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Vision for National Wastewater Surveillance

This chapter presents a vision for a national wastewater surveillance system that can be a critical asset for early detection of emerging pathogen outbreaks and for monitoring the spread and virulence of existing pathogens. Key elements of a robust national wastewater surveillance system are explained, along with criteria for expanding the pathogens monitored by such a system beyond SARS-CoV-2. Finally, the committee reviews spatial and temporal sampling approaches consistent with this national strategy for surveillance.

BENEFITS OF SUSTAINED NATIONAL WASTEWATER SURVEILLANCE

Investment in a robust national wastewater disease surveillance system is important to increase national preparedness for emerging infectious diseases and to monitor resurgences of known agents. The key advantage of wastewater surveillance is that it does not rely on clinical testing, thus enabling early detection of disease when clinical testing is not prevalent or when some patients exhibit mild or no symptoms and thus do not undergo clinical testing. Early detection of emerging infectious diseases is critical, as we can control diseases much more effectively when spread in the human population is limited. Thus, early detection can make the difference between the occurrence of a manageable disease outbreak and the progression of a full-blown epidemic or pandemic. The initial investment in wastewater surveillance infrastructure, developed as a result of the COVID-19 pandemic, has already been successful in identifying other emerging threats to public

health. For example, wastewater surveillance has enabled early detection of poliovirus outbreaks in New York and London as well as the recent spread of mpox (de Jonge et al., 2022; Nelson, 2022). This critical infrastructure and expertise can provide the foundation of a national wastewater infectious disease surveillance system.

An overriding lesson of the past 2 years is that an outbreak of an emerging pathogen will be followed by a period of remarkable uncertainty. Maintaining a national wastewater surveillance system ensures readiness to respond to evolving risks. Even well into the COVID-19 pandemic, given the unpredictability of SARS-CoV-2 variant emergence and spread, the value of these data to public health management continues to increase. To date, SARS-CoV-2 variants have emerged and spread in global “sweeps” whereby a new, highly transmissible variant that is capable of evading established immunity rapidly spreads. The emergence of new variants of concern is complicated by co-circulation of multiple variants or subvariants concomitantly (Elliot et al., 2022). Variations in human demographics, vaccination rates, and local environmental conditions are highly likely to lead to marked differences in variant/subvariant prevalence across communities (Saad-Roy et al., 2022), with impacts on the need for public messaging and data to inform infection prevention and to enhance hospital preparedness. Representative community-based wastewater surveillance, implemented on a national level with spatial and temporal representation in sampling, can provide data on SARS-CoV-2 trends and variant distribution in a practical and rigorous manner (see Chapter 2).

National wastewater surveillance also has value for monitoring known diseases that vary temporally and spatially. For example, early detection of influenza can provide critical data for healthcare systems and public health messaging in communities. Several illustrative, high-priority use cases that demonstrate the value of a national wastewater disease surveillance system are given in Box 3-1.

Finally, community-based wastewater surveillance has the potential to provide critical information necessary for understanding the virulence of emergent variants. Virulence—the severity of disease caused by a new variant—can be determined, for example, by dividing the number of hospitalized individuals by the total number of infections in the community. However, as testing for infection has moved from institutionally based testing (the results of which are reported to public health agencies) to at-home testing (in which a significant but unknown number of infections are unreported), a data gap has emerged and the required data for estimating variant virulence at the population scale have been lost. Wastewater surveillance may provide a means to understand trends in disease prevalence relative to trends in hospitalization for indications of virulence, and further scientific advances in using wastewater to estimate the number of infections in a community would enhance this value.

KEY CHARACTERISTICS OF A NATIONAL WASTEWATER SURVEILLANCE SYSTEM

The committee’s vision for a robust surveillance system includes five key characteristics: flexible, equitable, sustainable, integrated, and actionable.

Flexible

The system should have the flexibility to monitor multiple pathogens at the same time and pivot as needed to new pathogens of public health importance. Both the number of pathogens tracked and the scale of operation (e.g., frequency of sample collection, number of testing sites) should be flexible. For example, an emerging infectious disease threat might require that a system be adapted to test for new pathogens. Similarly, an outbreak of an existing disease in a given area might necessitate an increase in the frequency of testing in that area and expansion of testing to new sites to capture the temporal and spatial attributes of the outbreak.

Equitable

A robust, useful wastewater surveillance system should be as equitable across population demographics as possible. Equity requires a fair distribution of the benefits and burdens of public health interventions across individuals and communities. Although ethical analyses of public health surveillance systems often focus on their burdens and risks, such interventions can also confer important benefits on individuals and communities (WHO, 2017). For example, wastewater data can form the basis for allocating increased resources and outreach to such communities to improve

health (Hrudey et al., 2021; Ram et al., 2022; see Chapter 2). However, if a wastewater surveillance system is not equitable in its coverage, allocating resources using wastewater surveillance data may mean diversion of scarce public health funds away from certain vulnerable populations.

The potential for inequity is a particular concern for wastewater surveillance because there are presently large geographic differences in where surveillance is being conducted (and where it is possible to conduct surveillance) within the United States. Unsewered households (16 percent of the U.S. population; U.S. Census Bureau, 2022) and facilities are by necessity excluded from wastewater surveillance and many, but not all, lie within rural areas. A recent California study found disproportionately low availability of wastewater surveillance in designated “disadvantaged” communities and rural areas of the state (Medina et al., 2022). Moreover, as long as participation in wastewater surveillance remains voluntary on the part of state and local officials, who may decline to implement it for a variety of reasons including political considerations or limited capacity, use of wastewater surveillance could potentially widen existing disparities in how well residents of different states and counties are served by public health systems and programs (Adhikari and Halden, 2022).

An equitable wastewater surveillance system would invest resources in outreach efforts to engage officials from communities that are not currently participating. This engagement should include some assessment of barriers to participation followed by efforts to reduce these barriers when feasible and advantageous. Information about the logistics and advantages of wastewater surveillance for disease detection, as well as a playbook for starting up a local program, could lower barriers to broader participation. Dialogue may also reveal ethical, social, political, or legal worries that could be assuaged by learning from the experiences of existing wastewater surveillance programs.

A robust national wastewater surveillance system should include strategies by which data can be usefully extrapolated through statistical techniques to communities not covered by the system, including unsewered areas (e.g., based on mobility data and laborshed information on commuting and work patterns). Even with additional statistical analyses, wastewater surveillance data may not provide sufficient information about some regions. The use of multiple disease surveillance data sources can also help ensure equity of surveillance efforts with respect to unsewered households and communities, and regional public health agencies should take these data gaps into consideration when investing resources.

Sustainable

The COVID-19 pandemic highlighted the need for the U.S. public health system to maintain active vigilance in monitoring emerging disease

and threats from new pathogens in a way that is sustainable for decades to come. Sustainability of the system will require attention to two issues: financial support and operational burden. Implementing partners, including local utilities, wastewater treatment plants, academic research centers, and public health departments, that have been strained to provide wastewater surveillance services during a time of emergency cannot be expected to do so indefinitely without attention to the burdens that participation involves.

In addition to scaling up financial support, there may be a need to consider ways to scale down operational aspects of wastewater surveillance relative to the pandemic period while maintaining institutional capacity. For example, reduced frequency of sampling may feasibly provide limited baseline surveillance until an emerging threat or disease presence is detected, at which time sampling could be scaled up for more comprehensive coverage. Thus, a sustainable system is one with sufficient support—financial, technical, and logistical—to provide an “everyday” level of sampling and analysis and to scale up for particular pathogens as signals emerge that more intensive sampling or sampling of a different set of sites is needed.

Finally, a sustainable system will require outreach to policy makers and the public to demonstrate the societal value of a wastewater surveillance system to achieving public health outcomes. This, in turn, requires interpretable and actionable data.

Integrated

There are two key aspects of integration that are essential in a robust wastewater surveillance system: (1) collaboration and coordination across the participating partners (e.g., utilities, analytical laboratories, and public health departments), and (2) the analysis of data from different disease surveillance systems to ensure comprehensive understanding in supporting public health action. Collection of timely and accurate data requires collaboration across and within utilities collecting the samples, laboratories conducting the analysis, and the relevant local and national agencies using and disseminating the results. Data from all three of these system participants need to be fused to generate coherent information. For example, information from utilities and laboratories is needed to determine the accuracy of the data, assess whether there were other substances in the wastewater that could affect the validity of the results (e.g., if industrial chemicals were present that could cause degradation of the pathogen), and provide other information necessary to interpret the findings. Similarly, integration of efforts across different divisions within public health agencies (e.g., communicable disease, environmental health, and communications) is critical to support timely and effective action on the part of local, state, tribal, and federal stakeholders using wastewater surveillance system data. As the field

of wastewater surveillance is evolving, it is also important to integrate the latest advances and the expanded knowledge base from researchers (Hoar et al., 2022).

