Wastewater Methodological Notes

Wastewater data are normalized for fluctuations in human fecal content contribution. Two human fecal indicators are used: crAssphage and PMMoV. The specific fecal indicator measured for the sample varies based on the lab analyzing the sample. The ratio of SARS CoV-2 RNA to human fecal indicator is a unitless ratio indicating the intensity of coronavirus gene copies detected in the wastewater. Higher values indicate higher viral load and potentially higher transmission in the community that the sample is from. Trend plots use natural log-adjusted values for this intensity metric to reduce the influence of large spikes or declines in detection due to things like high intensity rain events that can make accurate detection difficult. Single samples show a snapshot in time for the level of coronavirus detected in wastewater, while several data points over time provide indicators of viral trends in a community. This dashboard provides two-week trend metrics to display if viral detection is increasing or decreasing by comparing samples taken over a fourteen-day window.

For some counties and jurisdictions, the natural log of gene copies is used to determine the two-week trend instead of intensity. The natural log of gene copies was found to correlate best with lab-confirmed case data and is suitable for sampling sites that do not collect fecal indicator data. Current sites using this method are in Genesee, Orleans, Allegany, and Suffolk Counties as well as New York City.

Comparing these trends to the observed case data is useful for confirming trends seen among clinical case data and provides additional information about viral transmission in communities. Wastewater samples are an excellent supplement to clinical testing and provide additional information about large populations quickly to understand and track the spread of COVID-19.

In March of 2022, we began reporting correlations between levels in wastewater and probability of community transmission. Low, Moderate, and High levels of SARS-CoV-2 in the wastewater correlate with estimated levels of community transmission and active case counts within the community contributing wastewater to the sample site:

As High is an expansive categorical result, we further break the high transmission category into quantiles. The quantiles use all historical data, by lab method, as multiple laboratories analyze wastewater samples. The historical data computes the quantiles that recent data is compared to. This returns a numerical percentile of where the recent data point falls along the historical data set.

Detection levels were determined based on statistical correlation between active case thresholds and wastewater detection. Detailed methods and documentation are available here. The figure below provides a visual representation of correlations between case thresholds and each detection level used.

Figure One: COVID-19 Incidence and Detection Levels

Estimates of the limits of detection (in terms of cases reported in the health system) of SARS-CoV-2 testing in wastewater relative to classification of transmission risk. Clear differentiation in the level of measured community-level COVID-19 incidence when categorizing wastewater results as quantifiable, detected but below the level of quantification, and not detected. Both “Substantial” and “High” are subsets of the “Quantifiable” category and will be reflected as “High” on dashboard visualizations. Size of the circles represents the number of individuals tested. Nondetection of SARS-CoV-2 RNA clusters around < 10 cases per 100,000.

Lab Methods

Labs that are contributing wastewater results in New York State use different methods. Comparison of data between and across sites analyzed by different labs may not correlate and is not recommended particularly for raw gene copies. If a method changes at the lab analyzing that site, past data may also not correlate.

Quadrant Biosciences is analyzing wastewater samples for most New York State counties. The limit of quantification for Quadrant's analysis method is 5 gene copies per milliliter. University at Buffalo - SUNY is analyzing wastewater samples for most of the Western Region of NYS. The limit of quantification for the method used by University at Buffalo - SUNY is 1 gene copy per milliliter. University at Buffalo - SUNY changed their methods to magnetic bead processing the week of April 17, 2022. It is recommended that data not be compared before and after this time point for University at Buffalo - SUNY sites. This is noted on the gene copies plot for those locations (e.g., Erie County WWTPs). Stony Brook University is analyzing wastewater samples for Suffolk County. Genesee and Orleans County Public Health Department is analyzing data for Genesee and Orleans Counties.

