A multi-step screening algorithm for fecal microbiota transplantation donors: integrating metagenomic and metabolomic profiling for improved safety and donor quality assessment.

BACKGROUND: Fecal Microbiota Transplantation (FMT) has emerged as a highly effective treatment for recurrent Clostridioides difficile infection. It is also a promising therapeutic approach for other microbiome-related disorders. However, the safety and efficacy of FMT depend critically on rigorous donor screening and selection protocols. In this study, we developed and evaluated an optimized donor screening and selection workflow that integrates multistep pathogen detection, metagenomic and metabolomic profiling, and clinical compatibility assessment to enhance FMT safety and donor quality assessment. METHODS: In this prospective cross-sectional study we evaluated 178 stool donor candidates using a novel screening algorithm, which combined extended medical history, blood and urine testing, stool metagenomics (16S rRNA gene sequencing) and metabolomics (short-chain fatty acids), and microbiological stool tests to exclude the stool samples with pathogens and ensure their microbial diversity. RESULTS: The FMT donor screening and selection workflow was developed and included four main steps: 1. Potential donors (n = 178) had to meet all the study inclusion criteria (47.8% of donors passed this screening step); 2. Potential donors (n = 85) underwent microbiological and microscopic stool examination (23% of initially enrolled donors passed this screening step). Most of the healthy donors were excluded at this selection step due to the abnormal values of Enterococcus spp. (47.1% of healthy donors had abnormal values), total number of Enterobacteriaceae and Bifidobacterium spp. (for both parameters 41.2% of healthy donors had abnormal values) abundance; 3. Potential donors (n = 41) underwent blood (total blood cell count, biochemical blood analysis) and urine (urinalysis) tests. Only 13 donors (7.3% of the initial cohort) proceeded to the final metabolomic analysis, resulting in the selection of 3 FMT donors (1.7% eligibility rate). CONCLUSIONS: This study establishes a data-driven, high-stringency donor selection framework that provides a rational basis for improving FMT safety and donor quality. Our findings advocate for the standardized implementation of advanced screening technologies, particularly SCFA metabolomics, as a critical quality control step in stool banking. While clinical validation of the efficacy-enhancing potential of this protocol requires prospective studies, the strong association between butyrate-producing microbiota and favorable FMT outcomes, combined with our previous clinical observations, supports the utility of our approach.