Integration with other relevant data is also key for data interpretation to drive public health actions. Wastewater data should not be interpreted in isolation and need to be integrated with information on epidemiological context—both to evaluate trends and hot spots and to understand the risk factors and population characteristics of the communities reflected in wastewater catchment areas. Integrated analysis and interpretation of wastewater data would consider these other important data sets and compare wastewater findings with existing epidemiological surveillance systems (e.g., syndromic surveillance, clinical data). Each of these systems has strengths and weaknesses, but wastewater data can be used in a complementary manner with data across different surveillance systems to understand the underlying population patterns. Seamless integration is particularly important for consistent and effective public health messaging and risk communication, especially when many independent entities are contributing to the collection and interpretation of wastewater data.

Actionable

The ultimate goal of a surveillance system is to produce actionable data for public health agencies and policy makers. In some cases, data from surveillance systems may even be used to inform decisions that individuals and families make about their risk behaviors (e.g., whether to travel to an area impacted by a disease outbreak). In order to be actionable, wastewater surveillance data must be timely, available, reliable, representative, and interpretable.

Timely information is critical for actions to contain infectious diseases. It is much easier to control a disease at the earliest point in an outbreak than when it is already widespread in the community. For early warning potential of wastewater surveillance to be realized, sample collection, analysis, and interpretation of data by public health decision makers must operate on a timescale that allows for informed and timely interventions. Timeliness is related to sustainability in that expeditious collection, analysis, and interpretation of wastewater data are unlikely to occur in the absence of sufficient human and financial resources.

Data are available when shared with public health agencies and others who can use the data to support decision making. The question of what availability should look like with respect to other stakeholders, including academic researchers, private companies, and the general public, is more complex. As is discussed in Chapter 4, increased data access requires thoughtful consideration of ethical concerns about privacy and potential

complexity or uncertainty in the interpretation of the data. It is clear from the nation’s experience during the COVID-19 pandemic, however, that knowledge generation and innovations can be accelerated when data are widely shared.

Reliability refers to consistent confidence that the results, with respect to magnitude and trend, represent the viral load and not a product of variability in the system from other factors. If results are not reliable, public health officials run the risk of taking costly actions to combat an emerging threat that does not exist or failing to detect one that does. In addition to financial costs, these outcomes can erode public trust in the public health surveillance system. Therefore, public health officials also need to understand clearly the strength of the evidence and limits on reliability, where they exist. To promote reliability, protocols used to generate the data need to be rigorously validated and reproducible, and the method performance should be openly reported. Reliability can be improved by integrating wastewater data with relevant data (i.e., syndromic data) to improve the sensitivity and specificity of information available and through additional scientific research.

Representative wastewater data capture information that is reflective of or is relevant to the population at risk. A key advantage of wastewater surveillance is that it does not rely on individuals seeking diagnostic testing for their symptoms or health conditions. However, to ensure that data from national wastewater surveillance are representative, sampling sites need to be representative of the nation’s population.

Finally, wastewater surveillance data should be interpretable in a public health context. The analysis methods and interpretation guidance should link the data with population patterns of disease so that public health officials and the public understand what the wastewater data imply for public health. This may necessitate different analytical approaches for different use cases. For example, the simple detection of an emerging pathogen might be actionable if the goal is to monitor for the introduction of a new or emerging disease. On the other hand, for a pathogen that is more established in a community (e.g., SARS-CoV-2, influenza), it would be valuable to develop an analytical approach that links temporal or concentration patterns of wastewater data to patterns of disease inferred from syndromic data (e.g., trends in cases, numbers of cases). Interpretability also naturally relies on integration across utilities, laboratories, and public health, as described above. To support data comparisons and interpretation across geographic areas or at the national scale, the wastewater surveillance should ideally generate data that can be standardized, either at the time of initial collection and analysis or through statistical adjustments.

A FRAMEWORK FOR IDENTIFYING CANDIDATE PATHOGENS FOR WASTEWATER SURVEILLANCE

Wastewater surveillance holds clear promise beyond SARS-CoV-2 and the COVID-19 pandemic. The question is not whether to sustain and expand wastewater surveillance efforts but how. Selection of candidate pathogens for surveillance necessitates careful consideration.

Application of a defined set of criteria can help guide and systematize evaluation of the most promising candidates for wastewater surveillance. Similar approaches have been proposed for prioritization of candidate pathogens for wastewater surveillance elsewhere (Eaton et al., 2021). Three closely linked key criteria are proposed here for evaluating potential targets for inclusion in a national wastewater surveillance panel (see Figure 3-1):

1. public health significance of the threat,
2. analytical feasibility for wastewater surveillance, and
3. usefulness of community-level wastewater surveillance data to inform public health action.

As discussed in Chapter 2, public health actions could include informing public health resource allocations, informing clinical resource allocations, and/or informing masking, social distancing, and stay-at-home policies. These three criteria are by no means exhaustive, but they form the key pillars of an initial assessment of the value and feasibility of potential expansions of wastewater surveillance. This assessment should be informed by the latest information on existing and emerging pathogens. In the face of limited resources, the criteria can help prioritize among potential candidates and guide efforts toward more in depth consideration of the most promising candidates for surveillance. These criteria can also be used to identify and prioritize research needs for promising candidates that lack key information or analytical methods needed for adoption in the National Wastewater Surveillance System (NWSS).

Criterion 1: Public Health Significance of the Threat

Pathogens considered for wastewater surveillance would need to pose a significant public health threat (actual or potential) to outweigh the cost and effort associated with the added workload for public utilities and public health jurisdictions. The U.S. Department of Health and Human Services, the National Security Council, and federal departments and agencies have coordinated on the development of national action priorities and strategies for surveillance based on public health significance (ASPR and NSC, 2018). Similarly, the World Health Organization (WHO) maintains lists of priority

pathogens and diseases, including antibiotic-resistant bacteria, as part of its public health emergency and preparedness strategies for international disease threats. U.S. states also maintain lists of priority conditions (e.g., notifiable conditions).

Drawing on work by these organizations, key parameters to evaluate whether a candidate pathogen for wastewater surveillance meets the criteria for public health significance include the following:

• What is the current or potential severity of disease in people? Morbidity, mortality, case fatality, health-adjusted life years lost, or years of livelihood lost are the main parameters characterizing the impact of pathogens in populations along a disease severity spectrum. Disease severity may change over time as new variants emerge, vaccines and treatments are developed or made more widely available, healthcare system capacity increases or decreases, or disease dynamics in the population shift (e.g., the age groups with the highest infection rates). Therefore, this determination should be revisited periodically.
• What is the current or potential distribution of disease in people? The extent of disease, as determined by measures of prevalence in the population, is an essential consideration. Surveilling for pathogens that cause rare diseases has limited relevance to public health and presents unique challenges to analytical feasibility (e.g., the quantity of a rare pathogen is unlikely to be detectable at community-level surveillance). The prospect of rapid community spread, with epidemic or pandemic potential, as well as the availability of effective vaccines or therapeutics, should also be weighed for candidates that are not currently widely distributed but have the potential to emerge. Similarly, it is important to consider how much uncertainty exists in our knowledge of the distribution of the disease; for example, diseases that are mostly asymptomatic but cause severe outcomes in a proportion of individuals may have higher uncertainty in the true underlying distribution of disease. Wastewater surveillance may provide an avenue to resolve such uncertainty and understand the extent of spread. Finally, the distribution of disease within the population should be assessed. The case for wastewater surveillance may be especially strong if vulnerable populations are missed or underrepresented in other surveillance approaches. Furthermore, if disease is significantly burdening population groups that have low access to vaccines and healthcare, the argument for heightened investment in surveillance and prevention is strengthened.

