Validation of Two Commercial Multiplex Real-Time PCR Assays for Detection of SARS-CoV-2 in Stool Donors for Fecal Microbiota Transplantation

Vincenzo Di Pilato,Maria Grazia Cusi,Claudia Gandolfo,Maurizio Sanguinetti,Cosmeri Rizzato,Alberto Antonelli,Gian Maria Rossolini,Mauro Pistello,Roberta Fais,Antonella Lupetti,Fabio Morecchiato,Luca Masucci,Gianluca Quaranta

doi:10.3390/microorganisms10020284

Abstract

Recurrent infection by Clostridioides difficile has recently been treated by fecal microbiota transplantation (FMT). As viable SARS-CoV-2 was recovered from stool of asymptomatic individuals, the FMT procedure could be a potential risk of SARS-CoV-2 transmission, thus underlying the need to reliably detect SARS-CoV-2 in stool. Here, we performed a multicentric study to explore performances of two commercially available assays for detection of SARS-CoV-2 RNA in stool of potential FMT donors. In three hospitals, 180 stool samples were spiked with serial 10-fold dilutions of a SARS-CoV-2 inactivated lysate to evaluate the Seegene Allplex™ SARS-CoV-2 (SC2) and SARS-CoV-2/FluA/FluB/RSV (SC2FABR) Assays for the detection of viral RNA in stool of FMT donors. The results revealed that both assays detected down to 2 TCID50/mL with comparable limit of detection values, SC2 showing more consistent target positivity rate than SC2FABR. Beyond high amplification efficiency, correlation between CT values and log concentrations of inactivated viral lysates showed R2 values ranging from 0.88 to 0.90 and from 0.87 to 0.91 for the SC2 and SC2FABR assay, respectively. The present results demonstrate that both methods are highly reproducible, sensitive, and accurate for SARS-CoV-2 RNA detection in stool, suggesting a potential use in FMT-donor screening.

Highlights

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease-2019 (COVID-19), has caused a pandemic affecting the world population at a global scale and remains a major public health threat [1].Even though people infected by SARS-CoV-2 exhibited a wide range of symptoms, COVID-19 is typically considered a respiratory disease, with primary manifestations including cough, sore throat, congestion, anosmia, and dyspnea
SARS-CoV-2-negative subjects were collected at Center 2 and pooled and diluted to a concentration of 50 mg/mL and 20 mg/mL
In order to evaluate a possible inhibitory effect exerted by an excessive stool matrix, which can negatively impact on rRT-PCR sensitivity and accurate pathogen detection, preliminary testing was performed using the SC2FABR assay and spiked dilution of SARS-CoV-2 viral-inactivated lysates into stool samples with a concentration of 50 mg/mL

Summary

Introduction

Even though people infected by SARS-CoV-2 exhibited a wide range of symptoms, COVID-19 is typically considered a respiratory disease, with primary manifestations including cough, sore throat, congestion, anosmia, and dyspnea. Even though the transmission of SARS-CoV-2 typically occurs through the respiratory tract, the recovery of viable viruses from stool of asymptomatic individuals and patients have been reported (i.e., sometimes well after their respiratory infection has cleared), as well as abundant gastrointestinal glandular cell ACE-2 expression (the target receptor for SARS-CoV-2) and active replication within enterocytes [2,5,6,7,8]. The detection of both viable SARS-CoV-2 and viral RNA in wastewater systems further underscore the potential role of the gastrointestinal tract in viral replication and shedding [9]. A fecal–oral transmission route for SARS-CoV-2 remains to be definitively demonstrated

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