. 2023 Aug 24;8(4):e0013223.

doi: 10.1128/msphere.00132-23. Epub 2023 Jun 20.

Longitudinal and quantitative fecal shedding dynamics of SARS-CoV-2, pepper mild mottle virus, and crAssphage

Peter J Arts¹, J Daniel Kelly^{2

3

4

5}, Claire M Midgley⁶, Khamal Anglin^{2

3}, Scott Lu^{2

3}, Glen R Abedi⁶, Raul Andino⁷, Kevin M Bakker⁸, Bryon Banman¹, Alexandria B Boehm⁹, Melissa Briggs-Hagen⁶, Andrew F Brouwer⁸, Michelle C Davidson¹⁰, Marisa C Eisenberg⁸, Miguel Garcia-Knight⁷, Sterling Knight¹, Michael J Peluso¹¹, Jesus Pineda-Ramirez^{2

3}, Ruth Diaz Sanchez^{2

3}, Sharon Saydah⁶, Michel Tassetto⁷, Jeffrey N Martin², Krista R Wigginton¹

Affiliations

¹ Department of Civil and Environmental Engineering, University of Michigan , Ann Arbor, Michigan, USA.
² Department of Epidemiology and Biostatistics, University of California , San Francisco, California, USA.
³ Institute for Global Health Sciences, University of California , San Francisco, California, USA.
⁴ Division of Hospital Medicine, UCSF , San Francisco, California, USA.
⁵ F.I. Proctor Foundation, University of California , San Francisco, California, USA.
⁶ National Center for Immunizations and Respiratory Diseases, Centers for Disease Control and Prevention , Atlanta, Georgia, USA.
⁷ Department of Microbiology and Immunology, UCSF , San Francisco, California, USA.
⁸ Department of Epidemiology, University of Michigan , Ann Arbor, Michigan, USA.
⁹ Department of Civil & Environmental Engineering, Stanford University , Stanford, California, USA.
¹⁰ School of Medicine, University of California , San Francisco, California, USA.
¹¹ Division of HIV, Infectious Disease, and Global Medicine, UCSF , San Francisco, California, USA.

PMID: 37338211
PMCID: PMC10506459
DOI: 10.1128/msphere.00132-23

Longitudinal and quantitative fecal shedding dynamics of SARS-CoV-2, pepper mild mottle virus, and crAssphage

Peter J Arts et al. mSphere. 2023.

. 2023 Aug 24;8(4):e0013223.

doi: 10.1128/msphere.00132-23. Epub 2023 Jun 20.

Authors

Affiliations

¹ Department of Civil and Environmental Engineering, University of Michigan , Ann Arbor, Michigan, USA.
² Department of Epidemiology and Biostatistics, University of California , San Francisco, California, USA.
³ Institute for Global Health Sciences, University of California , San Francisco, California, USA.
⁴ Division of Hospital Medicine, UCSF , San Francisco, California, USA.
⁵ F.I. Proctor Foundation, University of California , San Francisco, California, USA.
⁶ National Center for Immunizations and Respiratory Diseases, Centers for Disease Control and Prevention , Atlanta, Georgia, USA.
⁷ Department of Microbiology and Immunology, UCSF , San Francisco, California, USA.
⁸ Department of Epidemiology, University of Michigan , Ann Arbor, Michigan, USA.
⁹ Department of Civil & Environmental Engineering, Stanford University , Stanford, California, USA.
¹⁰ School of Medicine, University of California , San Francisco, California, USA.
¹¹ Division of HIV, Infectious Disease, and Global Medicine, UCSF , San Francisco, California, USA.

PMID: 37338211
PMCID: PMC10506459
DOI: 10.1128/msphere.00132-23

Abstract

Wastewater-based epidemiology (WBE) emerged during the coronavirus disease 2019 (COVID-19) pandemic as a scalable and broadly applicable method for community-level monitoring of infectious disease burden. The lack of high-resolution fecal shedding data for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) limits our ability to link WBE measurements to disease burden. In this study, we present longitudinal, quantitative fecal shedding data for SARS-CoV-2 RNA, as well as for the commonly used fecal indicators pepper mild mottle virus (PMMoV) RNA and crAss-like phage (crAssphage) DNA. The shedding trajectories from 48 SARS-CoV-2-infected individuals suggest a highly individualized, dynamic course of SARS-CoV-2 RNA fecal shedding. Of the individuals that provided at least three stool samples spanning more than 14 days, 77% had one or more samples that tested positive for SARS-CoV-2 RNA. We detected PMMoV RNA in at least one sample from all individuals and in 96% (352/367) of samples overall. CrAssphage DNA was detected in at least one sample from 80% (38/48) of individuals and was detected in 48% (179/371) of all samples. The geometric mean concentrations of PMMoV and crAssphage in stool across all individuals were 8.7 × 10⁴ and 1.4 × 10⁴ gene copies/milligram-dry weight, respectively, and crAssphage shedding was more consistent for individuals than PMMoV shedding. These results provide us with a missing link needed to connect laboratory WBE results with mechanistic models, and this will aid in more accurate estimates of COVID-19 burden in sewersheds. Additionally, the PMMoV and crAssphage data are critical for evaluating their utility as fecal strength normalizing measures and for source-tracking applications. IMPORTANCE This research represents a critical step in the advancement of wastewater monitoring for public health. To date, mechanistic materials balance modeling of wastewater-based epidemiology has relied on SARS-CoV-2 fecal shedding estimates from small-scale clinical reports or meta-analyses of research using a wide range of analytical methodologies. Additionally, previous SARS-CoV-2 fecal shedding data have not contained sufficient methodological information for building accurate materials balance models. Like SARS-CoV-2, fecal shedding of PMMoV and crAssphage has been understudied to date. The data presented here provide externally valid and longitudinal fecal shedding data for SARS-CoV-2, PMMoV, and crAssphage which can be directly applied to WBE models and ultimately increase the utility of WBE.

Keywords: PMMoV; SARS-CoV-2; crAssphage; fecal shedding; stool.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

**Fig 1**
SARS-CoV-2 RNA fecal shedding data. (a) Longitudinal plot of all SARS-CoV-2 N gene measurements. Empty symbols represent sample measurements below the LOB. (b) Boxplot and individual data points summarizing the peak fecal shedding magnitude for each individual with at least one sample above the LOB.

**Fig 2**
(a) The proportion of stool samples on each day after symptom onset where SARS-CoV-2 N gene was measured above the LOB. (b) A histogram showing the number of samples measured on each day after symptom onset.

**Fig 3**
Above LOB or LOD shedding measurements and histogram of PMMoV (top) and crAssphage (bottom) for all samples in this study, as measured by qPCR (crAssphage) or RT-ddPCR (PMMoV). In the box plots (left), each box summarizes all measurements available for an individual with the bar indicating the median shedding value. The histograms (right), summarize the distribution of shedding magnitude for the entire set of above detection limit measurements.

See this image and copyright information in PMC

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Longitudinal and quantitative fecal shedding dynamics of SARS-CoV-2, pepper mild mottle virus, and crAssphage

Affiliations

Longitudinal and quantitative fecal shedding dynamics of SARS-CoV-2, pepper mild mottle virus, and crAssphage

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

References

MeSH terms

Substances

Supplementary concepts

Grants and funding

LinkOut - more resources

Full Text Sources

Medical

Miscellaneous