There are several reasons why discordant results could be seen between the 2 split stool samples:
Differences in Stool Sample Prior to Stool Homogenization in Lab
- Need for Homogenization: To address this, laboratory protocols often require thorough homogenization of the entire stool specimen before splitting it into aliquots for analysis. Homogenization significantly lowers inter-aliquot variability, making the split samples more representative of the whole.
- Variability: Stool from different sections may show differing levels of specific organisms, leading to potentially dissimilar results between un-homogenized split samples.
-
Collection Bias: Patient collection methods can also introduce variability. Patients are typically instructed to collect samples from at least three to five different sections of the stool to ensure a representative sample for testing.
- Stool with hard and lump consistency (Bristol Stool Type 1-2) has more microbial composition variation between different collection sites in a single bowel movement. Therefore samples of different consistency will vary between Labs.
- The outer part of the stool sample will have a different ratio of aerobes/anaerobes because of an oxygen concentration gradient.
- Samples that are loose or watery (Bristol Stool Type 5-7) will have different microbial composition, increased fecal pH, and may cause a false positive for pancreatic elastase-1 (PE-1). Therefore samples of different consistency will vary between Labs.
- Microbiota can vary along with the type and amount of undigested fibers sampled in different aliquots.
- Presence of PCR inhibitors such as complex polysaccharides, bile salts, lipids, uric acid salts, chlorophyll, glycolipids, hemoglobin, and heparin. PCR inhibitors are frequent in stool samples. Un-homogenized stool can have variable PCR inhibitors in the sample which can contribute to false negative PCR results between Labs.
- Contamination with toilet water or urine will alter bacterial composition and diversity.
Differences in Temperature Changes in Transportation
Temperature change during shipping and receipt of specimen at Lab can account for significant variability:
- Repeated temperature fluctuation: Dramatic temperature change is a major stress for bacteria which can cause microbial death and DNA degradation.
- Duration of transportation time: Transportation time will impact microbial growth and decline.
- Frozen-thaw cycles: Frozen stool that has thawed and is refrozen can cause DNA degradation.
Differences in stool Sampling and DNA Isolation Kits
Different stool sampling, stabilization buffers, and DNA isolation kits can affect DNA yield, purity, and integrity, which in turn will impact bacterial diversity and bacterial composition results.
Differences in DNA Extraction Methods
Fecal DNA extraction includes a number of steps and methods, which can account for variability between Labs.
- Sample homogenization
- Cell lysis
- Non-DNA substances removal
- DNA purification
Conclusion
Understanding the variability of the microbiome requires longitudinal cohort studies and large-scale meta-analyses. Serial measurements over time using the same sampling and analytical methodology are best for assessing intra-individual variability.
Vibrant Gut Zoomer Validation Study- Key Findings
Gut Zoomer validation study using 212 spiked stool specimens and 1067 clinical and archived stool specimens.
- Overall sensitivity is 95.9% (95% CI 92.4–98.1)= 96% of people get true positive pathogen results
- Specificity is 100% (95% CI 99.9–100)= 100% of people get true negative pathogen results
- Stability study for Gut Zoomer collection methodology and transport revealed 100% concordance between the expected genotypes before and after shipping and handling
Yang, Y., Rajendran, V., Jayaraman, V. et al. Evaluation of the Vibrant DNA microarray for the high-throughput multiplex detection of enteric pathogens in clinical samples. Gut Pathog 11, 51 (2019.