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. 2021 Jun 9;12(6):e00363.
doi: 10.14309/ctg.0000000000000363.

Validation of High-Sensitivity Severe Acute Respiratory Syndrome Coronavirus 2 Testing for Stool-Toward the New Normal for Fecal Microbiota Transplantation

Ahmed Babiker  1   2 , Jessica M Ingersoll  2 , Max W Adelman  1 , Andrew S Webster  1 , Kari J Broder  2 , Victoria Stittleburg  1 , Jesse J Waggoner  1 , Colleen S Kraft  1   2 , Michael H Woodworth  1
Affiliations

Validation of High-Sensitivity Severe Acute Respiratory Syndrome Coronavirus 2 Testing for Stool-Toward the New Normal for Fecal Microbiota Transplantation

Ahmed Babiker et al. Clin Transl Gastroenterol. .

Abstract

Introduction: Mounting evidence demonstrates potential for fecal-oral transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The US Food and Drug Administration now requires SARS-CoV-2 testing of potential feces donors before the use of stool manufactured for fecal microbiota transplantation. We sought to develop and validate a high-sensitivity SARS-CoV-2 reverse transcriptase polymerase chain reaction (RT-PCR) procedure for testing stool specimens.

Methods: A modified extraction method was used with an RT-PCR assay adapted from the Centers for Disease Control and Prevention PCR protocol for respiratory specimens. Contrived specimens were created using pre-COVID-19 banked stool specimens and spiking in known concentrations of SARS-CoV-2-specific nucleic acid. The highest transcript concentration at which 2/2 or 1/2 SARS-CoV-2 targets were detected in 9/10 replicates was defined as the dual-target limit and single-target limit of detection, respectively. The clinical performance of the assay was evaluated with stool samples collected from 17 nasopharyngeal swab RT-PCR-positive patients and 14 nasopharyngeal RT-PCR-negative patients.

Results: The dual-target and single-target limit of detection were 56 copies/μL and 3 copies/μL, respectively. SARS-CoV-2 was detected at concentrations as low as 0.6 copies/μL. Clinical stool samples from known COVID-19-positive patients demonstrated the detection of SARS-CoV-2 in stool up to 29 days from symptom onset with a high agreement with nasopharyngeal swab tests (kappa statistic of 0.95, P value < 0.001).

Discussion: The described RT-PCR test is a sensitive and flexible approach for the detection of SARS-CoV-2 in stool specimens. We propose an integrated screening approach that incorporates this stool test to support continuation of fecal microbiota transplantation programs.

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Conflict of interest statement

Guarantor of the article: Michael H. Woodworth, MD, MSc.

Specific author contributions: M.H.W. conceived the study. A.B. and M.H.W. designed the study. A.S.W., K.J.B., and M.W.A. collected the samples. J.M.I. and V.S. performed the experiments and J.J.W. and C.S.K. provided laboratory supervision. A.B. and M.H.W. analyzed the data. A.B., C.S.K., J.M.I., and M.H.W. interpreted the data. A.B. and M.H.W. drafted the manuscript. All authors reviewed and critically revised the manuscript for important intellectual content.

Financial support: This work was supported in part by the Center for AIDS Research (P30 AI050409) and the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Award Number K23AI144036. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Potential competing interests: None to report.

Figures

Figure 1.
Figure 1.
Paired plot of stool CT and NP swab CT values for NP RT-PCR-positive patients (n = 17). CT, cycle threshold; NP, nasopharyngeal; RT-PCR, reverse transcriptase polymerase chain reaction.
Figure 2.
Figure 2.
Proposed SARS-CoV-2 assessments for potential feces donors before the release of material for FMT. Outlining initial screening questionnaire for exposures and symptoms for COVID-19 (a), followed by initial nasopharyngeal and stool testing of SARS-CoV-2 (b), followed by banking and storage of aliquots for SARS-CoV-2 testing of each sample for less than 60 days (c), followed by release of SARS-CoV-2 nasopharyngeal, and stool testing 2–4 weeks after last stool donation. Figure created with Biorender.com. COVID-19, Coronavirus 2019; FMT, fecal microbiota transplant; NP, Nasopharyngeal; RT-PCR, Reverse transcriptase polymerase chain reaction; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.
See this image and copyright information in PMC

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