title: 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 -80°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.
authors.affiliation: Metabolic Epidemiology Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA emily.vogtmann@nih.gov.
authors.forename: Emily
authors.identifier: http://orcid.org/0000-0003-1355-5593
authors.initials: E
authors.lastname: Vogtmann
authors.affiliation: Microbiome Program, Center for Individualized Medicine, Mayo Clinic, Rochester, Minnesota, USA.|Health Sciences Research, Mayo Clinic, Rochester, Minnesota, USA.
authors.forename: Jun
authors.initials: J
authors.lastname: Chen
authors.affiliation: Department of Public Health Sciences, University of Chicago, Chicago, Illinois, USA.
authors.forename: Muhammad G
authors.initials: MG
authors.lastname: Kibriya
authors.affiliation: Department of Population Health, New York University School of Medicine, New York, New York, USA.
authors.forename: Yu
authors.initials: Y
authors.lastname: Chen
authors.affiliation: University of Chicago Research Bangladesh, Dhaka, Bangladesh.
authors.forename: Tariqul
authors.initials: T
authors.lastname: Islam
authors.affiliation: University of Chicago Research Bangladesh, Dhaka, Bangladesh.
authors.forename: Mahbubul
authors.initials: M
authors.lastname: Eunes
authors.affiliation: University of Chicago Research Bangladesh, Dhaka, Bangladesh.
authors.forename: Alauddin
authors.initials: A
authors.lastname: Ahmed
authors.affiliation: University of Chicago Research Bangladesh, Dhaka, Bangladesh.
authors.forename: Jabun
authors.initials: J
authors.lastname: Naher
authors.affiliation: University of Chicago Research Bangladesh, Dhaka, Bangladesh.
authors.forename: Anisur
authors.initials: A
authors.lastname: Rahman
authors.affiliation: Department of Pediatrics, University of California San Diego, La Jolla, California, USA.
authors.forename: Amnon
authors.initials: A
authors.lastname: Amir
authors.affiliation: Biostatistics Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, Maryland, USA.
authors.forename: Jianxin
authors.initials: J
authors.lastname: Shi
authors.affiliation: Metabolic Epidemiology Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Christian C
authors.initials: CC
authors.lastname: Abnet
authors.affiliation: Microbiome Program, Center for Individualized Medicine, Mayo Clinic, Rochester, Minnesota, USA.|Department of Surgery, Mayo Clinic, Rochester, Minnesota, USA.
authors.forename: Heidi
authors.initials: H
authors.lastname: Nelson
authors.affiliation: Department of Pediatrics, University of California San Diego, La Jolla, California, USA.|Department of Computer Science and Engineering, University of California San Diego, La Jolla, California, USA.
authors.forename: Rob
authors.initials: R
authors.lastname: Knight
authors.affiliation: Microbiome Program, Center for Individualized Medicine, Mayo Clinic, Rochester, Minnesota, USA.|Health Sciences Research, Mayo Clinic, Rochester, Minnesota, USA.|Department of Surgery, Mayo Clinic, Rochester, Minnesota, USA.|Biomedical Engineering and Physiology, Mayo Clinic, Rochester, Minnesota, USA.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Chia
authors.affiliation: Department of Public Health Sciences, University of Chicago, Chicago, Illinois, USA.
authors.forename: Habibul
authors.initials: H
authors.lastname: Ahsan
authors.affiliation: Metabolic Epidemiology Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Rashmi
authors.initials: R
authors.lastname: Sinha
mesh_terms: D000328:Adult; D001419:Bacteria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D001459:Bangladesh; D005243:Feces / Q000737:chemistry / Q000382:microbiology*; D005260:Female; D005615:Freezing; D006801:Humans; D008297:Male; D064307:Microbiota; D008875:Middle Aged; D013048:Specimen Handling / Q000295:instrumentation / Q000379:methods*
keywords: feces; low/middle-income countries; microbiota; sampling methods; stability