Criterion 2: Analytical Feasibility for Wastewater Surveillance

Wastewater poses unique challenges to detection of pathogens (or pathogen biomarkers; see Box 1-1); therefore, the feasibility and optimization of detection methods needs to be considered fully when evaluating the usefulness of this type of surveillance for potential pathogen candidates. Key questions to assess include the following:

• Can the candidate pathogen be detected in wastewater? The pathogen must be excreted in urine/feces or otherwise routinely shed to wastewater for consideration.
• Are there cost-effective methods for sampling, concentration, and detection of the candidate pathogen? Sample acquisition and handling should be safe, effective, and readily achievable for public utilities, and detection methods should be precise and reproducible to enable standardization across sites and over time. Evaluation of detection methods should include specificity (i.e., the extent to which the agent is discernible from other targets) and sensitivity (i.e., the potential to detect the agent if present) in wastewater across a range of expected prevalence. Both rare and widespread pathogens pose unique challenges to detection and interpretation of surveillance data. Rare pathogens may fall below the level of detection at the community wastewater scale, while widespread pathogens require quantification rather than simple presence/absence for interpretation. Cost of sampling and testing could vary considerably for different candidate pathogens, with efficiencies achieved as individual specimens are tested for multiple pathogens. The costs for alternate (non-wastewater-based) surveillance methods for the candidate pathogen should be taken under consideration. If detection methods have not yet been optimized for wastewater, the feasibility and cost of method development should be considered.
• Can pathogens in wastewater be reliably calibrated to public health outcomes of interest in relevant populations? Although all infectious disease biomarkers will degrade in wastewater, the rate of degradation should be slow enough (or well-characterized enough) to facilitate reliable interpretation of the data. Other contributing sources of the agent in wastewater, such as animals or environmental sources, could reduce the usefulness of wastewater surveillance for public health decision making.

Criterion 3: Usefulness of Community-Level Wastewater Surveillance Data to Inform Public Health Action

Decisions about the expansion of wastewater surveillance should consider the usefulness of wastewater data relative to other types of surveillance data (e.g., existing, planned, or possible). Key questions in assessing the marginal value of wastewater information include the following assessments. In all of these, the objective is not to replace alternative sources of surveillance but to maximize the proportionate value of wastewater data with these other sources.

• How available are non-wastewater sources of data for the candidate pathogen? The extent to which other sources of data are readily available, cost-effective, and capable of informing public health actions should be weighed relative to the potential contributions of wastewater data. For many targets, samples and data could be available at clinics, hospitals, and other healthcare facilities through routine, syndromic-based, or targeted surveillance efforts. For other targets, the samples and data will not be available or it will not be cost-effective to obtain them. Air filter monitoring is increasingly available in public spaces for airborne pathogens and could serve as another source of samples and monitoring data, although air filter monitoring is typically conducted at smaller scale (typically building level) than community-level wastewater surveillance (Bhardwaj et al., 2021; Sousan et al., 2022). Digital disease surveillance (e.g., symptom-based monitoring through online searches, pharmacy data) is another source of information for monitoring disease symptoms (and disease distribution) in populations (Lu et al., 2019; Zhang et al., 2019). For novel or emerging pathogens that cannot be surveilled effectively in other settings, wastewater surveillance could have an especially important role in detection.
• What advantages, if any, do wastewater data have over alternative data sources? Even among pathogens for which healthcare data or other sources of information are available, the usefulness of that information may vary. For example, infected persons may be more or less likely to present for care. Wastewater data may have more consistent ascertainment (i.e., the fraction of infections captured in the data) than sources that rely on potentially changing behaviors around care-seeking and testing. If only syndromic data are available, wastewater surveillance data may be able to distinguish between different pathogens that lead to the same symptoms. Wastewater data may also shed light on key pathogen and disease parameters not ascertainable from other data sources. Wastewater

• samples can, for instance, distinguish among different variants, strains, and types of biomarkers if analytical techniques enable this level of specificity.
• Can wastewater data be used to bolster interpretation across data streams? Wastewater data are particularly useful in comparison with information emerging from other disease surveillance systems to increase confidence in the understanding of trends. Thus, an additional important metric to consider for wastewater surveillance is whether it could provide a functional alternative or a distinct data stream that can be used to validate signals for detection of emerging trends for a specific disease in a timely manner.
• To what extent can wastewater data expand the population represented by existing disease surveillance systems? Because wastewater surveillance data can be captured passively, without requiring individuals to seek care, participate in a study, or even exhibit symptoms, they may provide an opportunity to expand the population represented in existing data. Use of wastewater surveillance could be especially pertinent to unique, at-risk, or vulnerable communities that are likely to be missed through other surveillance approaches. Wastewater surveillance can also be particularly useful for diseases where mild or asymptomatic cases are common, or when clinical testing is not widely available or used.
• How likely is it that wastewater data could provide an earlier warning than other surveillance data that public health action is needed? This prospect will depend on several factors influencing the ability to monitor real-time trends in the community, including (1) the timing of the onset of pathogen shedding into wastewater; (2) the period of infectiousness; (3) the clinical course of disease (e.g., if individuals shed pathogen into wastewater before onset of symptoms); and (4) other time-varying parameters related to sample processing, detection, and reporting for a given pathogen. Lead time from reporting wastewater surveillance data to observed health outcomes (e.g., positive cases as measured in the same community, clinical disease in local healthcare settings, and local hospitalizations) will be relevant benchmarks for comparing wastewater surveillance data to other data sources for use in informing public health action.
• Are wastewater data efficient for informing public health action? It will be important to evaluate whether wastewater data are cost-effective as a replacement, as an additional source of data, or as the only available source of data. Cost-effectiveness should be weighed relative to public health importance and usefulness for decision making as prioritized above.

In some situations, the answers to these questions may be clear (e.g., if no other data about a given pathogen of interest exist). In others, determining whether to invest in wastewater surveillance data for a specific pathogen as part of a national surveillance system may require research studies to evaluate the quality, usefulness, and cost of wastewater data compared to or in tandem with other data sources. Regardless, these criteria and questions can guide how and when wastewater surveillance data may fill a gap or complement other forms of disease monitoring and public health data.

ILLUSTRATIVE APPLICATIONS OF CRITERIA

Putting the criteria outlined above into action requires careful consideration of the public health significance of the threat, the analytical feasibility of measuring the agent in wastewater, and understanding how this type of information might complement existing public health strategies to monitor the threat and inform decision making. Below, the committee presents several examples of agents of current public health concern and evaluates them against each of the criteria based on the state of the science at the time of writing. Both promising examples and examples of pathogens that do not currently meet the criteria are discussed for illustrative purposes on how the criteria could be applied. This is not intended to replace a thorough evaluation by the U.S. Centers for Disease Control and Prevention (CDC), including assessments of implementation costs relative to the value added beyond other available disease surveillance data. As the state of the science and infectious disease risk evolves, candidate pathogens will need to be reevaluated; pathogens that may not be good candidates for surveillance now might be well suited for wastewater surveillance in the future.

Promising Examples

The committee applied the criteria outlined above to several microbial threats that appear to be promising candidates for wastewater surveillance given their public health significance, analytical feasibility of measurement, and value above and beyond existing public health strategies: ongoing COVID-19 surveillance, influenza, antimicrobial resistance, and enterovirus D68 (EV-D68). In some cases, such as for ongoing COVID-19 surveillance, the criteria are mostly already met. In other cases, such as antimicrobial resistance, improvements in features such as analytical feasibility will be important to actualize a useful surveillance scheme.

Longer-Term Dynamics of COVID-19 and SARS-CoV-2 Variants

Criterion 1: Public health significance of the threat.

SARS-CoV-2 variants continue to emerge and escape established population immunity, posing a significant threat to public health. Although the numbers of hospitalizations and deaths have dropped relative to the number of infections, increased fitness for several of the emerging variants resulted in increased rates of transmission. This resulted in peak hospitalizations during the initial Omicron wave, but hospitalizations in most places in the United States have not risen comparably with subsequent omicron subvariants. Even with hospitalizations and death rates down, the health outcomes associated with SARS-CoV-2 infection in portions of the population have been significant (Ward et al., 2022), and the long-term health burden of “long COVID” continues to be poorly understood. Vaccination and natural infection with prior strains have helped to protect populations, but protection from infection seems to begin to wane after 3–4 months, and protection from severe disease may wane after 6–9 months (Dadras et al., 2022). Uptake of vaccine boosters has not been as consistent as it was for initial vaccination. Another complicating factor is that some newer variants at least partially evade immunity engendered by vaccination or prior infection and recovery. As a result, SARS-CoV-2 continues to be of major public health importance, but if cases of severe disease continue to drop and/or new variants of lower severity and possibly lower rates of long COVID emerge, this criterion will need reassessment.

Criterion 2: Analytical feasibility for wastewater surveillance.

SARS-CoV-2 wastewater surveillance has been implemented broadly, with demonstrated analytical feasibility of threat detection. As new variants have emerged, they have continued to be detectable in wastewater using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and RT-droplet digital PCR-based methods that are well established. Furthermore, sequencing-based methods are able to delineate the relative presence of different and even emerging variants in wastewater (Karthikeyan et al., 2022).

