key: cord-1013899-xc3zasd8
authors: Manzoor, S. E.; Zaman, S.; Whalley, C. M.; Inglis, D.; Bosworth, A.; Kidd, M.; Shabir, S.; Quraishi, N.; Green, C. A.; Iqbal, T. H.; Beggs, A. D.
title: Multi-modality detection of SARS-CoV-2 in faecal donor samples for transplantation and in asymptomatic emergency surgical admissions
date: 2021-02-03
journal: nan
DOI: 10.1101/2021.02.02.21250934
sha: dd2258fc534cf1cdb4ca611aed93321b3e5208d0
doc_id: 1013899
cord_uid: xc3zasd8

Introduction Faecal transplantation is an evidence based treatment for Clostridiodes difficile. Patients infected with SARS-CoV-2 have been shown to shed the virus in stool for up to 33 days, well beyond the average clearance time for upper respiratory tract shedding. We carried out an analytical and clinical validation of reverse-transcriptase quantitative (RT-qPCR) as well as LAMP, LamPORE and droplet digital PCR in the detection of SARS-CoV-2 RNA in stool from donated samples for FMT, spiked samples and asymptomatic inpatients in an acute surgical unit. Methods Killed SARS-CoV-2 viral lysate and extracted RNA was spiked into donor stool & FMT and a linear dilution series from 10-1 to 10-5 and tested via RT-qPCR, LAMP, LamPORE and ddPCR against SARS-CoV-2. Patients admitted to the critical care unit with symptomatic SARS-CoV-2 and sequential asymptomatic patients from acute presentation to an acute surgical unit were also tested. Results In a linear dilution series, detection of the lowest dilution series was found to be 8 copies per microlitre of sample. Spiked lysate samples down to 10-2 dilution were detected in FMT samples using RTQPCR, LamPORE and ddPCR and down to 10-1 with LAMP. In symptomatic patients 5/12 had detectable SARS-CoV-2 in stool via RT-qPCR and 6/12 via LamPORE, and in 1/97 asymptomatic patients via RT-qPCR. Conclusions RT-qPCR can be detected in FMT donor samples using RT-qPCR, LamPORE and ddPCR to low levels using validated pathways. As previously demonstrated, nearly half of symptomatic and less than one percent of asymptomatic patients had detectable SARS-CoV-2 in stool.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, which causes coronavirus disease 2019 (COVID-19), first emerged in Wuhan, China in late 2019 (1) and is responsible for nearly 1.5 million deaths worldwide. SARS-CoV-2 is primarily transmitted via respiratory droplets and direct contact routes between asymptomatic and symptomatic individuals. Although faeco-oral transmission has not been documented with this virus (2) , it is one of the main forms of transmission with other similar single-stranded RNA viruses, such as norovirus (3) . Multiple lines of evidence suggest that the gastrointestinal tract may form a reservoir for SARS-CoV-2 with the potential for infection and transmission (4) . The virus enters the host by binding its S1 "spike" glycoprotein to angiotensin-converting enzyme 2 (ACE2) in epithelial tissues which is avidly expressed in the ileum (5) . Furthermore, gastrointestinal symptoms such as diarrhoea are not uncommon in patients infected with SARS-CoV-2 affecting up to 40% patients admitted to hospital. Several studies have identified that over 40% of patients with detectable SARS-CoV-2 RNA from nasopharyngeal swabs, will also have detectable viral RNA in faecal samples (6) .

Moreover, faecal shedding of viral RNA has been shown to persist for up to 33 days after clearance from respiratory samples (6) . It is not known for certain whether SARS-CoV-2 present in faeces represents live and transmissible virus (7) , although early evidence suggests that this is possible in some and there remains uncertainty regarding the role of the gut in COVID-19 pathogenesis, potential for faeco-oral transmission of the virus and future outbreaks of infection in institutions such as hospitals and care homes.

