Challenges and Opportunities for Wastewater Monitoring of Influenza Viruses During the Multistate Outbreak of Highly Pathogenic Avian Influenza A(H5N1) Virus in Dairy Cattle and Poultry

The Centers for Disease Control and Prevention (CDC) began wastewater monitoring in September 2020 to provide additional information to health care providers and the public on the level of transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, and to inform public health actions.¹ This wastewater monitoring rapidly grew from a small number of states to a system in all 50 states that includes approximately 40% of the US population. Wastewater monitoring complements traditional clinical monitoring systems by detecting viruses being shed from infected individuals that might otherwise be missed; infections from asymptomatic individuals and those who do not seek or have access to clinical testing or care might be captured in wastewater-monitoring data from a given sewershed.²

Wastewater-monitoring data have been particularly valuable for managing COVID-19 because of the high rate of asymptomatic infections in humans contributing to SARS-CoV-2 transmission. Use of SARS-CoV-2 data from wastewater testing has been linked to successful public health actions, such as increasing uptake of vaccines through positioning of and communication about vaccination clinics.^3,4 The CDC and other public health and academic partners have expanded wastewater monitoring in the United States and globally to other pathogens, including influenza A viruses.^5–9 Although both influenza viruses and coronaviruses have zoonotic hosts, the animal hosts for SARS-CoV-2 appear significantly less likely to contribute to wastewater than do the dairy cattle and milk that are central to the current outbreak of highly pathogenic avian influenza (HPAI) A(H5N1) virus in animals.¹⁰

In the context of the current zoonotic outbreak of HPAI A(H5N1) virus in livestock, the CDC is monitoring for human infections using multiple systems, including public health laboratory testing to track influenza viruses such as novel viruses, trends in clinical laboratory data for influenza, and emergency department visits potentially associated with influenza.¹¹ There have been 14 instances of avian influenza A(H5) virus infection in people in the United States since 2022, 13 of which occurred between January and August 2024; these have been among people exposed to either infected poultry or infected dairy cattle. To use wastewater monitoring to complement traditional monitoring systems, it is urgent to better understand the meaning of influenza A virus levels and the H5 subtype in wastewater to inform public health emergency response action.

CURRENT STATUS OF WASTEWATER TESTING

Influenza A virus testing is being conducted on samples from more than 700 wastewater sites across 48 states, with testing occurring one to three times per week and data reported to CDC on a weekly basis. The CDC began publicly reporting wastewater data for influenza A viruses in May 2024 with an interim measure comparing the current level of influenza A virus in each wastewater site to the levels observed in the same wastewater site from October 1, 2023, to March 2, 2024.¹² This comparison period aligns with the human influenza season period before the detection of HPAI A(H5N1) virus in dairy cattle. This approach was implemented because the initial detections of influenza A(H5) virus subtype in wastewater coincided with large influenza A virus spikes occurring toward the end of the influenza season.¹³

By focusing on areas with unusually high influenza virus levels in wastewater (≥ 80th percentile) during a time when influenza infections were expected to be lower or decreasing, public health officials could quickly investigate the high influenza A virus levels. However, this interim measure limited the number of sites with sufficient data for analysis because it restricted analyses to sites that had begun testing for influenza A virus no later than October 1, 2023. Although data were submitted for more than 700 sites each week, less than half of the sites had sufficient data for analysis by this method because of the start date for their testing, the timeliness of data submission, or other missing information.

As we approach fall 2024, when seasonal influenza will begin to circulate more widely, the CDC is evaluating and validating an updated approach to monitoring influenza virus levels in wastewater over time in a manner that is similar to the wastewater viral activity measure used for SARS-CoV-2. In November 2023, the CDC initiated monitoring SARS-CoV-2 using the wastewater viral activity level, which allows aggregating data at the state, regional, and national levels rather than comparing each site’s data only to its own previous data.¹⁴ A similar approach to influenza A virus will allow clearer communication to the public by providing data on influenza levels at the state and national levels.

CHALLENGES WITH WASTEWATER TESTING

Testing for influenza A viruses in wastewater is challenging for a number of reasons, and the complexity has increased with the emergence of the HPAI A(H5N1) virus outbreak in dairy cattle.^15,16 First, among those infected with influenza A virus, the virus might only be intermittently shed in feces, and there are limited data on viral concentrations in feces and urine.¹⁷ Second, influenza A viruses infect humans but also are commonly found in animals and wild birds, such as waterfowl, which are the natural reservoir. Most wastewater systems in the United States are closed systems; however, open or combined wastewater systems receive storm water runoff, which could include inputs from wild birds. Some closed systems might also periodically receive inputs from sources accessible to wild birds.

Third, wastewater systems have a variety of inputs in addition to household sources (e.g., toilet, sink, and shower water). Some wastewater systems receive large volumes of input from milk- or meat-processing plants, inputs from poultry farms, and in some cases inputs from other agricultural sources that contain livestock waste. Current techniques do not allow determination of whether the source of the influenza A virus or subtype in wastewater is from an animal or a human source. Studies have shown that cattle have avian-specific receptors in both mammary and respiratory tissue and that the H5 virus can be found in very high levels in unpasteurized milk and in the lungs, muscle, and udder tissue.¹⁸ Detection of avian influenza A(H5) virus in wastewater is an important surveillance indicator and likely provides an indirect measure of the outbreak of HPAI A(H5N1) virus in dairy cattle in the United States, but it but does not provide clarity on whether any human infections are contributing viral particles.

