Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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

Author(s): Zouiouich S, Mariadassou M, Rué O, Vogtmann E, Huybrechts I, Severi G, Boutron-Ruault MC, Senore C, Naccarati A, Mengozzi G, Kozlakidis Z, Jenab M, Sinha R, Gunter MJ, Leclerc M

Publication: Cancer Epidemiol Biomarkers Prev, 2022, Vol. 31, Page 305-314

PubMed ID: 34782392 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of fecal specimen collection method and a variety of storage methods on the fecal microbiome using specimens that were collected during colorectal cancer screening.  Comparisons were made between matched fecal specimens that were frozen without any preservative and those stored frozen or at room temperature on GenSaver specimen collection cards or GenCollect specimen collection cards, mailed or stored at 4°C or 30°C for 0 or 7 d before freezing in OC-Auto Sampling tubes, or stored at room temperature for 0 or 7 days before freezing in Hemotrust tubes, One-Step FOB tubes, or Alfresa Pharma Specimen Collection Container A tubes. Participants undergoing colorectal cancer screening collected a fecal specimen at home (10 participants) or at work (9 participants). Within a few hours (exact number not specified), the specimens were manually homogenized and aliquoted into cryotubes, OC-Auto Sampling tubes, Hemotrust tubes, One-Step FOB tubes and Alfresa Pharma Specimen Collection Container A tubes, or on to GenSaver specimen collection cards, and GenCollect specimen collection cards. For each participant, a cryotube (gold standard) and one aliquot of each container type were immediately frozen at -80°C. Additionally, aliquots in OC-Auto Sampling tubes were: (1) mailed to a laboratory, (2) stored at 4°C for 7 d followed by room temperature storage for ≥4 h before freezing at -80°C, or (3) stored at 30°C for 7 d followed by room temperature storage for ≥4 h before freezing at -80°C. Aliquots in Hemotrust tubes, two One-Step FOB, and Specimen Collection Container A tubes were stored for 7 days at room temperature before being frozen at -80°C, tested for occult blood, and shipped at room temperature. Additional aliquots on GenCollect and GenSaver cards were stored in a closed cupboard at room temperature until extraction (average 70.2 d). All specimens were shipped prior to DNA extraction with the PowerFecal DNA Isolation Kit and sequencing with an Illumina MiSeq and the MiSeq Kit V2. Data was analyzed using Find, Rapidly, OTUs with Galaxy Solution (FROGS) v3.1.0 and statistical analysis was conducted using the DESeq2, icc, phyloseq and vegan R packages.

   Summary of Findings:

   Overall, the highest alpha diversity (Simpson and Shannon Indices) occurred in the specimen that was immediately frozen in a cryotube (gold standard), but the number of OTUs was highest in fecal specimens that were stored on GenSaver or GenCollect cards. In the linear mixed-effects model, the Shannon index was significantly lower than the gold standard specimen when stored in OC-Auto Sampling FIT tubes at −80°C (P=0.005), or in One-Step FOB tubes at −80°C or room temperature(P=0.03 and P<0.001, respectively), or in Specimen Collection Container A tubes at −80°C or room temperature (P<0.001, both). In MDS plots based on β-diversity metrics (Jaccard, Bray-Curtis, unweighted UniFrac, and weighted UniFrac), specimens grouped by patient source and not collection method/storage conditions. The percent variability in each β-diversity metric that was attributed to inter-individual differences was large when compared to the percent variability that was attributed to collection method/storage (54.68% versus 4.83% for Jaccard, 79% versus 6.9% for Bray-Curtis, 64.13% versus 4.68% for unweighted UniFrac and 71.28% versus 14.8% for weighted UniFrac). Regardless of collection method/storage, the most abundant phyla were Firmicutes followed by Bacteroidetes and Actinobacteria. At the genus level, the gold standard specimens had relatively fewer Faecalibacterium than any of the other collection methods/storage combinations. Other differences at the genus level, particularly in the abundance of Bacteroidetes, were observed but the differences displayed a high degree of inter-individual variability as well as differences between collection/storage conditions. 

   ICCs for specimens collected under the various collection/storage conditions with the gold standard specimens were variable for the relative abundance of Actinobacteria, Bacteroidetes, or Firmicutes as well as for the inverse Simpson index and weighted UniFrac. While ICCs were ≥0.89 between technical replicates of gold standard specimens, no other tube collection/storage condition achieved ICCs ≥75% for all of the metrics evaluated and no other collection storage condition yielded the highest ICC for all metrics. When compared to specimens in the same collection container that were immediately frozen, ICCs were high for specimens stored at room temperature (average 70.2 days) on GenCollect (0.91-0.99) or GenSaver cards (0.89-0.99) and for specimens stored at 4°C for 7 days in OC-Auto Sampling FIT tubes (≥0.78; except for the number of observed OTUs, ICC=0.65) or shipped in OC-Auto Sampling FIT tubes (0.76-0.94, except for relative abundance of Actinobacteria, ICC=0.65). ICCs were also high between specimens in Collection Container A tubes that were immediately frozen and those that were stored for 7 days at room temperature (ICCs 0.80-0.95; except for the relative abundance of Actinobacteria, ICC=0.58 and Firmicutes, ICC=0.66). The ICC between container-matched frozen specimens was lower and less consistent for specimens stored for 7 days at room temperature in Hemotrust tubes or One-Step FOB tubes and specimens stored for 7 days at 30°C in OC-Auto Sampling FIT tubes. ICCs were very high (≥0.75) for all genera between immediately frozen specimens and specimens stored at room temperature on GenCollect or GenSaver cards, but ICCs were lower for at least one genus for all other tube types.  

   Biospecimens

   • Bodily Fluid - Feces

   Preservative Types

   • Frozen
   • Other Preservative

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	Next generation sequencing

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Type of storage container	GeneCollect Card GenSaver Card OC-Auto Sampling tubes Hemotrust tubes One-Step FOB tubes Alfresa Pharma Specimen Collection Container A tubes
   Storage	Time at room temperature	0 days 7 days Average 70.2 d Average 70.2 days
   Storage	Storage temperature	-80°C 4°C Room temperature 30°C
   Storage	Between site transportation method	Mailed Not transported
   Storage	Storage duration	0 days 7 days
   Biospecimen Preservation	Type of fixation/preservation	FOBT Card Fecal immunochemical test (FIT) Frozen GeneCollect Card GenSaver Card OC-Auto Sampling tubes Hemotrust tubes Alfresa Pharma Specimen Collection Container A tubes One-Step FOB tubes

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