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 Infect...

Summary (Abstract)BackgroundFecal shedding of SARS-CoV-2 has raised concerns about transmission through fecal microbiota transplantation (FMT) procedures. While many tests have been authorized for diagnosis of COVID-19 using respiratory samples, no fully validated stool tests for detection of SARS-CoV-2 are currently available. We sought to adapt and validate an available test specifically for detection of SARS-CoV-2 in human stool.MethodsStool samples were spiked with inactivated SAR-CoV-2 virus for development and validation of the assay. A modified version of the CDC rRT-PCR SARS-CoV-2 test was used for detection of virus. Analytical sensitivity, assay reproducibility, and sample stability under a variety of storage conditions were assessed. We also performed the assay on stool samples collected from known COVID positive individuals.FindingsThe lower limit of detection (LoD) of the assay was found to be 3000 viral RNA copies per gram of original stool sample, with 100% detection across 20 replicates assessed at this concentration. Samples were relatively stable in all buffers tested at both 4°C and ambient temperature, with the exception of storage in STAR buffer at ambient temperature. Assay sensitivity was slightly diminished in low-copy-number samples after a single freeze-thaw cycle at −80°C. Thirty 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, we detected SARS-CoV-2 RNA in the stool of known COVID-19 positive individuals using this method.InterpretationThis is a sensitive, reproducible, and validated assay for detection of SARS-CoV-2 RNA in human stool with potential uses in FMT donor screening, sewage monitoring, and further research into the impact of fecal shedding on the epidemiology of this pandemic.FundingNational Institute for Allergy and Infectious Diseases, NIH. Center for Biologics Evaluation and Research, FDA.Research in ContextEvidence before this studySince the onset of the COVID-19 pandemic, multiple studies have documented shedding of SARS-CoV-2 RNA in feces and considered the potential for fecal-oral transmission of this virus. This potential risk led to the U.S. Food and Drug Administration issuing a safety alert that contained the recommendation that no stool donated after December 1, 2019 be used for manufacture of Fecal Microbiota for Transplantation (FMT) products in the United States until such a time as sufficient screening procedures could be put in place to mitigate this risk.Added value of this studyHere, we report the development and validation of an assay specifically meant for the detection of SARS-CoV-2 RNA in the stool of healthy individuals. While studies have reported detection of viral RNA in stool previously, this is the first publication of a validated assay designed for this purpose.Implications of all the available evidenceThe work presented here provides a validated SARS-CoV-2 stool assay with potential application to FMT donor screening protocols, sewage monitoring protocols, as well as research studies assessing the role of stool shedding and transmission on the epidemiology of COVID-19.

Characterization of live biotherapeutic product (LBP) batches typically includes a measurement of viability, such as colony forming units (CFU). However, strain-specific CFU enumeration assays can be complicated by the presence of multiple organisms in a single product with similar growth requirements. To overcome specific challenges associated with obtaining strain-specific CFU values from multi-strain mixtures, we developed a method combining mass spectrometry-based colony identification with a traditional CFU assay. This method was assessed using defined consortia made from up to eight bacterial strains. Among four replicate batches of an eight-strain mixture, observed values differed from expected values by less than 0.4 log10 CFU among all strains measured (range of differences, -0.318 to + 0.267). The average difference between observed and expected values was + 0.0308 log10 CFU, with 95% limits of agreement from -0.347 to 0.408 (Bland–Altman analysis). To estimate precision, a single batch of eight-strain mixture was assayed in triplicate by three different users, for a total of nine measurements. Pooled standard deviation values ranged from 0.067 to 0.195 log10 CFU for the eight strains measured, and user averages did not differ significantly. Leveraging emerging mass-spectrometry-based colony identification tools, a novel method for simultaneous enumeration and identification of viable bacteria from mixed-strain consortia was developed and tested. This study demonstrates the potential for this approach to generate accurate and consistent measurements of up to eight bacterial strains simultaneously and may provide a flexible platform for future refinements and modifications. Key points • Enumeration of live biotherapeutics is essential for product quality and safety. • Conventional CFU counting may not differentiate between strains in microbial products. • This approach was developed for direct enumeration of mixed bacterial strains simultaneously.

Antimicrobial resistant bacteria are an emerging and prevalent global threat with an urgent need for alternative therapies. Bacteriophage (phage) therapy is a promising approach to address these infections that has gained renewed interest in recent years. Despite this, questions remain regarding the therapeutic use of phages, including the impact that the immune response may have on phage therapy, particularly when this treatment is administered long-term or when reusing a specific phage treatment in a single individual. To investigate this, we developed a mouse model to assess phage treatment using a cocktail of five phages from the Myoviridae and Siphoviridae families that target vancomycin-resistant enterococcus (VRE) gut colonization. Phage cocktail treatment significantly reduced the intestinal bacterial burden of VRE in mice. We characterized innate and adaptive immune responses elicited against the phage cocktail after one and multiple exposures, respectively. While minimal innate responses were observed after phage administration, two courses of phage therapy induced phage-specific neutralizing antibodies and appeared to accelerate phage clearance from tissues. Interestingly, the myophages in our cocktail induced a more robust neutralizing antibody response than the siphophages. Proteins targeted by phage-specific antibodies were also identified from each phage family of the cocktail. Importantly, we show that this anti-phage immunity reduced the effectiveness of the phage cocktail in our murine model, leading to significantly higher fecal bacterial burden following repeat treatment. Collectively, this study shows the immune system has the potential to impede effectiveness of phage therapy and that the phage-specific immune responses can differ significantly between phages. These findings can help inform decisions about inclusion of specific phages in cocktails for future studies.