Screening FMT donors during the COVID-19 pandemic: a protocol for stool SARS-CoV-2 viral quantification

We read with interest the Correspondence by Christopher Green and colleagues¹ suggesting the need for a molecular test to screen faecal microbiota transplant (FMT) donors for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to prevent the potential risk of transmission. On March 12, 2020, the US Food and Drug Administration (FDA) issued safety alerts because of a death caused by transmission of drug-resistant Escherichia coli bacteraemia via FMT.² With more than 1 million people infected by SARS-CoV-2, screening policies for FMT donors should be stringent and scientifically validated. The presence of SARS-CoV-2 (including live virus) in stool of asymptomatic individuals implies that coronavirus disease 2019 (COVID-19) might be transmitted via the faecal route.3, 4 Development of stool tests has been slow, since real-time RT-PCR of respiratory samples is typically used to confirm the diagnosis of COVID-19.

At the time of writing, the FDA recommends that only FMT products generated from stool donated before Dec 1, 2019, should be used until proper testing and screening protocols are available.⁵ As described by Green and colleagues,¹ the University of Birmingham Microbiota Treatment Centre (Birmingham, UK) is not actively processing new donors until a validated SARS-CoV-2 stool test is available. The FMT centre at the Chinese University of Hong Kong (Hong Kong) is one of the largest providers of FMT in Asia and is the sole provider of FMT to the Health Authority of Hong Kong. Although the first case of COVID-19 was reported in Hong Kong on Jan 22, 2020, we quarantined all donor material donated since Nov 1, 2019, from use. We developed a screening protocol that combines a questionnaire to identify donors who might be at risk of SARS-CoV-2 infection with an RT-PCR assay for detecting SARS-CoV-2 in donor stool. The assay (panel ) allows SARS-CoV-2 viral quantification with a 3 h turnaround. We validated the assay in 81 stool samples from 21 confirmed SARS-CoV-2 cases and 114 stool samples from 114 asymptomatic non-infected individuals who had returned from high-risk areas. As per the diagnostic protocol of our local health authority, all COVID-19 cases had been confirmed by two RT-PCR tests targeting different regions of the RdRp gene in respiratory specimens. All 21 confirmed cases had positive stool tests (median two stool samples positive for SARS-CoV-2 per patient; viral load 2·9–7·1 log₁₀ copies per mL). No stool samples from the 114 asymptomatic individuals tested positive for SARS-CoV-2.

Panel. Protocol for SARS-CoV-2 viral quantification in stool samples.

Stool collection and viral DNA extraction

• •Collect stool in sterile plain bottles
• •Suspend 0·1 g stool in 1 mL viral transport medium (in 1:10 dilution)
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    Centrifuge at 4000 g for 20 min
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    140 μL aliquot of filtrate for following work
• •Extract viral RNA using spin column-based extraction method
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    Kit example: QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany), PureLink Viral RNA/DNA Mini Kit (ThermoFisher Scientific, Waltham, MA, USA)

SARS-CoV-2 viral load quantification

• •SARS-CoV-2 RNA quantified using RT-quantitative PCR with following settings:
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    Primer-probe set N1
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      2019-nCoV_N1-F: 5ʹ-GAC CCC AAA ATC AGC GAA AT-3ʹ
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      2019-nCoV_N1-R: 5ʹ-TCT GGT TAC TGC CAG TTG AAT CTG-3ʹ
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      2019-nCoV_N1-P: 5ʹ-FAM-ACC CCG CAT TAC GTT TGG TGGACC-BHQ1-3ʹ
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    Cycling conditions
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      One cycle: 25°C for 2 min, 50°C for 15 min, 95°C for 2 min
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      45 cycles: 95°C for 15 s and 55°C for 30 s

SARS-CoV-2 RT quantitative PCR data analysis

• •Samples are considered negative if cycle threshold values exceeded 39·9 cycles
• •Detection limit of real-time RT-PCR is 347 copies per mL

SARS-CoV-2= severe acute respiratory syndrome coronavirus 2.

We found that a single negative test, as in the current practice for screening other pathogens, is insufficient to exclude the presence of SARS-CoV-2 in stool. We recommend testing donors at multiple timepoints during the donation period, since the level of viral RNA present in stool can fluctuate around the margin of laboratory detection. Testing stool for SARS-CoV-2 should be done in appropriately equipped laboratories by trained staff; specimen handling would require biosafety level 2 laboratories or equivalent facilities.

Only with enhanced donor screening and validated stool tests for SARS-CoV-2 can we ensure safe and effective delivery of FMT to critically ill patients with recurrent and refractory Clostridioides difficile infection.