A wide range of methods for sample concentration, extraction, and analysis have been demonstrated to be “fit for purpose” for tracking SARS-CoV-2 detection trends in wastewater (Farkas et al., 2022; Maksimovic Carvalho Ferreira et al., 2022; Philo et al., 2022; Wehrendt et al., 2021; Zheng et al., 2022). However, several studies have demonstrated that performance varies considerably between methods (Chik et al., 2021; Pecson et al., 2021); thus, there is now some convergence upon optimal methods. Method standardization would bring greater reliability and validity to the data and also facilitate a broader interpretation of the data across different locations. However, standardization efforts risk stifling further innovation and improvement of methods. An alternative approach to standardization

that could provide similar strength without limiting future development is the adoption of performance validation standards (see Chapter 4).

Although detection of SARS-CoV-2 variants is technically feasible, one challenge to expanding this approach for emerging variants is that relatively little information exists on the relative shedding patterns and rates for different variants of SARS-CoV-2 and in the face of partial immunity engendered by prior infection or vaccination. Longitudinal studies in presumably infection- and vaccine-naive individuals infected with the original SARS-CoV-2 strain demonstrated that approximately half of individuals with mild-to-moderate disease shed SARS-CoV-2 ribonucleic acid (RNA) in their feces and that a subset of individuals continue to shed viral RNA for weeks to months after the original infection (Natarajan et al., 2022). Unfortunately, little is known about the dynamics of fecal SARS-CoV-2 shedding in individuals with some level of immunity (through vaccination and/or recovery from prior infection) and with new variants. This information gap confounds strong quantitative assessments. For example, it is possible that as the virus evolves, its shedding may increase, decrease, or completely cease in fecal samples.

Another consideration is that animals may also be infected by SARS-CoV-2; thus, animal waste may also contribute to both human-infecting and non-human-infecting variants that may be detected in wastewater, confounding the interpretation of the results. For example, variant typing in wastewater has identified so-called cryptic lineages containing mutations that have only rarely been detected in human clinical cases (Smyth et al., 2022), which might suggest a non-human contributing source (e.g., rats). A plausible alternative explanation is that these lineages derive from unsampled human infections (e.g., due to persistent shedding by immunocompromised individuals, or infections of different cell types). While these cryptic variants are of scientific interest, it is important to note that these variants currently represent only a minor fraction of the SARS-CoV-2 detected in wastewater. On the whole, analytical feasibility has improved for SARS-CoV-2 in wastewater, with methods available to detect multiple known and emerging variants. However, limitations in understanding shedding dynamics and the presence of non-human viral reservoirs do pose some challenges to interpreting results.

Criterion 3: Usefulness of community-level wastewater surveillance data to inform public health action.

SARS-CoV-2 wastewater surveillance is increasing in usefulness for informing public health action as SARS-CoV-2 begins to follow more of an endemic transmission pattern, with reduced clinical testing and at-home testing becoming much more common (see Chapter 2). This has led to increased use of wastewater surveillance to understand levels and trends, given its consistent ascertainment. On the

other hand, declining rates of hospitalization and death suggest that case (or infection) levels (whether measured clinically or via wastewater) may be less useful to inform action, with hospitalizations and other severity indicators taking a larger role. Nonetheless, given that changes in transmission precede changes in hospitalizations or deaths, wastewater (and case) data remain useful as a leading indicator. Additionally, a major use case for wastewater surveillance of SARS-CoV-2 is variant detection and monitoring, which has been useful for predicting upcoming increases in case load as one variant replaces another as the dominant strain circulating in a population. Wastewater surveillance data continue to be used for public health decision making, staging of resources, and planning and thus are clearly actionable. However, if severity continues to drop as the virus becomes endemic, the actionability of wastewater trend data correlated to case data may diminish. As the infrastructure for wastewater surveillance becomes more mature, the relative cost-effectiveness is expected to improve.

Summary.

In summary, disease (both acute and long-term) caused by SARS-CoV-2 is still a significant public health concern, particularly for emerging variants. SARS-CoV-2 variant measurement in wastewater is analytically feasible, although detailed information on how shedding may vary in different variants and by immune status (prior infection, vaccination) is, at present, lacking, and non-human reservoirs may also contribute to variants detected in wastewater. SARS-CoV-2 remains a good candidate for ongoing wastewater surveillance, because such surveillance provides information that is likely to be strongly complementary to the more limited clinical testing and variant sequencing that are currently being performed.

Influenza

Criterion 1: Public health significance of the threat.

Influenza is a prevalent seasonal disease that affects humans and is a significant public health threat. Seasonal drift of the genome of influenza caused by accumulation of point mutations allows influenza to pose an annual public health concern for humans. These annual seasonal outbreaks result in an average of 35,000 deaths and 200,000 hospitalizations in the United States, and between 290,000 and 650,000 deaths globally (Rolfes et al., 2018; Thompson et al., 2004). The 1918–1919 influenza pandemic resulted in 50 to 100 million global deaths. Influenza is unquestionably a significant public health threat. Beyond humans, influenza viruses also infect a number of additional host species with significant potential for enzootic and epizootic transmission. Avian influenza can be characterized as either low pathogenicity avian influenza or high pathogenicity avian influenza (HPAI). HPAI is a devastating agent in commercial bird flocks that can result in the loss of tens of

millions of birds and associated finances. Cross-species co-infection with multiple influenza strains can also result in a reassortment of the viral RNA segments, leading to antigenic shift and human pandemic potential for the virus (Kim et al., 2018).

Criterion 2: Analytical feasibility for wastewater surveillance.

The influenza pathogen can, for the most part, be readily detected in clinical samples using molecular methods such as RT-PCR. These methods have been extended to wastewater, demonstrating that surveillance is analytically feasible. Influenza can be detected in a variety of body fluids. Despite predominant transmission by the respiratory route, influenza is also associated with gastrointestinal symptoms, has been detected in the feces of some infected patients, and thus is likely to be present in wastewater (Wolfe et al., 2022; Ye et al., 2016). Avian influenza can be transmitted by the fecal-oral route in birds (Alexander, 2007). The virus is enveloped and thus somewhat biochemically similar to SARS-CoV-2, so it may also be expected to partition to wastewater solids as does SARS-CoV-2 (Ye et al., 2016). Concentration and recovery methods that have been demonstrated as “fit for purpose” for SARS-CoV-2 should also work for influenza, although this requires performance validation. Detecting and differentiating specific influenza viruses, including those that emerge seasonally via antigenic drift and those that emerge from antigenic shift and have a high pandemic potential, should be achievable using existing molecular approaches and next-generation sequencing.

Influenza virus possesses a single-stranded, negative-sense, segmented RNA genome, and this segmented nature poses a potential complication in strain typing, as multiple strains are likely to be present in wastewater. However, this is no greater problem than delineating different variants of SARS-CoV-2 and may be overcome with next-generation sequencing approaches.

One potentially complicating factor is the wide availability of a live attenuated influenza vaccine (e.g., Flumist). This vaccine replicates in the body and is likely excreted to wastewater through feces or respiratory secretions. The potential for live vaccine to confound wastewater surveillance would need to be evaluated.

Although additional research is warranted to better understand the frequency, level, and pattern of shedding to wastewater; the persistence of the virus in wastewater; and the performance of wastewater methods for broader community surveillance, influenza outbreaks have already been monitored by wastewater surveillance on a local or institutional scale (Mercier et al., 2022; Wolfe et al., 2022). As a result, there is strong evidence for the analytical feasibility of detecting influenza in wastewater.

Criterion 3: Usefulness of community-level wastewater surveillance data to inform public health action.

Influenza infections are prevalent, and because only a small subset of infected individuals present for clinical care and are captured by public health surveillance systems, wastewater surveillance data are expected to be of high use in informing public health action. Furthermore, dominant strains that infect humans often change on a seasonal basis. Although some clinical typing of strains is done, this information may lag behind initial upticks in case rates. Thus, new strains of influenza may first be detected through wastewater surveillance instead of clinical typing; furthermore, once a new strain of influenza is found to be circulating in a population, wastewater surveillance could supplement clinical and syndromic surveillance to provide early warning of spread of the virus to new regions. Even tracking of seasonal flu within populations using wastewater surveillance has the potential to inform public health decisions on communication to the public and distribution and staging of resources (i.e., vaccine clinics, hospital staffing).

Summary.

