Nationwide WastewaterSCAN June 2025 Update

Hi everyone,

This newsletter contains updates on the WastewaterSCAN program with wastewater samples analyzed by the lab through June 25, 2025. Below you will find information on the program in terms of participating partners, review of targets being measured, as well as current wastewater categories (low, medium, high) of the infectious disease targets.

Best,

WastewaterSCAN Team

WWSCAN Participant Stats:

• 147 plants sampling
• >37 million people covered through sampling efforts
• 40 states

The methods for our assays are in the public domain and links for these are provided at the end of the newsletter.

WWSCAN Published Body of Work document:

• We are excited to share a document that provides an overview of the program’s peer-reviewed scientific literature, organized by topics and themes. Click here to access the WastewaterSCAN published body of work.

WWSCAN FAQs:

• Frequently Asked Questions on Measurements of Candida auris (C. auris) in Wastewater Solids
• Frequently Asked Questions on WWSCAN Measurements of H5 Marker in Wastewater Solids
• Frequently Asked Questions on WWSCAN Measurements of Hepatitis A Virus (HAV) RNA in Wastewater Solids
• Frequently Asked Questions on WWSCAN Measurements of Measles RNA in Wastewater Solids
• Frequently Asked Questions on WWSCAN Measurements of Mpox clade Ib in Wastewater Solids

WWSCAN Factsheets and Guidance Documents can be found here.

As a reminder, WWSCAN's categorize sites based on Levels + Trends + Frequency of detection. We adopted this methodology back in 2023 as a way to clearly communicate what wastewater is telling us about community disease circulation. The wastewater categorization helps us quickly understand if the recent measurements for a disease fall into a low, medium, or high category and are determined based on a combination of the following variables:

• Trends tell us how concentrations are changing  (↑, ↓, -) over the last 21 days
• Levels tell us whether concentrations are relatively lower or higher (based on national tertiles over last 365 days)
• Frequency of detection indicates how often a pathogen is detected in wastewater (important for pathogens that are not commonly detected)

There are 3 Archetypes for WWSCAN Pathogens:

• Pathogens that are commonly detected (regardless of season - includes SARS-CoV-2, Norovirus)
• Seasonal pathogens (rarely detected outside of season/outbreaks - includes Influenza A + B, RSV, HMPV, EV-D68)
• Pathogens that are not commonly detected (regardless of season - includes C. auris, HepA, Measles, Mpox clade Ib, Mpox clade II)

WWSCAN recently updated the ‘Pathogens that are not commonly detected’  logic to include a 'Not detected' category (see table below for reference).

To learn more about WWSCAN's categorization logic, please see: https://data.wastewaterscan.org/about/#8

SARS-CoV-2 N gene RNA median concentration across all sites in past 3 weeks was 23,836 copies/gram. Below is the population weighted average SARS-CoV-2 N gene concentrations (normalized by PMMoV) is shown in black from all participating WWSCAN sites across the country over the last 365 days.

Nationally, SARS-CoV-2 is in the 'HIGH' category with aggregated measurements showing medium concentrations and a upward trend over the last 3 weeks.

Below are the current SARS-CoV-2 wastewater categories for all WWSCAN sites (recall the category is determined by both the trend and the level):

• 95 are in the LOW Wastewater Category
• 38 are in the MEDIUM Wastewater Category
• 10 are in the HIGH Wastewater Category
• 4 do not have enough data to calculate

Sequencing of SARS-CoV-2 from wastewater is available for a select number of individual sites. The sequencing protocol is available on protocol.io. The plots below show the relative proportions of different variants inferred from sequencing the entire genome of SARS-CoV-2. Results are based on sequencing of 2 samples per week, combined to provide a weekly value.

Sequencing data is also available in aggregate across all sites in WWSCAN with sequencing data. That plot is shown below and also suggests that LP.8.1 (shown in brown) is the most abundant variant across all sites at 28.5%, followed by LF.9 (shown in yellow) is at 16.2%. XFG (shown in orange) at 10.2% and NB.1.8.1 (shown in mauve) is at 3.4%.