Fourth, we do not know how much avian influenza A(H5) virus is needed to result in a positive H5 subtype detection in a wastewater system (i.e., sensitivity) and how that might vary by the size of the population served by a single sewershed, which can range from thousands to millions. And fifth, differences in laboratory approaches and reporting approaches can complicate interpretation¹⁹; the CDC is currently validating an influenza A H5 subtype molecular detection assay for use in wastewater with the intention of sharing the protocol and recommending its use as a reference standard testing assay before the start of the 2024/2025 influenza season.

Despite these challenges, wastewater monitoring for influenza A virus and H5 subtype and close coordination with local public health officials to better understand the likely sources of influenza A(H5) virus in wastewater can help refine methods for use in the upcoming influenza season. For example, detection of H5 in wastewater could trigger additional monitoring or testing of animals, milk, or humans.

WASTEWATER TESTING FOR HPAI A(H5N1)

A report released recently in the CDC’s Morbidity and Mortality Weekly Report highlights the findings and follow-up public health investigations for high levels of influenza A virus and H5 subtype detection in wastewater.²⁰ The H5 testing in wastewater used a quantitative test, but public health investigations were triggered by any qualitative detection of H5 in wastewater.¹³

In three out of four states with detections of high levels of influenza A virus in wastewater, there were consistent findings from other human clinical influenza-monitoring systems reflecting late seasonal influenza epidemic activity in certain communities. There was no evidence of avian influenza (HPAI) A(H5N1) virus detected in people in the states with high influenza A virus levels in wastewater. By contrast, eight of nine states with HPAI A(H5) virus detections in wastewater occurred in states with reported HPAI A(H5N1) virus–infected herds.

And, despite H5 testing of wastewater occurring in 41 states, only nine states had one or more sites with an H5 detection during the nine-week period of the investigations with a high correlation between reported infected herds and detection of H5 in wastewater. States with H5 detections in wastewater identified potential contributing sources of animal inputs, including dairies or milk-processing plants, meat-processing plants, and other sources of agricultural inputs.

Some processing sites received and processed milk or meat products from outside their state, further complicating the interpretation. Testing specific areas in a sewer network (subsewershed testing) following initial detection of influenza A(H5) virus in wastewater supported the hypothesis of milk-processing plants contributing to these detections.¹³ Review of existing human surveillance system data in jurisdictions with H5 detections in wastewater did not identify unusual human influenza activity, providing further evidence that these H5 detections were likely of animal origin.

LESSONS LEARNED AND PLANNING

The current outbreak of HPAI A(H5N1) virus in dairy cattle, poultry, and other animals as well as human cases identified among those with exposure to infected animals requires a One Health approach to monitor and protect human and animal health, and wastewater monitoring can be an important additional approach in this endeavor.²¹

Wastewater monitoring is a relatively new public health tool that can complement our existing health-monitoring systems to help us better track infectious diseases and guide public health actions, such as alerting clinicians about viruses circulating in the community, positioning and increasing uptake of vaccines, and alerting the public of periods of increased risk and consideration for taking personal protective measures. It provides valuable data on virus levels at the community level, even when individuals may not have symptoms or seek clinical testing, and therefore is not limited to detecting the more severe cases that require medical attention. However, wastewater monitoring is not a one-size-fits-all solution, and the value of wastewater monitoring in addition to other health-monitoring systems will depend on the jurisdiction and the specific disease to be monitored, including the following:

• 1.the strength and coverage of other monitoring systems;
• 2.the proportion of infected individuals who are asymptomatic, with wastewater monitoring having added importance in detecting asymptomatic infections given those individuals are unlikely to be seen in health care settings or tested, and the relative proportion of transmission accounted for by asymptomatic versus symptomatic individuals as well as the proportion of symptomatic individuals who do not seek health care or receive testing;
• 3.the role of animal reservoirs and animal product inputs to sewersheds and future laboratory techniques that might allow public health officials to distinguish human from animal sources in wastewater;
• 4.gaps in available information (e.g., likely zoonotic sources that have not been tested) on the state of an outbreak that can be at least partially addressed with findings from wastewater testing; and
• 5.the distribution of wastewater-monitoring sites in areas relevant to the pathogen being addressed.

For the current outbreak of HPAI A(H5N1) virus in dairy cattle, the consideration of these factors will vary widely among jurisdictions and likely among different areas in a jurisdiction. Monitoring for increases in influenza A virus and H5 subtype detections during times of low seasonal influenza activity, such as the summer months, can help improve planning for how to use wastewater testing efficiently with limited resources during the respiratory illness season. This approach also helps fill in critical information gaps that could slow our response to the ongoing threat from HPAI A(H5N1) viruses.

To ensure an effective wastewater-monitoring system that informs public health actions during the upcoming fall through winter respiratory illness season, it is important to incorporate the following components: (1) testing wastewater for influenza A virus and subtypes: this will facilitate detection and monitoring of different influenza viruses circulating in the human or animal population, providing insights into disease dynamics; (2) deployment of wastewater testing in high priority locations: by strategically focusing health-monitoring resources on areas with a higher risk of disease transmission or where outbreaks in people may be more likely to occur (e.g., areas with identified infected dairy cattle herds or poultry farms), we can obtain targeted data that reflect broader trends and help us prioritize public health actions; and (3) timely data integration to address knowledge gaps: wastewater monitoring can provide additional insights into disease patterns and transmission dynamics by including those who are not tested or seen in a health care setting, allowing us to track changes in viral activity and fill information gaps that may exist in monitoring systems that rely on individuals seeking and having access to care.

By incorporating these elements into national wastewater monitoring, we can improve our ability to monitor respiratory illnesses, inform public health actions, and prepare for and respond to future outbreaks.