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The copyright holder for this preprint this version posted February 3, 2021. ; https://doi.org/10.1101/2021.02.02.21250934 doi: medRxiv preprint Faecal microbiota transplantation (FMT) involves the transplantation of processed faecal samples (8) from healthy donors to individuals with disease associated with imbalance in the gut microbiome. In recent years FMT has transformed the treatment of patients with Clostridiodes difficile infection (9), especially in those with recurrent or refractory disease. Although FMT donors and their stool donations undergo screening to good manufacturing procedure (GMP) standards for pathogens of potential significance, such as multi-drug resistant Enterobacteriaceae, some risks of this remain (10) . During the current pandemic many stool banks have need to stop providing FMT due to the potential risk of transmission of SARS-CoV-2 from asymptomatic donors and, earlier this year the Federal Drug Administration mandated that only stool donated prior to 1 st December 2019 could be used for FMT.

A recent international consensus paper recommenced direct stool testing for the presence of SARS-CoV-2 would be needed for safe FMT supply in the COVID-19 era 2020 (11, 12) .

In this paper we present the first the results of testing FMT donor stool for the presence of SARS-CoV-2 virus using an internally developed assay based on an existing CE-IVD marked product as well as various novel diagnostics that have been developed in response to the pandemic. We also report using this to test asymptomatic acute patients admitted to hospital with COVID-19 as part of the 'second spike' of the pandemic in 2020 in order to understand the prevalence of faecal SARS-CoV-2 detection in this cohort.

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Stool samples were collected under an existing gut microbial profiling study with ethical approval from Yorkshire & The Humber -Bradford Leeds Research Ethics Committee (16/YH/0100). Samples were obtained from 12 symptomatic COVID-19

patients admitted to hospital who tested positive for SARS-CoV-2 RNA with PCR testing on naso-pharyngeal swabs and a further 97 asymptomatic patients presenting to the Surgical Assessment Unit of Sandwell and West Birmingham NHS Trust from September-November 2020 (a time of increasing prevalence).

Samples used for spiking experiments were taken from pre-existing stocks at the University of Birmingham Microbiome Treatment Centre FMT bank. These were from a batch collected and stored in 2017, prior to the emergence of the SARS-CoV-2 virus. These samples were collected in readiness for use in a trial of FMT in inflammatory bowel disease (STOP-COLITIS 2015-005753-12). For this study, FMT was manufactured over a 10-day donation period and retention and stool study samples were collected and stored in accordance with an MHRA approved GMP process REC and ethical approval (17/EM/0274).

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The copyright holder for this preprint this version posted February 3, 2021. ; https://doi.org/10.1101/2021.02.02.21250934 doi: medRxiv preprint Quantitative PCR was used to analyse both from COVID-19 patients and pre-COVID FMT following "spiking", with varying concentrations of SARS-CoV-2 inactivated lysate (cell culture in Qiagen AL buffer). Purified RNA extracted from an aliquot of the lysate was also analysed using quantitative PCR as an additional positive control to determine whether RNA could be recovered and detected, and to determine the limit of the assay. A negative control of nuclease-free water was incorporated into the assay.

Live SARS-CoV-2 England/2/2020(VE6-T) virus was isolated from infected VE6-T cells, then inactivated in Qiagen lysis buffer AL and frozen at -80 o C. Viral RNA was purified from 500μL of cell lysate using a QIAmp Qiagen kit, then diluted serially to 10 -5 in nuclease free water. Viral cell lysate was also diluted to 10 -5 serially ten-fold in nuclease free water.

This dilution series of both Lysate and RNA were then used to spike aliquots of faecal stool and FMT samples in duplicate. Extraction control samples consisting of 10-fold dilutions of cell lysate and RNA were also prepared.

RNA was extracted from 0.2-0.25g of stool. All stool and FMT 'spiked' samples, along with the extraction control samples were lysed using bead beating PM1 buffer (containing guanidinium thiocyanate, Qiagen) to inactivate the virus prior to extraction under containment level 2+ (CL2+ ) conditions. A RNeasy PowerMicrobiome kit (Qiagen, Hilden) was then used for viral RNA extraction and purification of total RNA from stool with on-column DNase treatment. A subset of samples underwent quality assessment using an Agilent Tapestation 2200 with . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 

