Validation and testing of a method for detection of SARS-CoV-2 RNA in healthy human stool

Abstract

Summary (Abstract)BackgroundFecal 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.MethodsStool 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.FindingsThe 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°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.InterpretationThis 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.FundingNational Institute for Allergy and Infectious Diseases, NIH. Center for Biologics Evaluation and Research, FDA.Research in ContextEvidence before this studySince the onset of the COVID-19 pandemic, multiple studies have documented shedding of SARS-CoV-2 RNA in feces and considered the potential for fecal-oral transmission of this virus. This potential risk led to the U.S. Food and Drug Administration issuing a safety alert that contained the recommendation that no stool donated after December 1, 2019 be used for manufacture of Fecal Microbiota for Transplantation (FMT) products in the United States until such a time as sufficient screening procedures could be put in place to mitigate this risk.Added value of this studyHere, we report the development and validation of an assay specifically meant for the detection of SARS-CoV-2 RNA in the stool of healthy individuals. While studies have reported detection of viral RNA in stool previously, this is the first publication of a validated assay designed for this purpose.Implications of all the available evidenceThe work presented here provides a validated SARS-CoV-2 stool assay with potential application to FMT donor screening protocols, sewage monitoring protocols, as well as research studies assessing the role of stool shedding and transmission on the epidemiology of COVID-19.

Standardized and optimized preservation, extraction and quantification techniques for detection of fecal SARS-CoV-2 RNA

“…In most studies, stool samples are collected and stored without a preservative [10][11][12] . They are then resuspended in PBS in a 1:5 ratio (w:v) prior to RNA extraction.…”

“…In parallel, we also tested how these preservation methods interact with three different extraction kits -a) MagMAX viral/pathogen nucleic acid isolation kit (MM; Applied Biosystems), a magnetic-bead based protocol that has been successfully used with respiratory samples 27 , b) QiaAMP viral RNA minikit (QA; Qiagen), a column-based protocol that is used in many studies of fecal SARS-CoV-2 RNA [10][11][12] , c) Quick-RNA viral kit (ZV; Zymo Research), another column based protocol that is rated to be compatible with the ZY stool collection kit. All three of these extraction kits are scalable to a high-throughput format and therefore easily adaptable to clinical laboratories and other large-scale efforts.…”

“…Further, the variability in techniques used across studies makes meta-analysis difficult, hindering our overall understanding of the disease. While there is heterogeneity, the majority of existing studies collect and store stool without any preservative [10][11][12] , dilute stool in PBS at the time of RNA extraction, and employ the QIA-Amp Viral RNA kit from Qiagen for RNA isolation. Unfortunately, the efficiency of these strategies in preserving and extracting SARS-CoV-2 RNA is unknown and…”

An optimised protocol for detection of SARS-CoV-2 in stool

“…These data are consistent with previous reports that highlighted the higher sensitivity of the N1 assay compared to the N2 assay 27 . This LoD corresponds to 1000 vp g -1 for N1 and 2000 vp g -1 for N2 and to the best of our knowledge is the lowest LoD so far described 17,24 . It is worth noting that for practical reasons we took roughly half of the initial faecal slurry for RNA extraction (500-600 μL of the 1 mL in which 100 mg stool was homogenised).…”

“…However, the faecal matrix has properties distinct from those of respiratory samples 20–22 and this makes the reliable detection of SARS-CoV-2 challenging. Recently, a few methods have been described for the detection of SARS-CoV2 in stool samples 17,23,24 . However, the potentailly low concentration of SARS-CoV2 in faeces and the unique features of the sample matrix require optimized protocols to improve the recovery of viral RNA and increase our ability to detect the virus in stool samples.…”