Overview of the Ohio Wastewater 

Monitoring Network

Rebecca Fugitt, MS, RS

Ohio Department of Health

Nichole Brinkman, PhD

US EPA Office of Research and Development

The views expressed in this presentation are those of the author(s) and do not necessarily represent the views or policies of the U.S. Environmental Protection 

Agency. Any mention of trade names or commercial products does not constitute EPA endorsement or recommendation for use.
Ohio Wastewater Monitoring Network 

(OWMN)

• Goal 

– Monitor trend of SARS-CoV-2 

RNA at specific locations (vs 

compare sites)

– Serve as early indicator of 

COVID-19 community spread

– Prioritize resources

• Statewide network 

– Started July 2020

– leveraged expertise and 

resources

• Ohio Universities 

• US EPA-ORD

• 67 locations twice a week

• Sequencing to screen for possible 

presence of SARS-CoV-2 variants

– Variants of Concern (VOC)

– Variants of Interest (VOI)

https://coronavirus.ohio.gov/wps/portal/gov/covid-19/dashboards/otherresources/wastewater
Public Health Application

• To serve as an early warning of infection in communities and an understanding of case 

trends

• The focus is on trends or significant changes in the number of viral gene copies 

detected.

• Currently action is taken when at least 3 samples show a sustained increase of at least 

10-fold (1 log)

• State actions when increases are observed:

– Notify the local health district and utility

– Provide information on how to interpret the data and link to message toolkit

– Notify the state pandemic testing team for linkages to establish pop-up testing sites

– Provide case data by sewershed to local health district (this extraction to be provided soon)

• Participation in the CDC National Wastewater Surveillance System

• Toolkit link: https://coronavirus.ohio.gov/wps/portal/gov/covid-19/healthcareproviders-and-local-health-districts/for-local-health-districts-and-governments
Accomplishments

• Built statewide network that represents wastewater flow from nearly 5 

million residents

• Almost 1 year of weekly data collected

• All data is publicly available on the Ohio coronavirus dashboard and is 

updated daily

• Provided nearly 500 warnings to local health communities

• Expanded to include genomic sequencing of wastewater to pair with clinical 

data and inform public health decisions
Lab Method Logistics

• Twice weekly samples 

• Report data within 2 days of 

sample receipt

• No prescribed method; labs 

decide

• Supply chain shortages

• Low target concentration

• Sample hold time: 4°C - 72 

hours

• No sample pasteurization

Sample 

Concentration

Nucleic Acid 

Extraction

RT-qPCR/ 

RT-ddPCR

Genome/Genetic 

Sequencing

Quantitative 

Analysis

Qualitative 

Analysis
Quality Assurance/Quality Control

• Matrix Spike to assess method recovery efficiency

– Coronavirus recommended: human (OC43), murine (MHV), bovine (BCoV)

• Inhibition control to monitor for PCR amplification inhibition

• RT-qPCR standards/RT-ddPCR positive control

• Human fraction measurements

– crAssphage

– Pepper mild mottle virus

• Monthly Interlaboratory Method Validation

– Pick a site with sufficient concentration of SARS-CoV-2

– Each lab gets 0.5 L

– Each lab processes and analyzes sample

– Report data to Project Coordinator
OH Network Lab Methods 

LAB LOD 

(copies/L) Processing Method Nucleic Acid Extraction

Quantitative 

Analysis 

Method

RT-PCR Standard 

Curve/ Control Inhibition Control Matrix Spike Fecal Indicator

A 850 Centrifugation, 

filtration

Qiagen RNeasy PowerWater 

Kit RT-qPCR DNA plasmid Dilution MHV crAssphage

B 135 Centrifugation, 

filtration Qiagen Allprep DNA/RNA Kit RT-qPCR DNA plasmid Dilution BCoV crAssphage

C 133

Tween, solids removal 

hollow fiber 

ultrafiltration 

(InnovaPrep)

Qiagen PowerMicrobiome 

Kit RT-ddPCR DNA plasmid Luciferase Control RNA OC43 crAssphage

D 7,440 Filtration Trizol, garnet bead beating, 

alcohol precipitation RT-qPCR DNA plasmid Luciferase Control RNA BCoV PMMoV

E 500 Centrifugation, 

filtration

Trizol and RNA purification 

kit RT-qPCR Synthetic RNA Luciferase Control RNA BCoV crAssphage

F 3,000

Promega, add 

protease, supernatant 

through GFA/silica 

column

Promega Wastewater Large 

Volume TNA Capture Kit RT-qPCR DNA plasmid Promega probe OC43 PMMoV

G 231 Centrifugation, 

filtration

Qiagen RNeasy PowerWater 

Kit/Trizol-chloroform RT-ddPCR SARS-CoV-2 genomic RNA

Luciferase Control 

RNA OC43 crAssphage

H 891 Acidification, 

Filtration, extract filter

Qiagen Allprep PowerViral 

DNA/RNA Kit RT-qPCR Synthetic RNA Mouse lung RNA OC43 PMMoV
OH Network Lab Methods 