Influenza A (IAV) RNA was detected in ~22% of samples in past 3 weeks. IAV RNA median concentration across all sites in the past 3 weeks was below the limit of detection. Below is the population weighted average IAV concentrations (normalized by PMMoV) is shown in black from all participating WWSCAN sites across the country over the last 365 days.

Nationally, Influenza A is in the 'LOW' Wastewater Category with >50% of sites now in seasonal offset of IAV in wastewater.

Below are the current Influenza A wastewater categories for all WWSCAN sites (recall the category is determined by both the trend and the level when in seasonal onset):

• 138 are in the LOW Wastewater Category
• 4 are in the MEDIUM Wastewater Category
• 1 are in the HIGH Wastewater Category
• 4 do not have enough data to calculate

H1 Marker in Influenza A (IAV) The H1 marker in influenza A has been detected in ~5% of samples over the last 3 weeks. The H1 median concentration across all sites in the past 3 weeks was below the limit of detection. You can find a heat map of positive detections here.

H3 Marker in Influenza A (IAV) In the last 3 weeks, the H3 marker in influenza A was detected in ~1% of samples. You can find a heat map of positive detections here.

H5 Marker in Influenza A (IAV) As of June 25, 2025, WWSCAN sites across 28 states (AK, AR, CA, CO, CT, DE, FL, IA, ID, IL, KS, KY, MA, ME, MD, MI, MN, NC, NE, NH, NJ, NV, SD, TN, TX, UT, VA and VT) have seen positive detections for the H5 marker in influenza A. Since prospective monitoring began, 1,016/25,656 samples have tested positive (~3.9% detection rate). In June 2025, there were only 2 positive H5 marker detections (1 in NJ and 1 in VA). You can find a heat map of positive detections here.

Influenza B (IBV) RNA was detected in ~18% of samples in past 3 weeks. IBV RNA median concentration across all sites in the past 3 weeks was below the limit of detection. Below the population-weighted average IBV RNA concentrations (normalized by PMMoV) is shown in black from all participating WWSCAN sites across the country over the last 365 days.

Nationally, Influenza B is in the 'LOW' Wastewater Category with >50% of sites now in seasonal offset of IBV in wastewater.

Below are the current Influenza B wastewater categories for all WWSCAN sites (recall the category is determined by both the trend and the level when in seasonal onset):

• 136 are in the LOW Wastewater Category
• 0 are in the MEDIUM Wastewater Category
• 7 are in the HIGH Wastewater Category
• 4 do not have enough data to calculate

Respiratory syncytial virus (RSV) RNA was detected in ~7% of samples in past 3 weeks. RSV RNA median concentration across all sites in the last 3 weeks was below the limit of detection. Below the population-weighted average RSV RNA concentrations (normalized by PMMoV) is shown in black from all participating WWSCAN sites across the country over the last 365 days.

Nationally, RSV is in the 'LOW' Wastewater Category with >50% of sites now in seasonal offset of RSV in wastewater.

Below are the current RSV wastewater categories for all WWSCAN sites (recall the category is determined by both the trend and the level when in seasonal onset):

• 143 are in the LOW Wastewater Category
• 0 are in the MEDIUM Wastewater Category
• 0 are in the HIGH Wastewater Category
• 4 do not have enough data to calculate

HMPV (human metapneumovirus) RNA was detected in ~46% of samples in the last 3 weeks. The median HMPV RNA concentrations across all sites in the last 3 weeks was below the limit of detection. Below is the population weighted average HMPV RNA concentrations (normalized by PMMoV) shown in black from all participating WWSCAN sites across the country over the last 365 days.

Nationally, HMPV is in the 'LOW' Wastewater Category with >50% of sites now in seasonal offset of HMPV in wastewater. ~24% of sites are still in seasonal onset for HMPV.

