Comparison of Fecal Collection Methods for Microbiota Studies in Bangladesh

Abstract

To our knowledge, fecal microbiota collection methods have not been evaluated in low-and middle-income countries. Therefore, we evaluated five different fecal sample collection methods for technical reproducibility, stability, and accuracy within the Health Effects of Arsenic Longitudinal Study (HEALS) in Bangladesh. Fifty participants from the HEALS provided fecal samples in the clinic which were aliquoted into no solution, 95% ethanol, RNAlater, postdevelopment fecal occult blood test (FOBT) cards, and fecal immunochemical test (FIT) tubes. Half of the aliquots were frozen immediately at Ϫ80°C (day 0) and the remaining samples were left at ambient temperature for 96 h and then frozen (day 4). Intraclass correlation coefficients (ICC) were calculated for the relative abundances of the top three phyla, for two alpha diversity measures, and for four beta diversity measures. The duplicate samples had relatively high ICCs for technical reproducibility at day 0 and day 4 (range, 0.79 to 0.99). The FOBT card and samples preserved in RNAlater and 95% ethanol had the highest ICCs for stability over 4 days. The FIT tube had lower stability measures overall. In comparison to the "gold standard" method using immediately frozen fecal samples with no solution, the ICCs for many of the microbial metrics were low, but the rank order appeared to be preserved as seen by the Spearman correlation. The FOBT cards, 95% ethanol, and RNAlater were effective fecal preservatives. These fecal collection methods are optimal for future cohort studies, particularly in low-and middle-income countries. IMPORTANCEThe collection of fecal samples in prospective cohort studies is essential to provide the opportunity to study the effect of the human microbiota on numerous health conditions. However, these collection methods have not been adequately tested in low-and middle-income countries. We present estimates of technical reproducibility, stability at ambient temperature for 4 days, and accuracy comparing a "gold standard" for fecal samples in no solution, 95% ethanol, RNAlater, postdevelopment fecal occult blood test cards, and fecal immunochemical test tubes in a study conducted in Bangladesh. Fecal occult blood test cards and fecal samples stored in 95% ethanol or RNAlater adequately preserve fecal samples in this setting. Therefore, new studies in low-and middle-income countries should include collection of fecal samples using fecal occult blood test cards, 95% ethanol, or RNAlater for prospective cohort studies.

Reproducibility of assessing fecal microbiota in chronic constipation

“…Similar approaches have also been used to study the reproducibility and stability of various fecal sampling methods. [35][36][37][38][39] Overall, the agreement between two fecal samples for all measures of alpha and beta diversity in healthy people and patients with 57 (0.28-0.86) 0.89 (0.81-0 which, to speculate, may be more pronounced in people with normal compared to slow colonic transit. The fecal microbiota, stool consistency, and colonic transit are known to be univariately associated with each other.…”

“…The reason we chose the ICC measure over other measures is that ICC is directly related to statistical power, a key issue in the study design. Similar approaches have also been used to study the reproducibility and stability of various fecal sampling methods …”

Comparison of Methods To Collect Fecal Samples for Microbiome Studies Using Whole-Genome Shotgun Metagenomic Sequencing

“…A major direction for future microbiome research is implementation of fecal sample collections in large-scale, prospective epidemiologic studies. Evidence from 16S rRNA gene amplicon sequencing studies suggests that the chosen fecal collection method impacts multiple estimates of microbial composition (9)(10)(11)(12); thus, development of standard fecal collection protocols is required, especially for pooling of microbial data across studies. Ideal fecal sample collection methods preserve the original microbial signature of the fecal sample, stabilize microbial DNA, and prevent bacterial growth during room-temperature storage for multiple days, which is typical under field conditions (10,(13)(14)(15)(16).…”

“…Only one small study (n ϭ 8) (20) investigated stability and "gold-standard" concordance of WGSS fecal samples, collected using 100% ethanol or RNAlater. That study did not consider other common, established methods for fecal sample collection such as fecal occult blood test (FOBT) cards and fecal immunochemical tests (FIT), which were recently shown to be reproducible, stable, and relatively concordant with the "goldstandard" collection method in 16S rRNA gene studies (10)(11)(12). FOBT cards and FIT tests are used widely in colorectal cancer screening, providing opportunities for establishing prospective cohorts using discarded tests.…”

Library Preparation and Sequencing Platform Introduce Bias in Metagenomic-Based Characterizations of Microbiomes

“…Due to the challenge of replicating results, one must not over-emphasize the results from exploratory research and keep in mind with the maturation of metagenomics, that there is a need to continually validate the robustness and ability to replicate results. (20,21). With the improvement of reference databases and bioinformatics tools, the validation is an ongoing process (22–25).…”