Influenza is a good candidate for potentially expanded wastewater surveillance. Flu-like diseases caused by influenza are a significant public health concern because certain strains cause significant disease, especially in immunocompromised populations, and influenza has previously caused pandemics. Influenza measurement in wastewater is analytically feasible, and wastewater detection has already been demonstrated to be useful in limited studies, although use of a live-attenuated vaccine may complicate interpretation of influenza measurements in wastewater. Wastewater surveillance may provide a complementary source of information to existing methods for influenza strain and case rate tracking using clinical information that enables better understanding of overall case rate as it includes individuals with less severe or no symptoms. Furthermore, wastewater data might serve as a leading indicator of the emergence of new influenza strains in some settings.

Antimicrobial Resistance

Criterion 1: Public health significance of the threat.

Antimicrobial resistance is a critical threat in medicine and has been declared as 1 of the top 10 health threats facing humanity by the WHO (EClinicalMedicine, 2021).¹ CDC’s Antibiotic Resistant Threats in the United States, 2019 estimates that there are 2.8 million resistant infections annually in the United States, responsible for 35,900 deaths (CDC, 2019). In addition, Clostridioides difficile infection, attributable to antibiotic disruption of the gut microbiome, is estimated

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¹ See https://www.who.int/news-room/fact-sheets/detail/antimicrobial-resistance.

to cause an additional 12,800 deaths each year (CDC, 2019). Newly emergent threats such as drug-resistant Aspergillus and Candida auris infections highlight the continually evolving nature of the threat. The direct healthcare costs associated with six common multidrug-resistant bacterial pathogens were determined to be $4.6 billion per year, with C. difficile representing another $1 billion (CDC, 2019; Nelson et al., 2021). Globally, an estimated 4.95 million deaths were associated with antibiotic-resistant bacterial infections in 2019 (AMC, 2022). Thus, antimicrobial resistance is of great public health significance (see also NASEM, 2022).

Criterion 2: Analytical feasibility for wastewater surveillance.

Detection of antimicrobial resistance in wastewater is technically feasible, although some challenges need to be overcome to make surveillance robust, extensible, and reliable. Detection of antimicrobial resistance is routinely performed in clinical bacterial and fungal isolates using culture and sensitivity-testing approaches. Additionally, molecular-based (PCR) methods are also used to amplify and detect mutations in genes that confer antibiotic resistance as well as mobile genetic elements that can confer antimicrobial resistance. Many of the molecular methods that are used to detect antimicrobial resistance may be adaptable for wastewater surveillance. Genes or transcripts encoding antimicrobial resistance mechanisms are commonly present at detectable levels in wastewater, including those of CDC-listed “urgent” and “serious” threats. The gut microbiome has been demonstrated to be an important reservoir of pathogens that infect other tissues in humans (e.g., bacteria can translocate from the gut to the blood and cause bloodstream infections; see Tamburini et al., 2018). Thus, shedding of pathogens and their antimicrobial resistance genes would be expected to occur often in individuals harboring these pathogens. The wastewater resistome (defined as the collection of antimicrobial resistance genes and mutations detected in wastewater) would be predicted to be relatively stable over time (Brinch et al., 2020), suggesting that infrequent sampling and testing could be used to assess shifts in the prevalence of antimicrobial resistance over time. Detection of antimicrobial resistance at the point of wastewater treatment provides a community-wide analysis of current prevalence and trends.

The wastewater resistome will reflect the predominant resistance genes in the community microbiome, predominantly the human gastrointestinal microbiome—although human non-intestinal microbiomes and animal microbiomes may also contribute to detectable levels depending on the community. One challenge to using wastewater surveillance for the presence, absence, and abundance of antimicrobial resistance genes is that it reflects genes that are present in both pathogens and commensal organisms (which are carried without causing disease in most individuals). Thus, the wastewater resistome as detected by amplification of specific resistance

genes or transcripts will largely reflect commensals circulating in a predominantly healthy population. Linking resistance genes to specific pathogens is certainly feasible but, in most cases, will require either long-read sequencing or bacterial culture on selective media followed by resistance gene amplification—more costly, labor-intensive approaches. Thus, neither approach is amenable to routine wastewater surveillance but may be useful if triggered by specific hospital or community disease outbreaks (see Box 3-2).

Many clinically relevant antimicrobial resistance genes are carried on plasmids, which are pieces of DNA that replicate separately from the bacterial chromosome and may be transferred between related and sometimes unrelated organisms. Detection of these antimicrobial resistance elements may be relevant whether they are found in commensal organisms or in pathogens. This is because the overwhelming majority of multiantibiotic resistance is carried on mobile plasmids that can move from non-pathogenic commensal bacteria to pathogenic bacteria. Additionally, even commensal

bacteria can cause severe infections in certain immunocompromised individuals when carried into a hospital or acute care nursing facility. CDC has prioritized carbapenem-resistant Acinetobacter and carbapenem-resistant Enterobacterales as “urgent threats” and extended spectrum beta-lactam-resistant Enterobacterales as a “serious threat” (CDC, 2019). The genetic determinants responsible for encoding these resistances are carried on mobile plasmids and are commonly detected within commensal bacteria within communities—thus amenable to wastewater surveillance and actionable in terms of informing healthcare facilities of the risk in susceptible patients. This value is supported by research that has shown that wastewater detection roughly parallels detection of resistance in hospitals within the wastewater catchment (Parnanen et al., 2019).

Criterion 3: Usefulness of community-level wastewater surveillance data to inform public health action.

Wastewater surveillance data on antimicrobial resistance are likely to be useful to inform public health action, though the exact ways in which they complement existing data sources and result in specific actions have yet to be defined. Thus, while promising, wastewater surveillance of antimicrobial resistance may not yet be ready to put into use at this time. Although the wastewater resistome is expected to be relatively stable over time, there are specific use cases when targeted sampling and analysis might be highly valuable. An example is the detection of a previously unknown multidrug-resistant infection in a clinical setting (see Box 3-2). Relatedly, as new resistant bacteria have frequently been detected outside the United States (e.g., NDM-1 beta-lactamase, which confers broad resistance to beta-lactam antibiotics; Yong et al., 2009), targeted screening at community wastewater facilities linked to or serving international points of entry would provide an early awareness signal to healthcare facilities and clinical laboratories. In U.S. clinical laboratories, detection of resistance is largely automated and screens for currently known resistance patterns based on “antibiograms,” which use local or regional aggregate data. The emergence of a new resistant mechanism would not be included in automated testing and requires use of a non-automated test. Awareness of emergence detected in wastewater would alert clinical laboratories to screen for resistant bacteria otherwise undetectable based on historical antibiogram data. Analysis of human waste collected from incoming aircraft has provided geographically defined patterns of resistant determinants and may represent a source of introduction into a community (Nordahl Petersen et al., 2015). These data would complement data from the Global Sewage Surveillance program that has been testing sewage for antimicrobial resistance markers in 60 countries since 2016 (Aarestrup and Woolhouse, 2020; Hendriksen et al., 2019).

Summary.

Fecal shedding of pathogens and their antimicrobial resistance genes is expected to occur, and their measurement in wastewater is analytically feasible. However, the high prevalence and range of antimicrobial resistance genes present in commensal organisms may make it difficult to identify relevant increases of antimicrobial resistance genes above the very high background rate that is detected. Antimicrobial resistance is of high public health significance, and if analytical advances allow more rapid and cost-effective mapping of resistance to specific pathogens, surveillance in wastewater would be of even higher value. Furthermore, as antimicrobial resistance frequently emerges in “hot spots” outside the United States and then spreads globally, wastewater surveillance at sentinel sites such as airports may serve as an early warning signal for additional screening of newly emergent antimicrobial-resistant pathogens. In summary, antimicrobial resistance is a promising candidate for future wastewater surveillance system development, though some challenges and exact applications have to be further investigated and defined.

Enterovirus D68

Criterion 1: Public health significance of the threat.

EV-D68 is a non-polio enterovirus that is of moderate public health significance. EV-D68 has circulated in the United States since at least 1962 and has been linked to biennial seasonal outbreaks in the fall since 2014 (Helfferich et al., 2019). The virus is associated with a range of clinical illness, ranging from mild acute respiratory disease to a severe polio-like paralysis (acute flaccid myelitis, AFM) (Sooksawasdi Na Ayudhya et al., 2021). Although the virus is a relatively rare cause of significant respiratory disease, the unprecedented severe cases in young children have made this a virus of significant public health concern, despite the lack of clear information on its prevalence, distribution, and transmissibility. This virus is spread through respiratory droplets via direct person-to-person contact and indirect contact through touching of contaminated surfaces, raising concern for high transmissibility. The virus is expected to demonstrate a high degree of asymptomatic infection.

Criterion 2: Analytical feasibility for wastewater surveillance.