Below are the current HMPV wastewater categories for all WWSCAN sites (recall the category is determined by both the trend and the level):

• 107 are in the LOW Wastewater Category
• 8 are in the MEDIUM Wastewater Category
• 28 are in the HIGH Wastewater Category
• 4 do not have enough data to calculate

Enterovirus D68 (EV-D68) RNA was detected in ~9% of samples collected in the last 3 weeks across WWSCAN sites (with most of the positive detections being in California). The median concentration across all sites was below the limit of detection in the last 3 weeks. Below is the population-weighted average EV-D68 RNA(normalized by PMMoV) shown in black from all participating WWSCAN sites across the country over the last 365 days.

Nationally, EV-D68 is in the 'LOW' Wastewater Category with <50% sites showing onset of EV-D68 in wastewater.

Below are the current EV-D68 wastewater categories for all WWSCAN sites (recall the category is determined by both the trend and the level):

• 139 are in the LOW Wastewater Category
• 2 are in the MEDIUM Wastewater Category
• 2 are in the HIGH Wastewater Category
• 4 do not have enough data to calculate

Parvovirus (Parvo) DNA - In mid May 2025, we began prospectively monitoring for Parvovirus. Parvovirus B19 DNA was detected in ~47% of samples collected in the last 3 weeks across WWSCAN sites.

The chart below shows each WWSCAN plant in Texas as rows, and days as columns (labeled at the bottom axis). White indicates no samples, blue is a non-detect for Parvo DNA and red is a detection for Parvo DNA.

As a reminder, we are not currently calculating wastewater categories for Parvovirus. In order to calculate wastewater categories we need a longer time series of data to examine trends and levels.

We recently published a paper on 'Community Infections Linked with Parvovirus B19 Genomic DNA in Wastewater, Texas, USA, 2023–2024'. This paper covers retrospective analysis conducted at two WWTPs in Texas prior to WWSCAN rolling out the assay to all participating WWSCAN sites. You can find the paper linked here.

Norovirus GII (HuNoV GII) RNA was detected in 100% of samples analyzed in the last 3 weeks. The median concentrations across all sites in the last 3 weeks is 3,265,759 copies per gram, down ~51% from May 2025. Below is the population weighted average HuNoV GII RNA concentrations (normalized by PMMoV) shown in black from all participating WWSCAN sites across the country over the last 365 days.

Nationally, Norovirus is in the 'LOW' Wastewater Category with aggregated measurements showing low concentrations and downward trend over the last 3 weeks.

Below are the current Norovirus wastewater categories for all WWSCAN sites (recall the category is determined by both the trend and the level):

• 108 are in the LOW Wastewater Category
• 27 are in the MEDIUM Wastewater Category
• 8 are in the HIGH Wastewater Category
• 4 do not have enough data to calculate

Human Adenovirus Group F (HAdV-F) RNA - In mid May 2025, we began prospectively monitoring for HAdV-F.

We have detected HAdV-F RNA in 100% of samples from all WWSCAN sites in the last 3 weeks. As a reminder, we are not currently calculating wastewater categories for HAdV-F. In order to calculate wastewater categories we need a longer time series of data to examine trends and levels.

Rotavirus (Rota) RNA- In mid May 2025, we started measuring Rotavirus again. We previously paused measurements of Rota in December 2024 in order to being measuring Mpox clade Ib.

We have detected Rota RNA in ~98% of samples from all WWSCAN sites in the last 3 weeks. As a reminder, we are not currently calculating wastewater categories for Rota. In order to calculate wastewater categories we need a longer time series of data to examine trends and levels.

Below is the is the population weighted average Rota RNA concentrations (normalized by PMMoV) shown in black from all participating WWSCAN sites across the country over the last 365 days. Note measurements of Rota RNA were paused for a 6 month period (indicated by the dashed red lines in the chart).

Candida auris (C.auris) DNA - We have detected C. auris DNA in ~4.5% of samples from all WWSCAN sites in the last 3 weeks. Nationally, C. auris is in the 'LOW' category due to limited detections.

The chart below shows plants in Michgan as rows, and days as columns (labeled at the bottom axis). White indicates no samples, blue is non-detect for C. auris DNA and red is detect for C. auris DNA.

