Validation and Testing of a Method for Detection of SARS-CoV-2 RNA in Healthy Human Stool

Background: Fecal shedding of SARS-CoV-2 has raised concerns about transmission through fecal microbiota transplantation (FMT) procedures. While many tests have been authorized for diagnosis of COVID-19 using respiratory samples, no fully validated stool tests for detection of SARS-CoV-2 are currently available. We sought to adapt and validate an available test specifically for detection of SARS-CoV-2 in human stool. Methods: Stool samples were spiked with inactivated SAR-CoV-2 virus for development and validation of the assay. A modified version of the CDC rRT-PCR SARS-CoV-2 test was used for detection of virus. Analytical sensitivity, assay reproducibility, and sample stability under a variety of storage conditions were assessed. We also performed the assay on stool samples collected from known COVID positive individuals. Findings: The lower limit of detection (LoD) of the assay was found to be 3000 viral RNA copies per gram of original stool sample, with 100% detection across 20 replicates assessed at this concentration. Samples were relatively stable in all buffers tested at both 4 o C and ambient temperature, with the exception of storage in STAR buffer at ambient temperature. Assay sensitivity was slightly diminished in low-copy-number samples after a single freeze-thaw cycle at -80°C. Thirty contrived SARS-CoV-2 samples were tested by a second laboratory and were correctly identified as positive or negative in at least one of two rounds of testing. Additionally, we detected SARS-CoV-2 RNA in the stool of known COVID-19 positive individuals using this method. Interpretation: This is a sensitive, reproducible, and validated assay for detection of SARS-CoV-2 RNA in human stool with potential uses in FMT donor screening, sewage monitoring, and further research into the impact of fecal shedding on the epidemiology of this pandemic. Funding: National Institute for Allergy and Infectious Diseases, NIH. Center for Biologics Evaluation and Research, FDA. Declaration of Interests: None to declare.