Data for New York City's five boroughs are analyzed by the City Health Department. Real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) was used for SARS-CoV-2 N1 gene copy determinations for samples collected August 31, 2020-March 7, 2023. Starting with samples collected on March 12, 2023, digital reverse transcription quantitative polymerase chain reaction (dPCR) has been used. Due to differences in methodology, dPCR viral load values are ~10-20 times higher than RT-qPCR values. Previously, we displayed the raw data for dPCR starting in May 2023, however, due to the change, levels in NYC appeared much higher than they actually were. To address this, we have temporarily adjusted NYC’s data to make it comparable to older data using a linear adjustment that translates the dPCR data to levels comparable to RT-qPCR data. These new levels are now displayed and present estimates for SARS-CoV-2 levels found in wastewater. The method change is represented on the raw gene copy plots for NYC sites with a vertical dashed line on March 12, 2023.

Beginning on March 3, 2024, the NYC Health Department started using a new extraction method and dPCR assay. The new assay measures Nucleocapsid gene copies. The method change is noted in trend figures by a vertical dashed line on March 3, 2024. Historical data are scaled up to make comparisons between old and new samples easier. High percentile groupings for NYC sites are based on data collected from August 31, 2020 to the present.

New York City data are reported weekly. For more information on methods used for SARS-CoV-2 detection in New York City, please visit this website. For access to New York City historical data, please visit this website.

Starting in January 2025, regions in the state are transitioning over to our State Public Health Laboratory, the Wadsworth Center, for wastewater surveillance testing. This transition is occurring in a stepwise fashion, beginning with the Capital Region in January 2025 and continuing with other regions through the summer of 2025. The affected regions and their respective counties include:

• Capital Region (Albany, Columbia, Greene, Rensselaer, Saratoga, Schenectady, Warren, Washington)
• Mohawk Valley (Fulton, Hamilton, Herkimer, Montgomery, Oneida, Schoharie) 
• Southern Tier (Broome, Chemung, Chenango, Delaware, Otsego, Schuyler, Steuben, Tioga, Tompkins) 
• North Country (Clinton, Essex, Franklin, Jefferson, Lewis, St. Lawrence) 
• Central (Cayuga, Cortland, Madison, Onondaga, Oswego) 
• Finger Lakes (Livingston, Monroe, Ontario, Seneca, Wayne, Wyoming, Yates) 
• Mid-Hudson (Dutchess, Orange, Putnam, Rockland, Sullivan, Ulster, Westchester) 
• Western New York (Cattaraugus, Chautauqua, Allegany, Erie, Niagara) 
• Long Island (Nassau, Suffolk)

As part of this transition, the Wadsworth Center has adopted a different extraction and quantification methodology than those previously used by regional laboratories. Specifically, Wadsworth has implemented the CERES Nanotrap method in conjunction with digital droplet PCR (ddPCR) for SARS-CoV-2 concentration measurements. Due to differences in methodology, viral load values obtained through this new method may not be directly comparable to values from previous laboratory methods.

To address these differences and ensure consistency in reported data, adjustments may be applied to historical values to facilitate comparisons between old and new methodologies. Method changes are indicated in trend figures by vertical dashed lines corresponding to the date of transition for each region.

This transition is expected to be completed in the summer of 2025. For access to historical wastewater surveillance data, please visit NY Open Data.

Wastewater samples are collected at participating treatment plants usually at influent points where the pipes run into the treatment plant.

Sewershed boundaries are created from several sources including physical maps provided by treatment plant operators, existing GIS data from participants, and digitized using NYS parcel data. Boundaries indicated the estimated service area for the treatment plant providing information on the community represented by each wastewater sample.

Treatment plant locations are from the NYS GIS data clearinghouse and available here.

Samples with detected levels of SARS-CoV-2 are genetically sequenced to identify the multiple variants or lineages of SARS-CoV-2 and to estimate the relative proportions of these variants and lineages. Wastewater samples contain multiple lineages of SARS-CoV-2, which differs from patient samples. Because of this complexity, the sequence data is analyzed by a lineage decomposition mixture model to assess the different lineages that make up the sample and the relative proportions of these lineages. These samples are distributed to the NYS SARS-CoV-2 Genetic Sequencing Laboratory Consortium, which consists of:

• NYSDOH Wadsworth Center
• New York Medical Center
• SUNY Upstate
• University at Buffalo SUNY
• University of Rochester

Following sequencing, whole-genome sequence data files are sent to Syracuse University for data interpretation and dissemination.