Detection of EV-D68 in wastewater is expected to be analytically feasible, though the few existing methods for its detection in wastewater are not quite as developed as they are for other enteroviruses such as poliovirus. Enteroviruses commonly infect humans, and existing clinical methods that leverage RT-PCR for virus detection are used to detect viral strains of particular concern in patients. Thus, it should be relatively straightforward and feasible to expand these existing approaches to develop a wastewater surveillance approach for the virus. In fact, a recent study by Tedcastle et al. (2022) described

detection of EV-D68 in wastewater samples in the United Kingdom; furthermore, studies from Israel and Scotland have already demonstrated wastewater surveillance for EV-D68 (Erster et al., 2022; Majumdar et al., 2019; Weil et al., 2017).

In addition to RT-PCR approaches for virus detection, sequencing of the viral envelope protein 1 (VP1) region of the viral genome allows EV-D68 to be distinguished from other enteroviruses. Thus, it is feasible to develop a next-generation sequencing approach to both detect and characterize the virus in wastewater. Such an approach would require amplification of a specific region of the genome (amplicon) and subsequent sequencing of that region to both detect and differentiate this virus from other closely related enteroviruses. As a non-enveloped RNA virus that can likely be detected using standard RNA extraction and either sequencing or RT-qPCR or droplet digital RT-PCR methods, EV-D68 would be easily adaptable to cost-effective inclusion in a wastewater surveillance panel.

Like other enteroviruses, EV-D68 infects the gut and thus may be detected in stool. However, the level and magnitude of shedding is not clear. Although little is reported on the stability or persistence of EV-D68 or components of the virus such as RNA or antigens in wastewater, as a non-enveloped virus, EV-D68 and its RNA would be expected to be relatively stable. Past studies on other enteroviruses have shown that they may be stable for days to months in wastewater. At least one prior study in Israel used both clinical and wastewater-based disease surveillance in the investigation of EV-D68 (Erster et al., 2022). Furthermore, the biennial pattern of outbreaks since 2014 suggests an ability to design a study to calibrate the environmental signal to clinical outcomes. No animal reservoirs of EV-D68 have been described; thus, no other contributing sources to wastewater are expected other than human infections.

Criterion 3: Usefulness of community-level wastewater surveillance data to inform public health action.

Wastewater surveillance data about EV-D68 are expected to be highly complementary to the limited existing public health data on this pathogen and are expected to inform public health action. Because EV-D68 infection often is asymptomatic or minimally symptomatic and severe respiratory disease or AFM as a consequence of EV-D68 is not reportable from a public health perspective, little is known about the prevalence and seasonality of this virus in the community. Thus, inclusion in wastewater surveillance could contribute significantly to understanding the biennial pattern of outbreaks and raise awareness for the virus as an etiologic agent in the face of other more commonly circulating viruses. Identification of an uptick in EV-D68 could, for example, help to warn hospitals to watch for clinical manifestations of the virus. It also could help to stage resources for epidemiological investigation.

Summary.

The diseases caused by EV-D68 are a significant public health concern. EV-D68 measurement in wastewater is analytically feasible, and shedding occurs at a high enough rate that detection has already been demonstrated to have value in limited studies. Given the lack of alternative methods to measure EV-D68 prevalence and burden in different communities, wastewater surveillance is expected to be of high possible usefulness. In summary, EV-D68 is a promising candidate for expanded and improved wastewater surveillance.

Examples of Pathogens That Are Not Currently Applicable or Need More Data

Applying the criteria outlined above, many microbial threats do not currently meet the level of public health significance, analytical feasibility of measurement, and adequate value above and beyond existing public health strategies required to be considered for broad implementation. As an example of how the criteria above might be applied to evaluate such candidates, two examples—Candida auris and prions—are explored below in detail. In these examples, it is evident that some criteria are fully or partially met, but others fall substantially short. Both of these currently questionable candidates are evaluated below to illustrate how to apply these three criteria in public health decision making as it relates to candidate selection.

Candida auris

Criterion 1: Public health significance of the threat.

Candida auris is an emerging fungal pathogen (yeast) that can cause a range of infections from mild superficial infections to severe invasive infections. C. auris is of high public health significance as it has been linked with serious outbreaks in healthcare settings and is frequently resistant to commonly used antifungal drugs. C. auris is a significant risk for those in clinical settings and nursing homes, particularly those undergoing invasive procedures, but infections have been detected across age groups. Due to the multidrug resistance, this is a significant pathogen of concern. However, most cases have been linked to hospital- or care facility-based exposures, and little is known about colonization of individuals outside the clinical setting. C. auris seems to be highly transmissible in a clinical setting as evidenced by reported outbreaks. It is expected to be spread by person-to-person contact in healthcare settings but has been shown to be persistent in the environment and may also be indirectly spread by contaminated surfaces in healthcare settings. The disease may be severe, with death resulting in as many as one in three

cases.²C. auris now has international distribution, having been reported in half of the U.S. states and more than 30 countries.

Criterion 2: Analytical feasibility for wastewater surveillance.

It is unclear whether existing analytical methods and the level of shedding of C. auris into wastewater are adequate for detecting this microbial threat in wastewater. Detection of C. auris in the clinic is performed by fungal culture and mass spectrometry, or by molecular PCR-based methods that amplify and then sequence the D1–D2 region of the 28S ribosomal RNA gene (rRNA) or the internal transcribed spacer (ITS) region of the rRNA. Although culture-based methods are likely not expandable to wastewater surveillance, amplicon sequencing-based methods may be valuable for detecting C. auris, if it is present at a high enough concentration to be detected. Recent research has demonstrated that Candida species can be detected in hospital wastewater (Mataraci-Kara et al., 2020), although whether such organisms can be detected in community wastewater treatment facilities remains unknown. Thus, although some likely portable molecular methods exist for detection, considerable research is necessary to demonstrate the feasibility of methods, and the presence and persistence of the yeast in wastewater. Currently no methods have been described for the isolation or detection of C. auris from wastewater. Direct extraction of the fungal nucleic acid from wastewater may be feasible, but it is not clear whether C. auris would be present at high enough levels to allow detection.

The presence of C. auris in human stool is not well described in humans, although mouse studies suggest that fungal burden in stool is higher for some invasive strains of C. auris than for other strains (Abe et al., 2020). Because the fungus can colonize the skin of individuals, C. auris shed with sloughed skin (e.g., from hand washing or showering) may be a source of loading into wastewater. Based on the available data describing known niches for C. auris infection and colonization in humans, no other contributing sources of C. auris to wastewater have been demonstrated. The best available data also suggest that the concentrations of C. auris in wastewater are likely rather low, which might challenge detection, even with amplification-based methods. Furthermore, it is unclear whether the agent is stable or perhaps can replicate in wastewater, though it has been demonstrated to persist on environmental surfaces.

Finally, because colonization can occur in the absence of infection, it is unclear whether wastewater data would be correlated with clinical outcomes, and no data exist to suggest that levels in wastewater relative to clinical outcomes would be consistent over time. That being said, as

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² See https://www.cdc.gov/fungal/candida-auris/candida-auris-qanda.html.

colonization rates increase, wastewater data may correlate with increased exposure for susceptible hosts and thus increased infections.

Criterion 3: Usefulness of community-level wastewater surveillance data to inform public health action.

Information on C. auris presence and abundance in different geographic communities is expected to be of high usefulness, as little other information is available regarding the distribution and abundance of this organism. C. auris became a notifiable condition in 2018 in the United States. At present, limited institutional screening for C. auris is being carried out, mostly in individuals who are strongly suspected of being colonized with the fungus. As infection with the agent can range from mild to severe, it is expected that the current rates of C. auris infection that are reported are an underestimate of the actual population-based burden of infection or colonization with the pathogen. Thus, wastewater surveillance data, if accurate and quantitative, might be helpful. Furthermore, very little is known about community prevalence of the agent outside clinical settings. It is possible that wastewater surveillance could help inform a better understanding of the distribution of the agent, but it is still not clear how it would be actionable. For the time being, most cases of C. auris seem to occur in hospital and long-term acute care settings; thus, more proximal and local wastewater surveillance at these institutions may be preferable to broad-scale regional wastewater surveillance.

Summary.

C. auris is a questionable candidate for wastewater surveillance at present, given limitations in analytical feasibility of detection and the fact that it is unclear how much this organism is shed into wastewater. The infections caused by C. auris are a significant public health concern, and the broad-scale antifungal resistance of this pathogen makes it an agent of particular concern. Additionally, there is a lack of alternative, thorough methods to measure C. auris prevalence and burden outside of hospital and long-term acute care facilities where patient screening might be performed. If research and development resolve the uncertainties and challenges for C. auris detection in wastewater in low-prevalence settings, wastewater surveillance might be of moderate to high value, particularly if the population prevalence of C. auris increases in coming years.

