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A method for detection of SARS-CoV-2 RNA in healthy human stool: a validation study.
Coryell, Michael P; Iakiviak, Mikhail; Pereira, Nicole; Murugkar, Pallavi P; Rippe, Jason; Williams, David B; Heald-Sargent, Taylor; Sanchez-Pinto, L Nelson; Chavez, Jairo; Hastie, Jessica L; Sava, Rosa L; Lien, Christopher Z; Wang, Tony T; Muller, William J; Fischbach, Michael A; Carlson, Paul E.
  • Coryell MP; Division of Bacterial, Parasitic and Allergenic Products, Office of Vaccines Research and Review, Center for Biologics Evaluation and Research, US Food and Drug Administration, Silver Spring, MD, USA.
  • Iakiviak M; Department of Bioengineering and ChEM-H, Stanford University, Stanford, CA, USA.
  • Pereira N; Department of Bioengineering and ChEM-H, Stanford University, Stanford, CA, USA.
  • Murugkar PP; Department of Bioengineering and ChEM-H, Stanford University, Stanford, CA, USA.
  • Rippe J; Division of Infectious Diseases, Department of Pediatrics, Ann & Robert H Lurie Children's Hospital, Chicago, IL, USA.
  • Williams DB; Division of Infectious Diseases, Department of Pediatrics, Ann & Robert H Lurie Children's Hospital, Chicago, IL, USA.
  • Heald-Sargent T; Division of Infectious Diseases, Department of Pediatrics, Ann & Robert H Lurie Children's Hospital, Chicago, IL, USA.
  • Sanchez-Pinto LN; Northwestern University Feinberg School of Medicine, Chicago, IL, USA.
  • Chavez J; Division of Infectious Diseases, Department of Pediatrics, Ann & Robert H Lurie Children's Hospital, Chicago, IL, USA.
  • Hastie JL; Northwestern University Feinberg School of Medicine, Chicago, IL, USA.
  • Sava RL; Division of Infectious Diseases, Department of Pediatrics, Ann & Robert H Lurie Children's Hospital, Chicago, IL, USA.
  • Lien CZ; Northwestern University Feinberg School of Medicine, Chicago, IL, USA.
  • Wang TT; Division of Bacterial, Parasitic and Allergenic Products, Office of Vaccines Research and Review, Center for Biologics Evaluation and Research, US Food and Drug Administration, Silver Spring, MD, USA.
  • Muller WJ; Division of Bacterial, Parasitic and Allergenic Products, Office of Vaccines Research and Review, Center for Biologics Evaluation and Research, US Food and Drug Administration, Silver Spring, MD, USA.
  • Fischbach MA; Division of Viral Products, Office of Vaccines Research and Review, Center for Biologics Evaluation and Research, US Food and Drug Administration, Silver Spring, MD, USA.
  • Carlson PE; Division of Viral Products, Office of Vaccines Research and Review, Center for Biologics Evaluation and Research, US Food and Drug Administration, Silver Spring, MD, USA.
Lancet Microbe ; 2(6): e259-e266, 2021 06.
Article in English | MEDLINE | ID: covidwho-1164728
Semantic information from SemMedBD (by NLM)
1. Laboratory Procedures METHOD_OF Detection
Subject
Laboratory Procedures
Predicate
METHOD_OF
Object
Detection
2. Feces PART_OF Homo sapiens
Subject
Feces
Predicate
PART_OF
Object
Homo sapiens
3. Laboratory Procedures DIAGNOSES 2019 novel coronavirus
Subject
Laboratory Procedures
Predicate
DIAGNOSES
Object
2019 novel coronavirus
4. Laboratory Procedures USES Detection
Subject
Laboratory Procedures
Predicate
USES
Object
Detection
5. Buffers COEXISTS_WITH Cary Blair Medium
Subject
Buffers
Predicate
COEXISTS_WITH
Object
Cary Blair Medium
6. Assay METHOD_OF Detection
Subject
Assay
Predicate
METHOD_OF
Object
Detection
7. Detection COEXISTS_WITH Donor Selection
Subject
Detection
Predicate
COEXISTS_WITH
Object
Donor Selection
8. Laboratory Procedures METHOD_OF Detection
Subject
Laboratory Procedures
Predicate
METHOD_OF
Object
Detection
9. Feces PART_OF Homo sapiens
Subject
Feces
Predicate
PART_OF
Object
Homo sapiens
10. Laboratory Procedures DIAGNOSES 2019 novel coronavirus
Subject
Laboratory Procedures
Predicate
DIAGNOSES
Object
2019 novel coronavirus
11. Laboratory Procedures USES Detection
Subject
Laboratory Procedures
Predicate
USES
Object
Detection
12. Buffers COEXISTS_WITH Cary Blair Medium
Subject
Buffers
Predicate
COEXISTS_WITH
Object
Cary Blair Medium
13. Assay METHOD_OF Detection
Subject
Assay
Predicate
METHOD_OF
Object
Detection
14. Detection COEXISTS_WITH Donor Selection
Subject
Detection
Predicate
COEXISTS_WITH
Object
Donor Selection
ABSTRACT