LAB LOD 

(copies/L) Processing Method Nucleic Acid Extraction

Quantitative 

Analysis 

Method

RT-PCR Standard 

Curve/ Control Inhibition Control Matrix Spike Fecal Indicator

A 850 Centrifugation, 

filtration

Qiagen RNeasy PowerWater 

Kit RT-qPCR DNA plasmid Dilution MHV crAssphage

B 135 Centrifugation, 

filtration Qiagen Allprep DNA/RNA Kit RT-qPCR DNA plasmid Dilution BCoV crAssphage

C 133

Tween, solids removal 

hollow fiber 

ultrafiltration 

(InnovaPrep)

Qiagen PowerMicrobiome 

Kit RT-ddPCR DNA plasmid Luciferase Control RNA OC43 crAssphage

D 7,440 Filtration Trizol, garnet bead beating, 

alcohol precipitation RT-qPCR DNA plasmid Luciferase Control RNA BCoV PMMoV

E 500 Centrifugation, 

filtration

Trizol and RNA purification 

kit RT-qPCR Synthetic RNA Luciferase Control RNA BCoV crAssphage

F 3,000

Promega, add 

protease, supernatant 

through GFA/silica 

column

Promega Wastewater Large 

Volume TNA Capture Kit RT-qPCR DNA plasmid Promega probe OC43 PMMoV

G 231 Centrifugation, 

filtration

Qiagen RNeasy PowerWater 

Kit/Trizol-chloroform RT-ddPCR SARS-CoV-2 genomic RNA

Luciferase Control 

RNA OC43 crAssphage

H 891 Acidification, 

Filtration, extract filter

Qiagen Allprep PowerViral 

DNA/RNA Kit RT-qPCR Synthetic RNA Mouse lung RNA OC43 PMMoV

5 8 2 3 4 3 2

Number of different procedures employed at the various processing/analysis steps
Validation Results (April 2021)

100

1000

10000

100000

1000000

A B C D E F G H

N2 RNA Copies/L

Lab

N2 N2-Adjusted

N2 RNA concentrations from 8 labs span >2 orders of magnitude
RT-ddPCR Mutation Assays

0%

20%

40%

60%

80%

100%

0

2000

4000

6000

8000

10000

12000

4/4/2021 4/11/2021 4/18/2021 4/25/2021 5/2/2021

Percent of RNA Molecules 

N2 RNA Molecules/L

Date

N501Y

N501 501Y N2

0%

20%

40%

60%

80%

100%

0

2000

4000

6000

8000

10000

12000

4/4/2021 4/11/2021 4/18/2021 4/25/2021 5/2/2021

Percent of RNA Molecules 

N2 RNA Molecules/L

Date

del 69-70

69-70WT 69-70del N2

https://outbreak.info

RNA sequences with deletions of nucleotides that result 

in absence of spike aa 69-70 increases over time

RNA sequences with nucleotides that change spike aa 

501 increases over time
Genome/Genetic Sequencing

• Pooled sample

– Cannot assemble a genome

– Focus on mutations that 

cause amino acid 

substitutions, signatures of 

VOC/VOI

• Genome/Genetic Sequencing

– 3 labs, different methods

– Tiled amplicon approach

– Short read seq via Illumina

– Short term - spike amino 

acid changes for CDC’s 

VOC/VOI

– Report (for each site)

• Read depth

• Number of alternative 

alleles

4/5/21 4/12/21 4/19/21 4/19/21 4/26/21 4/26/21

Sample

Spike Substitution

Frequency of Substitution
OH Network Insights

• How do current practices (eg. methods, protocols, technologies, best practices, etc.) successfully 

contribute toward comparable, high quality data/results/decisions?

– Using a consistent method, trends of SARS-CoV-2 RNA in a sewershed can be evaluated

– Implementation of Quality Control parameters allow for confidence in lab measurements

– Frequent communication/regular meetings facilitate interlab discussion and troubleshooting

• How do current practices compromise efficiency and reduce confidence in data/results/decisions?

– Too many labs/methods result in measurement variation

– Varied experience leads to measurement variation

– Supply shortages lead to method changes

• What is needed to increase comparability and confidence in data and results?

– Standardized methods/procedures

– Standardized quality control samples/reagents

– Statistical models to quantify uncertainty

• What types of standards could potentially help to fill these needs?

– Matrix Spike

– Extraction controls

– RT-qPCR standards 

– RT-ddPCR controls

– Inhibition controls

– Sequencing controls
Research Team and Partners

EPA/ORD

Maitreyi Nagarkar

Chloe Hart

Scott Keely

Emily Wheaton

Michael Jahne

Eunice Varughese

Jay Garland

Brian Morris

Ana Braam

Barry Wiechman

Sara Okum

Utilities

Metropolitan Sewer District of 

Greater Cincinnati

Bruce Smith

City of Dayton

Chris Clark, Walter Schroder

City of Marion

Brittany Bauer

City of Portsmouth

Tommy Stewart

Montgomery County

Jim Davis

City of Hamilton

Mark Smith

City of Springfield

Jeff Yinger

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

University Labs

Ohio State University

University of Toledo

Kent State University

University of Akron

Bowling Green State University

Commercial Lab

LuminUltra