Below are the C. auris wastewater categories for all WWSCAN sites (recall the category is determined by the frequency of detection):

• 131 are in the NOT-DETECTED Wastewater Category
• 12 are in the LOW Wastewater Category
• 0 are in the MEDIUM Wastewater Category
• 0 are in the HIGH Wastewater Category
• 4 do not have enough data to calculate

Hepatitis A (Hep A) RNA - We have detected Hep A RNA in ~8% of samples from all WWSCAN sites in the last 3 weeks. Nationally, HepA is in 'LOW' category due to limited detections.

The chart below shows each WWSCAN plant in Southern California as rows, and days as columns (labeled at the bottom axis). White indicates no samples, blue is a non-detect for Hep A RNA and red is a detection for Hep A RNA.

Below are the current HepA wastewater categories for all WWSCAN sites (the category is determined by the frequency of detection):

• 121 are in the NOT-DETECTED Wastewater Category
• 22 are in the LOW Wastewater Category
• 0 are in the MEDIUM Wastewater Category
• 0 are in the HIGH Wastewater Category
• 4 do not have enough data to calculate

Measles (MeV) RNA - In mid-May 2025, we began prospectively monitoring for MeV. Since monitoring began, 8 sites across 6 states have had at least 1 positive detection of MeV in wastewater samples collected. Nationally, MeV is in the 'LOW' category due to limited detections.

Below is a map of sites with MeV detections in wastewater since prospective monitoring began.

Below are the current MeV wastewater categories for all WWSCAN sites (the category is determined by the frequency of detection):

• 140 are in the NOT-DETECTED Wastewater Category
• 3 are in the LOW Wastewater Category
• 0 are in the MEDIUM Wastewater Category
• 0 are in the HIGH Wastewater Category
• 4 do not have enough data to calculate

Mpox clade Ib DNA - In mid December 2024 we began prospective monitoring for Mpox clade Ib. You can find a heat map of the Mpox clade Ib data here. In the last 3 weeks, there were no positive detection of Mpox clade Ib. Nationally, Mpox clade Ib is in the 'NOT-DETECTED' category due to no detections.

Below are the Mpox clade Ib wastewater categories for all WWSCAN sites (recall the category is determined by the frequency of detection):

• 143 are in the NOT-DETECTED Wastewater Category
• 0 are in the LOW Wastewater Category
• 0 are in the MEDIUM Wastewater Category
• 0 are in the HIGH Wastewater Category
• 4 do not have enough data to calculate

Mpox clade II DNA - There was 1 positive detection of Mpox clade II in the last 3 weeks. Nationally, Mpox clade Ib is in the 'LOW' category due to limited detections.

Below are the Mpox clade II wastewater categories for all WWSCAN sites (recall the category is determined by the frequency of detection):

• 142 are in the NOT-DETECTED Wastewater Category
• 1 are in the LOW Wastewater Category
• 0 are in the MEDIUM Wastewater Category
• 0 are in the HIGH Wastewater Category
• 4 do not have enough data to calculate

Protocols (Pre-analytical methods and SARS-CoV-2 analytical methods including controls):

• High Throughput RNA Extraction and PCR Inhibitor Removal of Settled Solids for Wastewater Surveillance of SARS-CoV-2 RNA
• High Throughput pre-analytical processing of wastewater settled solids for SARS-CoV-2 RNA analyses
• Quantification of SARS-CoV-2 variant mutations (HV69-70, E484K/N501Y, del156-157/R158G, del143-145, LPPA24S, S:477-505, and ORF1a Del 141-143) in settled solids using digital RT-PCR
• High Throughput SARS-COV-2, PMMoV, and BCoV quantification in settled solids using digital RT-PCR

Pre-prints and peer-reviewed publications provided WWSCAN methods: 

• SARS-CoV-2 
• Influenza, Human metapneumovirus, Respiratory syncytial virus, Human parainfluenza
• Mpox clade Ib
• Mpox clade II
• Norovirus GII
• Adenovirus group F, Rotavirus
• Candida auris
• Hepatitis A
• Enterovirus D68
• Parovirus

If you have questions about the website or anything else, please email: wwscan_stanford_emory@lists.stanford.edu