BACKGROUND:

Faecal shedding of SARS-CoV-2 has raised concerns about transmission through faecal microbiota transplantation procedures. Validation parameters of authorised tests for SARS-CoV-2 RNA detection in respiratory samples are described in product labelling, whereas the published methods for SARS-CoV-2 detection from faecal samples have not permitted a robust description of the assay parameters. We aimed to develop and validate a test specifically for detection of SARS-CoV-2 in human stool.

METHODS:

In this validation study, we evaluated performance characteristics of a reverse transcriptase real-time PCR (RT-rtPCR) test for detection of SARS-CoV-2 in human stool specimens by spiking stool with inactivated SARS-CoV-2 material. A modified version of the US Centers for Disease Control and Prevention RT-rtPCR SARS-CoV-2 test was used for detection of viral RNA. Analytical sensitivity was evaluated in freshly spiked stool by testing two-fold dilutions in replicates of 20. Masked samples were tested by a second laboratory to evaluate interlaboratory reproducibility. Short-term (7-day) stability of viral RNA in stool samples was assessed with four different stool storage buffers (phosphate-buffered saline, Cary-Blair medium, Stool Transport and Recovery [STAR] buffer, and DNA/RNA Shield) kept at -80°C, 4°C, and ambient temperature (approximately 21°C). We also tested clinical stool and anal swab specimens from patients who were SARS-CoV-2 positive by nasopharyngeal testing.

FINDINGS:

The lower limit of detection of the assay was found to be 3000 viral RNA copies per g of original stool sample, with 100% detection across 20 replicates assessed at this concentration. Analytical sensitivity was diminished by approximately two times after a single freeze-thaw cycle at -80°C. At 100 times the limit of detection, spiked samples were generally stable in all four stool storage buffers tested for up to 7 days, with maximum changes in mean threshold cycle values observed at -80°C storage in Cary-Blair medium (from 29·4 [SD 0·27] at baseline to 30·8 [0·17] at day 7; p<0·0001), at 4°C storage in DNA/RNA Shield (from 28·5 [0·15] to 29·8 [0·09]; p=0·0019), and at ambient temperature in STAR buffer (from 30·4 [0·24] to 32·4 [0·62]; p=0·0083). 30 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, SARS-CoV-2 RNA was detected using this assay in the stool and anal swab specimens of 11 of 23 individuals known to be positive for SARS-CoV-2.

INTERPRETATION:

This is a sensitive and reproducible assay for detection of SARS-CoV-2 RNA in human stool, with potential uses in faecal microbiota transplantation donor screening, sewage monitoring, and further research into the effects of faecal shedding on the epidemiology of the COVID-19 pandemic.

FUNDING:

National Institute of Allergy and Infectious Diseases, US National Institutes of Health; Center for Biologics Evaluation and Research, US Food and Drug Administration.
Subject(s)

Full text: Available Collection: International databases Database: MEDLINE Main subject: SARS-CoV-2 / COVID-19 Type of study: Diagnostic study / Prognostic study Limits: Humans Language: English Journal: Lancet Microbe Year: 2021 Document Type: Article Affiliation country: S2666-5247(21)00059-8

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Full text: Available Collection: International databases Database: MEDLINE Main subject: SARS-CoV-2 / COVID-19 Type of study: Diagnostic study / Prognostic study Limits: Humans Language: English Journal: Lancet Microbe Year: 2021 Document Type: Article Affiliation country: S2666-5247(21)00059-8