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Droplet digital real-time PCR assays using target RNA, primers (450nM), fluorescent-labelled probes (200nM) and Bio-Rad ddPCR supermix were prepared. For use in this protocol, the E gene primers and 6-FAM labelled probe (used in the WHO E gene assay test (14) ) were used with the One step RT-ddPCR Advanced Kit (BioRad Laboratories). Samples were fractioned into 20,000 nanolitre sized water-in-oil droplets using QX200 Droplet generator (BioRad Laboratories). The droplets were transferred in a 96-well plate to a thermal cycler where PCR amplification of the template occurs within each droplet. Following PCR each droplet was analysed individually on the QX200 Droplet Reader (BioRad Laboratories) and the fraction of PCR-positive and PCRnegative droplets in the original sample were counted using QuantaSoft software.

The data were analysed using Poisson distribution statistics to determine the concentration of target DNA in the original sample in absolute copies/ml.

The LamPORE SARS-CoV-2 assay utilises a combination of reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) and Nanopore sequencing technology (Oxford Nanopore Technologies, Oxford), as described previously (16, 17). For the SARS-CoV-2 the target regions N2, E1 and ORF1a genes each span approximately 180bp of the viral genome. Briefly, the viral RNA genome was reverse transcribed into cDNA which was then amplified using strand-displacement polymerase to generate concatenated copies of the original target region. The DNA sequences produced (a 2kb concatamer with sequence for 180bp target region) were then aligned against the SARS-CoV-2 genome using a custom algorithm.

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In order to understand the utility of a rapid assay to detect SARS-CoV-2 in stool, extracted faecal RNA underwent testing using the Optigene RNA-LAMP kit targeted against ORF1ab under manufacturer's instructions for use.

To evaluate the analytical sensitivity of the assay and determine the analytical limit of detection, virus derived from in vitro cell culture was prepared in two different ways.

Lysate was created, by adding equal volumes of cell culture supernatant containing live virus to a Buffer AL (Qiagen), a lysis buffer preparation containing guanidinium thiocyanate designed to both inactivate the virus rendering it safe to handle, and to stabilise RNA to protect it from RNAse mediated degradation.

It is recognised that re-extraction of purified RNA reduces the yield of RNA recovered, and unprotected RNA spiked into a matrix such as stool or FMT preparation is likely to degrade rapidly. To prevent this lysate preparation was also spiked into the stool and FMT preparations. The lysate is less vulnerable as viral RNA is associated with proteins that serve to shield the virus and the addition of guanidinium thiocyanate even at low quantities serves to reduce the activity of RNAse degradative enzymes.

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The copyright holder for this preprint this version posted February 3, 2021. ; Furthermore, the preparation method for extracting the RNA from the stool and FMT specimens requires mechanical homogenisation, which serves to further fragment and degrade RNA, even in samples such as that introduced in the lysate. When fragment analysis was carried out, we found RNA integrity number (RIN) of <2 in all samples, suggesting RNA processed in this way is highly degraded.

As anticipated spiked RNA into faecal samples yielded poor results (Tables 2 & 3) , while the lysate was detectable at 3/5 dilutions. RNA extracted from the FMT and stool where lysate was used as the 'spike in', was then tested using RT-ddPCR to determine the copy number recovered in this experiment. The lowest copy number recoverable from this dilution series was 8 copies of virus genome per reaction (or 0.4 copies per microliter of recovered RNA), equating to a limit of detection for the workflow (extraction and ViaSure RT-qPCR) of 8 copies per microliter of recovered RNA. The approximate limit of detection based on this data can be expressed as copies per gram of faecal preparation and is 204 copies of virus in 1g of faecal preparation using the following calculation:

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Of the 12 symptomatic COVID-19 patients who provided stool samples (Table 1, The potential for faeco-oral asymptomatic transmission of SARS-CoV-2 has significant implications for FMT programmes globally. With FMT services on pause, treatment of patients with recurrent and refractory CDI remain sub-optimal with a likely consequential impact on morbidity, mortality and health care resource.