Prions

Criterion 1: Public health significance of the threat.

Prion diseases are rare but can be very severe, and as such, they are of moderate overall public health significance in most settings. Prions are transmissible proteins with an abnormal conformation. They can trigger an abnormal folding of native cellular proteins in the brain, resulting in transmissible spongi-

form encephalopathy diseases (TSEs). These diseases primarily target the central nervous tissue and are usually rapidly progressive and fatal. Both human and animal TSEs exist, with concerns about the potential for cross-species transmission. In animals, the spread of the proteins is believed to be through bodily fluids, either via direct contact or indirectly through a contaminated environment. In humans, most TSEs are genetically inherited or arise spontaneously within an individual, but there are types of prion disease that result from infections acquired from others or the environment. Specifically, transmission of the rare but severe Kuru disease and variant Creutzfeldt-Jakob disease is believed to be through ingestion of infected meat. Due to the rare nature of the human disease and the poor transmissibility of the agent, most TSEs are unlikely to pose a broad and significant public health concern. However, chronic wasting disease in deer may pose a risk to hunters and others handling or consuming felled deer, and many states in the northern Midwest have surveillance for chronic wasting disease using neural tissue from killed deer.

Criterion 2: Analytical feasibility for wastewater surveillance.

There are substantial challenges to the analytical feasibility and robust and reliable detection of this agent in wastewater. Diagnosis of prion disease is made by immunocytochemical or protein-based detection methods performed on brain biopsies. PCR-based methods are not applicable because the agent is protein based. Prion-protein detection can be carried out by standard biochemistry methods such as immunoblotting (Nicholson, 2015; Yokoyama, 1999). To date, no methods have been described for the detection of prions in wastewater. Detection of prion proteins in wastewater might be possible through antigen-based detection (e.g., enzyme-linked immunosorbent assay, ELISA) in matrices that have prion protein present. Alternatively, there is a protein misfolding cyclic amplification assay (Green and Zanusso, 2018), but it is not clear how this would be feasible to adapt to wastewater. Furthermore, it is unclear how the prions would be isolated from wastewater samples for analysis.

Although there are reports of fecal shedding of prions by deer and goats (Cheng et al., 2016; Haley et al., 2011; Krüger et al., 2009; Miller and Williams, 2004; Safar et al., 2008; Tennant et al., 2020; Terry et al., 2011), there are no reports of fecal or other shedding of prions in humans (although it is plausible). It is not clear that animal prions could be differentiated from human prions in wastewater, though presumably methods such as ELISA can use antibodies that differentiate between the different proteins that cause animal versus human prion diseases. Should differentiation of prions from animal versus human sources be challenging, it is important to note that animal wastes are unlikely to be a large contributing source in most wastewater systems, although prions from rodents, domestic pets,

and wastewater flow from slaughterhouses could be contributing sources. Prions are extremely stable in the environment; they resist degradation, disinfection, and treatment processes. If present in sewage, they would be expected to persist.

Criterion 3: Usefulness of community-level wastewater surveillance data to inform public health action.

At present, wastewater surveillance data on prions are not expected to significantly contribute to informing public health action. Most TSEs are not transmitted but rather are inherited or arise spontaneously within an individual. Due to the fact that TSEs are rapidly progressing conditions, infected individuals would likely seek out medical care, and as a notifiable condition, information would be conveyed to state and national public health agencies. It is also unclear how any wastewater surveillance data could be broadly acted upon by public health agencies due to the rarity of the disease.

Summary.

The diseases caused by prions are severe and important but of low current prevalence with poor transmissibility and therefore of lower general public health concern. Prion measurement in wastewater is not currently analytically feasible, although biochemical methods exist that could enable method development, and the data are currently expected to be of limited value for public health management of prion diseases.

VISION FOR AN EFFECTIVE FRAMEWORK FOR DETERMINING TEMPORAL AND SPATIAL RESOLUTION

When developing a vision for a national wastewater surveillance program, a key consideration is its spatial and temporal resolution—that is, how frequently and from which locations samples should be collected to provide optimal cost-effectiveness and usefulness of the data. The temporal and spatial resolution of sampling needs to be determined based on the objectives of the program (e.g., the use cases in Box 3-1), which will be a function of the pathogen(s) of interest, the analytical methods, and both the epidemiology and pathogenesis of the disease. A typical set of objectives for the overall surveillance program could be determination of infection and disease prevalence and ascertainment of “hot spots” and/or “hot moments.” The relative variability of a target pathogen in space and time and the value of understanding this variability needs to be balanced with the expense of collecting and analyzing at different sites and times. Temporal and spatial variability will be discussed, followed by a potential path forward to design an overall sampling strategy.

Temporal Variability

Temporal variation is an important consideration in determining a sampling frequency that is affected both by changes in flow within a sewer system as well as the timescales involved in changes in disease transmission patterns. For example, different pathogens have different speeds at which an outbreak might progress or seasonal patterns that affect the needed frequency of sampling over the course of the year. Each of these sources of temporal variation will affect the sampling frequency needed to capture useful wastewater surveillance information for different pathogens.

It is well known that influent flow rate and composition coming into wastewater treatment plants fluctuate owing to daily and weekly variations in contributions to the sewer system, which can affect pathogen measurements in wastewater (Wade et al., 2022). Additionally, the relative extent of these variations tends to be larger in smaller treatment plants owing to an averaging effect that occurs in the collection system (Tchobanoglous et al., 2003). In wastewater collection from combined sewers or older leaky sewers, there could also be variability with storm events.

As an example, Li et al. (2021) took daily samples of liquid and solids from raw wastewater following the imposition of more stringent public health measures in August 2020 (see Figure 3-2). There was clearly a downward trend, corresponding to attenuation of community spread of COVID-19, but there were also daily fluctuations about the trend of about 1–2 logs. Mendoza Grijalva et al. (2022) conducted hourly sampling for SARS-CoV-2 in the influent to a treatment plant in Contra Costa, California. They observed a greater likelihood of detection during periods of peak diurnal flow (under dry weather conditions) and recommended collaboration with the wastewater utility to ascertain timing of usual peaks. The fluctuations suggest the reasonableness of daily composites, preferably flow weighted, to achieve detection, although this finding needs to be checked for any new pathogens that are studied. The frequency of sampling would need to be determined based on the use case and the pathogen of concern.

Infectious disease transmission also changes at a range of different timescales, depending on the characteristics of the population and the pathogen (Anderson and May, 1992; Delamater et al., 2019). Population characteristics such as the level of population movement, population density, and age and other demographic factors that influence susceptibility and immunity may all affect transmission patterns, causing outbreaks and other changes in transmission patterns to occur over different timescales. For example, an outbreak could proceed rapidly through a highly susceptible, dense population but move much more slowly through a population with lower contact rates and higher immunity. Additionally, each of these

factors may change over time, adding additional complexity to the choice of sampling frequency. Furthermore, pathogens have different transmission routes, incubation periods, infectious periods, and other natural history characteristics that mean that the timescale of changes in transmission for different pathogens can vary widely, with some having outbreaks that span days or a week or two, and others that move through a population over the course of months. Different pathogens have different temporal distributions in when and how long they are shed, and how that relates to the clinical manifestation of disease (e.g., some pathogens are shed before onset of symptoms, some may continue to be shed after symptoms resolve). Further, these temporal shedding patterns may evolve over time as the pathogen evolves.

All of these factors will impact how rapidly an outbreak will spread and how frequently samples should be taken to determine important changes in disease transmission. For example, changes that occur over months may only need weekly sampling, while a rapidly evolving outbreak where action is needed more immediately might require semiweekly or daily sampling. The useful sampling frequency for a given pathogen may vary as a function of the season or upon detection (e.g., less frequent sampling and analysis that shifts to frequent sampling if a pathogen is found).

Spatial Variability

Spatial factors can result in variability in wastewater surveillance data over regional and national scales. For example, samples from wastewater treatment plants serving larger populations do not provide the same level of granularity as those from a plant serving smaller populations (Sharara et al., 2021), although they do capture a wider population and may avoid some of the noise and variability of plants serving smaller populations. Even if the wastewater treatment plants sampled served the same population size, multiple demographic and environmental factors are spatially heterogeneous, including

• annual precipitation;
• proportion of domestic versus nondomestic flow;
• proportion of flow attributable to hospital, healthcare, and congregate living facilities;
• combined versus separate sewers;
• population demographic and socioeconomic factors (e.g., socioeconomic status, social vulnerability, and urban/rural patterns);
• proportion of people who commute to work or school from outside the sewershed; and
• spatial heterogeneity in disease spread patterns.

