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)

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

Correction factors: Fecal input, Flow

crAssphage

PMMoV

Flow (MGD)

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

Depth of SNVs at different dilutions

Dayton11/23/20

Taylor Creek11/11/20

Full conc1:21:41:8

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