long-term quantitative monitoring and preliminary
TRANSCRIPT
Long-term quantitative monitoring and preliminary
sequencing of SARS-CoV-2 in wastewater in Cincinnati, OHMaitreyi Nagarkar, Nichole Brinkman, Scott Keely, Michael
Jahne, Jay Garland
Research Team & PartnersOhio Monitoring Network:
Hamilton County Public Health Department: Chris Griffith
Ohio Water Resources Center: Zuzana Bohrerova
Ohio Department of Health: Rebecca Fugitt
Ohio EPA: Brian Hall, Tiffani Kavalec
Ohio State University
University of Toledo
Kent State University
University of Akron
Case Western University
US EPA:Nichole Brinkman
Chloe Hart
Emily Wheaton
Scott Keely
Michael Jahne
Eunice Varughese
Jay Garland
Brian Morris
Ryan Kauchak
Ana Braam
Sara Okum
Barry Wiechman
Jacob Botkins
Utilities:
Metropolitan Sewer District of Greater Cincinnati: Bruce Smith
City of Dayton: Chris Clark, Walter Schroder
City of Marion: Steve Morris
City of Portsmouth: Tommy Stewart
Montgomery County: Jim Davis
City of Hamilton: Mark Smith
City of Springfield: Jeff Yinger
Wastewater Monitoring of SARS-CoV-2• SARS-CoV-2 is shed in feces of some patients, for
variable amounts of time
• Viral RNA can persist for at least a few days (longer than infectious virus)
• Wastewater surveillance is being used worldwide at different scales
• Wastewater sequencing is now being used to detect mutations associated with variants
Chen et al (2020), Ling et al, Med. J. Chin. (2020); Bivins et al, ES&T Letters 2020
Project Objectives• Consistent, high-frequency (1-2x/week) monitoring of SARS-CoV-2 RNA in wastewater at >12 sites
in Southwest Ohio (in conjunction with a broader statewide network)• Report data weekly; the state uses it for rapid public health response
• Optimizing methods for sample processing, nucleic acid extraction (maximum recovery, low inhibition), and sequencing
• Inter-laboratory comparisons across the Ohio network
• Modeling the relationship between RNA detected in wastewater and cases within the sewershed including assessment of normalization factors
• Better characterizing how sewershed size, sewer type, composition of wastewater, and other factors impact measurements
• Sequencing weekly samples to track mutations / variants
Sample Processing Methods
24-hr composite sample, 225 ml
Centrifuge3000 x g, 15 min
Membrane filtration, 0.45µm
Supernatant
OC43 spike
FilterPellet
0.2 ml “Direct” sample
Ultrafiltration
Retentate
DNA & RNA Extraction (Trizol-Chloroform or Kit)
QuantificationRT-ddPCR: SARS-CoV-2 (N1, N2), OC43, PMMoV, Inhibition control;
ddPCR: crAssphage, HF183
• Turnaround time ~ 3 days; reported to OH network
• Eliminated UF
• No significant difference found when wastewater sample held at 4oC up to 4 days
• Up to fivefold reduction observed when there was a freeze-thaw cycle (-70oC)
+
DNA & RNA Extraction (Trizol-Chloroform or Kit)
QuantificationRT-ddPCR: SARS-CoV-2 (N1, N2), OC43, PMMoV, Inhibition control;
ddPCR: crAssphage, HF183
• Turnaround time ~ 3 days; reported to OH network
• Eliminated UF
• No significant difference found when wastewater sample held at 4oC up to 4 days
• Up to fivefold reduction observed when there was a freeze-thaw cycle (-70oC)
Sequencing
Viral DNA and RNA were primarily solids-associated
crAssphage
N1 & N2
PMMoV
• 90% of DNA/RNA found in pellet or filter fraction, thus discontinued use of UF fraction
Co
pie
s/ex
trac
t
Site SiteSite
Ohio Wastewater Monitoring Network: Dashboard
https://coronavirus.ohio.gov/wps/portal/gov/covid-19/dashboards/other-resources/wastewater
Increase >100%
Increase 50- 100%
Steady
Decrease <= -50%
Individual site example: Mill Creek
N2 copies/L Total cases (7 day moving
avg)
Mill Creek
Aug 20 Apr 21
Million gene copies/day
https://coronavirus.ohio.gov/wps/portal/gov/covid-19/dashboards/other-resources/wastewater
Objectives of OH Network•Serve as an early warning of infection in communities or congregate settings.
•Provide information that can help local communities more quickly intervene with protective measures to slow disease spread.
•Help communities measure the effectiveness of such interventions (quarantine/face coverings/business limitations).
•Develop methodologies/predictive models to translate viral loads detected for comparison with other data, such as rates or percentage of infection in communities.
•Where possible, compare results to previously collected data on prevalence in specific communities to better understand factors affecting disease spread.
•Determine impacts on disproportionately affected communities or communities where risk of infection is greater.
From https://coronavirus.ohio.gov/wps/portal/gov/covid-19/dashboards/other-resources/wastewater
Sampling at different spatial resolutions
Mill Creek:180 MGD5% Industrial40% Combined
Taylor Creek:3 MGD0% Industrial0% Combined
Mill CreekLick Run
Taylor Creek
Sequencing: SARS-CoV-2 gRNA pilot• Amplicon sequencing with Swift
Biosciences kit (345 primers across the genome)
• Lower concentration of cDNA in gRNA + WW (2 Ct difference)
• The expected 3 SNVs (Single Nucleotide Variants) between Washington State isolate and Wuhan isolate were detected in both (none against WA reference sequence)
• >98% genome coverage
gRNA
gRNA in WW
2000X
2000X
Sequencing: Preliminary results from current wastewater samples• Tested pellets and filters separately
• Pellets had issues with cDNA synthesis; N2 undetected by qPCR in all but one sample
• Pellets yielded very few sequence reads (2-500 in run where most samples had >100,000 reads
• Filter fractions from two sites had 98 - 99.78% genome coverage across a dilution series where the minimum concentration was ~12355 gene copies/L
Dayton
Taylor Creek
Mismatches between amplicon primers and OH clinical genome sequences over time
Percent matching
99%
97%
95%
93%
3/30/20 6/28/20 10/26/20 2/28/21
Figure: S. Keely
Future Directions• Sampling with Ohio Wastewater Monitoring
Network continued through at least June 2021• How does vaccine deployment affect
wastewater measurements?
• Developing Bayesian models that include fecal indicator abundances, flow, rainfall, sample recovery, time lags, etc. to better understand the relationship between clinical cases in a sewershed and gene copies/L of SARS-CoV-2 RNA
• Continue sequencing to track mutations associated with existing and new variants