC profiles between CD and UC patients were not distinguishable (0.57 (0.45–0.69), 0.87). Baseline VOC profiles of responders did not differ from non-responders (0.70 (0.58–0.83), 0.1). In conclusion, faecal VOC profiles of paediatric IBD patients differ significantly from those of CGIs.

Background and Aims: Therapeutic drug monitoring (TDM) of methotrexate (MTX) is challenging due to its pharmacokinetics and short plasma half-life. Intracellular MTX–polyglutamates (PG1–5), which accumulate over time, have not been assessed in pediatric inflammatory bowel disease (IBD). This study aimed to evaluate erythrocyte MTX-PG as a potential TDM tool in pediatric IBD. Methods: In this cross-sectional study, MTX-PG concentrations were measured in erythrocytes of children with IBD on stable low-dose MTX for at least 12 weeks using stable-isotope dilution liquid chromatography–tandem mass spectrometry. The influence of administration route, MTX dosage, and anthropometrics on MTX-PG concentrations was examined. Results: Seventy-eight patients were included, showing MTX-PG3 as the predominant subspecies (median 27.0 nmol/L) with a median MTX-PGtotal of 74.8 nmol/L. A higher MTX dose correlated significantly with elevated levels of MTX-PG3, MTX-PG4, MTX-PG5, and MTX-PGtotal (P < .01). Adjusted for body surface area, MTX dose remained significantly associated with higher MTX-PG concentrations (P < .01). However, comparison by administration route was limited due to a few patients on subcutaneous MTX (n = 4). Conclusions: We observed high interindividual variability in the reached erythrocyte MTX-PG concentrations. Body surface adjusted or unadjusted MTX dosage showed a positive linear correlation with erythrocyte MTX-PG concentrations in children with IBD. This is a prerequisite for TDM and provides a strong basis for further research into the relation between TDM of MTX, efficacy, and toxicity.