Comparison of Fecal Sample Collection Methods for Microbial Analysis Embedded within Colorectal Cancer Screening Programs

Background Colorectal cancer (CRC) screening programs with fecal sample collection may provide a platform for population-based gut microbiome-disease research. We investigated sample collection and storage methods impact on the accuracy and stability of the V3-V4 region of the 16S rRNA genes and bacterial quantity across seven different collection methods (i.e., no solution, two specimen collection cards, and four types of fecal immunochemical test (FIT) used in four countries) among 19 healthy volunteers. Methods Intraclass correlation coefficients (ICCs) were calculated for the relative abundance of the top three phyla, the most abundant genera, alpha-diversity metrics, and the first principal coordinates of the beta-diversity matrices to estimate the stability of microbial profiles after storage for 7 days at room temperature, 4{degree sign}C, 30{degree sign}C. Additionally, screening for the presence of occult blood in the stool, and accuracy was compared to samples frozen immediately with no solution (i.e. the putative gold standard). Results When compared to the putative gold standard, we observed significant variation for all collection methods. However, inter-individual variability was much higher than the variability introduced by the collection method. Stability ICCs were high ({greater than or equal to}0.75) for FIT tubes that underwent CRC screening procedures. The relative abundance of Actinobacteria (0.65) was an exception and was lower for different FIT tubes stored at 30{degree sign}C (range, 0.41-0.90) and room temperature (range, 0.06-0.94). Conclusions Paper collection cards and different types of FIT are acceptable tools for microbiome measurements. Impact Our findings inform on the utility of commonly used fecal sample collection methods for developing microbiome-focused cohorts nested within screening programs.