Availability of a validated SARS-CoV-2 stool assay for donor screening would facilitate safe restart of FMT production and supply. Through our optimised methodology we have shown that using the VIASURE assay we can detect SARS-CoV-2 in stool samples containing more than 200 viral copies per gram. This is comparable to the widely used nasopharyngeal swab testing (14) . As faecal viral shedding persists long after clearance from the upper respiratory tract (for up to 33 days in stool), the direct testing of faecal samples arguably is necessary. It is notable that in the spiking experiments whereas RNA was detectable in lysate spiked samples in neither donor stool nor prepared FMT was directly spiked RNA . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 3, 2021. ;  detectable. This is probably due to RNA destruction during the extraction process whereas in lysate the genetic material is protected within virions. Furthermore, for the first time, in the current work we have been able to demonstrate the efficacy of a Nanopore technology platform in faecal samples. This exciting development opens the possibility of rapid near-subject testing of stool samples with potential for applications beyond the current COVID-19 pandemic.

Our limited patient data demonstrates that about 40% of symptomatic patients admitted with acute COVID-19 test positive for SARS-CoV-2 in faecal samples and this is congruent with other published data (6) . Although the main impetus behind our work is to ensure that we have a suitable stool assay to resume our national FMT service, the finding of SARS-CoV-2 in stool highlights the intriguing potential role of the gastro-intestinal tract in the pathogenesis of the disease. We also have shown that SARS-CoV-2 cannot be detected in the stool of asymptomatic patients presenting with acute surgical emergencies in an area with high incidence of SARS-CoV-2. While true faecal-oral transmission and infectivity of stool samples from COVID-19 patients has yet to be confirmed, there may be sufficient circumstantial evidence to suggest that this is likely to be the case in certain circumstances.

In previous work examining antibody development against SARS-CoV2 about a quarter of hospital staff had diarrhoea and there was an association between this and the likelihood of antibody detection in serum (18). There is accumulating evidence that the gut mounts an active immunoglobulin response to the virus (19) and it is suggested that the gut IgA response may impact on the efficacy of the body's response to vaccination. In vitro work from several laboratories have shown . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 3, 2021. ;  that intestinal epithelium is readily infected suggesting that the intestine is a potential site of SARS-CoV-2 replication (20) with upregulation of viral response genes.

intestinal inflammation based on measurement of faecal calprotectin(21). In contrast, a large retrospective cohort reported an intriguing suggestion that gastro-intestinal involvement may be associated with a more benign outcome of infection (22).

Although these data are subject to question in light of the retrospective study design there was a signal of a comparatively 'anti-inflammatory' peripheral cytokine response in patients with gastrointestinal symptoms.

Interestingly, in our study of asymptomatic patients presenting to an acute surgical unit during the peak of the 2 nd wave of SARS-CoV-2 infection in the UK, we found no positivity either via NP swab or in stool, despite a proportion of them having diarrhoea on admission. A single patient was identified who was previously positive for SARS-CoV-2 who had detectable virus in their stool 25 days after initial positivity.

Together these findings suggest that faecal transmission in the absence of a positive nasopharyngeal swab is unlikely to be an issue, and that faecal transmission is not an important route in SARS-CoV-2 in asymptomatic patients.

The practice of FMT, while revolutionary for the treatment of CDI (9) and showing promise in IBD (23), has recently been stalled as a result of transmission of infective pathogens to patients who came to harm as a result (10) . Recent cases include drug resistant Escherichia coli (Enteropathogenic Escherichia coli and Shigatoxinproducing Escherichia coli) and related to inadequate screening of donated stool samples.

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The copyright holder for this preprint this version posted February 3, 2021. ;  It is therefore imperative that donated FMT stool samples are carefully screened for SARS-CoV-2 and only used once tested negative (12). The Chinese University of Hong Kong has an active FMT program and, similar to our approach, they have adopted a similar approach of careful enhanced donor screening and PCR testing of stool samples in order to perform FMT safely (24).

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The copyright holder for this preprint this version posted February 3, 2021. ; is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 3, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 3, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 3, 2021. 

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Screening faecal microbiota transplant donors for SARS-CoV-2 by molecular testing of stool is the safest way forward

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VIASURE SARS-CoV-2 Real Time PCR Detection Kit