The advanced bioinformatics package Freyja is used to process sequence data and estimate the relative abundances of sublineages. This estimation is based on the measurement of single nucleotide variant frequency and sequencing depth at each position in the genome, providing a reliable estimate of the true lineage abundances in the sample.

While the map shows the single highest relative abundance for the most recent samples, relative abundances displayed in the sequencing stacked bar chart are calculated by aggregating all relative abundances by week. These can be viewed at sewershed, county, region, or statewide levels, which will provide a more detailed look, or aggregated spatial level, which gives a broader picture. Relative abundances below 5% are grouped in the "Other" category.
 

Samples are taken at each WWTP at different times during the week then sent to a laboratory for extraction and quantification of viral particles in the sample. Quantification data are the first results available and usually available in 1 to 4 days after the sample is collected. Sequencing data takes longer to generate results because the sample extracts are sent to different laboratories around the state for viral genome sequencing of SARS-CoV-2. The shipping time is 1 to 2 days, but then the samples need to be sequenced, uploaded and processed. The typical lag time for sequencing data is a total of 7 to 21 days, however it can take longer if there are unexpected delays anywhere along the way. Data for quantification of SARS-CoV-2 and data for sequencing will therefore have different dates for what is considered most recent for the location. The most recent data available will always be displayed for each dashboard.

Intensity of SARS CoV-2 RNA in wastewater - the natural log-adjusted ratio of SARS-CoV-2 RNA copies detected to total human fecal indicator detected.

Natural log of raw gene copies - SARS-CoV-2 RNA copies are natural log transformed to provide a more linear fit to the data for calculating trends over time.

Sewershed - a term used to refer to the service area of a treatment plant. Sewersheds can represent the entire service area for a plant or a portion of the service area. These smaller portions are sometimes called catchments and represent sampling at manholes or pump stations before the influent reaches the primary treatment facility.

crAssphage - bacteriophage commonly excreted from humans which is used to determine the relative level of SARS-CoV-2 RNA in the wastewater. The ratio of crAssphage to SARS-CoV-2 helps estimate if there is small amount of SARS-CoV-2 or if detection levels indicate greater infection in the population. It is one of the fecal indicators used to normalize data and calculate the intensity value.

PMMoV - pepper mild mottle virus - common virus found in peppers that is excreted in human waste. It is one of the human fecal indicators used to normalize wastewater sample results to adjust for different population sizes.

WWTP - wastewater treatment plant

Variant - A variant is a viral genome (genetic code) that may contain one or more mutations.

Lineage - A lineage is a group of closely related viruses with a common ancestor. SARS-CoV-2 has many lineages; all cause COVID-19.

Assay - A test for a specific chemical, microbe, or an effect

Wastewater Surveillance - Strategic sampling and testing of wastewater to detect pathogens or other health targets to better understand disease burden and spread within a community.

• New York State Department of Environmental Conservation
• Syracuse University
• SUNY ESF
• SUNY Upstate Medical University
• Quadrant Biosciences
• University at Buffalo - SUNY
• Stony Brook University
• Genesee and Orleans County Public Health Department
• CDC National Wastewater Surveillance System

• David Larsen, Department of Public Health, Syracuse University: Supervision, method development, data analysis
• Hyatt Green, Department of Environmental and Forest Biology, SUNY-ESF: Method development, data analysis
• Dustin Hill, Department of Public Health, Syracuse University: Web development, data analysis, data visualization
• Mary Collins, Department of Environmental Studies, Center for Environmental Medicine and Informatics, SUNY-ESF: Web development, data visualization
• Christopher Dunham, Director Research & Decision, Syracuse University: Web development, data management, quality assurance.

For questions about wastewater data and results, contact Dr. David Larsen at [email protected]