These factors may vary at the sewershed or sub-sewershed level (see Box 1-3) or may vary at a range of spatial scales from census tract to regional or state level (e.g., regional laborshed and commuting patterns, or spatial variability in contact patterns that influence disease transmission). This variability in the different epidemiological and wastewater features across space can affect the optimal design of a wastewater surveillance system, and the choice of a spatial scale and resolution needed to accurately capture trends and patterns may vary by pathogen and use case (see example use cases in Box 3-1). For example, if a disease tends toward highly localized, distinct outbreaks or clusters (highly spatially heterogeneous), and population contact patterns mean that there is relatively little spatial

mixing of the population, one may need a highly spatially resolved wastewater surveillance system to localize transmission hot spots in a way that is actionable for public health. On the other hand, for SARS-CoV-2 and other pathogens that tend to show broad-scale community spread or where population contact/mobility is high, it may be sufficient to have fewer, potentially larger sewersheds that are monitored and provide information about overall population trends in an actionable way (although analysis of CDC’s National Wastewater Surveillance System [NWSS] data would be needed to determine the optimal sampling plan).

Data from the current sampling program and related efforts that measure different pathogens across multiple sites (e.g., mpox, influenza, respiratory syncytial virus [RSV]) could be analyzed to ascertain how spatial variability in the factors listed above may impact the understanding of disease transmission for different use cases (e.g., to discern trends, detect new outbreaks). This information would assist in the design of a cost-effective, representative sampling framework for the nation and help assess the value of an adaptive framework, wherein some combination of spatial scales is regularly monitored but then more finely resolved as needed once a pathogen or trend of interest is detected.

Considerations for Designing a Representative Sampling Framework

A foundational question in the design of a representative strategy is the articulation of the objectives and their desired weighting. As stated by Cheng et al. (2020),

The allocation of wastewater surveillance sampling effort over time and space can also be viewed as an optimization question. Two other questions then arise in the design of a sampling program that has the objective of discerning prevalence and trends:

1. How “dense” in space should the sampling sites be, and how many should be set up?
2. How frequently should each site be sampled?

The temporality/frequency of sampling will depend on the dynamics of the particular pathogen and disease and the objective of the surveillance system. As shown in the Honolulu COVID-19 data in Figure 3-2, the decline in wastewater concentration with time during a “lockdown” was discernible with daily sampling and likely would have been discernable had the sampling been twice or thrice a week. The question for another pathogen would be whether a more rapid discernment of trend has value in terms of public health actionability that would justify the increased effort.

The density of sampling in space depends upon how readily more sparse allocation of efforts can discern prevalence and trends compared to a denser allocation. This may differ in parts of the United States due to demographic differences over geographic areas. In some cases, community mobility may be sufficient so that populations who reside in unsewered areas or in small separate sewersheds may contribute to a large city’s wastewater system where they work or attend school, such that centralized wastewater surveillance could still sample this population to some degree. Additionally, statistical tools may be useful to extrapolate available data to unsampled areas. As shown by Fairchild et al. (2013), in the case of clinical influenza sampling in Iowa, it is possible for sparser (and hence less expensive) networks to provide data of similar information value to denser networks. The NWSS and similar data could be analyzed to shed light on these factors.

Consideration of equity of sampling with attention to environmental justice populations needs to be embedded in site selection. Equitable action to improve public health requires information from a disease surveillance system that represents and resolves patterns for a range of populations across sociodemographic groups, particularly those who may not be as well-reflected in traditional clinical surveillance methods that may be impacted by issues of access to and availability of care, issues of trust, and issues of cost. There is the potential for wastewater surveillance to provide a more fully representative lens through which to conduct public health surveillance, as it does not require active participation by individuals. Such data could be used independently or compared with clinical data to understand disease epidemiology. Importantly, there is also an ethical trade-off relating to the spatial scale at which wastewater surveillance is conducted: sampling at the larger community level helps avoid stigmatization of particular neighborhoods and minimize threats to individuals’ privacy, but examining smaller sewersheds could help target resources and useful public health efforts to the areas that need them most (see Chapter 4 for additional discussions of ethics and privacy).

A full national picture of disease distribution will require comparative analysis of wastewater data with other sources of information. As discussed previously in this chapter, there are significant populations that are not con-

nected to municipal wastewater systems, and these unsewered populations tend to be more rural. Rural areas also tend to have lower median incomes and reduced access to health services than sewered populations [Indiana ACIR, 2019; Long et al., 2018]. These findings underscore the importance of finding alternative approaches for disease surveillance in these communities to avoid potential health disparities. Furthermore, not all sewered jurisdictions would institute a wastewater surveillance system, due to cost, staffing, or cost-effectiveness considerations or unwillingness to participate. Therefore, community-based wastewater surveillance should be viewed as only one tool in national disease surveillance. An aggregate disease surveillance program can achieve equity, even if any single component, such as wastewater surveillance, may not cover all components. Design of the best complementary tools for the unsewered population is important but beyond the scope of this study.

The bottom line is that the temporal and spatial resolution of a sampling program should be subject to intentional design, should be informed by preliminary and ongoing data, and may differ with different pathogens and use cases. Without thorough statistical analysis of existing data, CDC cannot be sure that, moving forward, its investments are appropriate to achieve representative information.

Sentinel Sites

Wastewater-based surveillance for SARS-CoV-2 was initially set up and continues to be carried out in a range of settings, from highly localized efforts at universities or prisons to aggregated and more standardized surveillance spanning hundreds of wastewater treatment plants across the nation. Although this report focuses on the importance of a national wastewater surveillance system, hyperlocal surveillance at selected sentinel sites in certain circumstances would be an integral element of a true national system. A sentinel site here refers to a location where enhanced or specific surveillance should take place because it represents the “front line” of entry to a larger community. Wastewater infectious disease surveillance information gathered as close as possible to where targets of interest enter a community may have a differentiated value from that obtained from a non-selective community wastewater surveillance system. For example, wastewater surveillance at major U.S. airports and ports of entry would identify initial cases for pathogens from other regions in travelers entering the country (Agrawal et al., 2022; Medema et al., 2020). At these sites, an abundance of global travelers in a localized setting could serve to enrich targets of interest that may be too dilute to detect in larger wastewater surveillance systems prior to significant disease transmission. Furthermore, it may not be cost-effective to perform surveillance on a broad range of

global targets of interest at a national level across a broad network, but focused surveillance of a much larger number of targets could be carried out at these more limited sentinel sites, while serving the interests of the nation as a whole.

Given the unique function of sentinel sites within a larger public health system, these sites may differ from regular network sites within a national system both in terms of the types of pathogens targeted and the frequencies at which sampling would occur. Sentinel sites could serve to screen for a wide range of potential pathogens or diseases of concern in other countries, including emerging pathogens. Initial pathogen detection at one or more sentinel sites could trigger expanded wastewater surveillance at communities or broad-scale national surveillance, as appropriate for the pathogen detected. Because of the need to be responsive to the emergence of potential threats in a timely manner, the frequency of sampling at these sites would benefit from being more frequent and/or dynamic in response to global public health information. Sentinel sites could thus be activated with rapid upscaling of sampling efforts under specific circumstances that would justify enhanced screening.

The process of selecting sentinel sites depends largely on the target that is under consideration. Large airports with a high flux of global travelers may be appropriate sentinel sites for global surveillance of pathogens and antimicrobial-resistance genes that are prevalent outside of the United States. Temporarily ramped up sentinel wastewater surveillance should also be considered to coincide with large-scale gatherings that could facilitate disease introductions or transmission, such as at international sporting events. However, these are not the only types of sentinel sites that should be considered. Because emerging pathogens and antimicrobial-resistance genes can arise from animals, another type of sentinel site that would be important to evaluate is animal-intensive areas such as livestock and poultry farms and large zoos (if runoff from animal enclosures is collected into the sewershed). Because of the need for a variety of sentinel sites based on these targets, it would be advisable to include among these sentinel sites both major cities and small, rural wastewater treatment plants. As diseases emerge, or after they have emerged, correlating local wastewater surveillance data with case and hospitalization data would inform how these types of surveillance systems should scale.

In the committee’s vision for a national community-based wastewater surveillance system, the development of sentinel sites has clear additive value. These sites may provide early warning of emerging threats in the United States before they reach the general population and could directly inform subsequent scaling of surveillance for new or re-emerging pathogens in the broader national surveillance system.

CONCLUSIONS AND RECOMMENDATIONS

Wastewater surveillance is and will continue to be a valuable component of the nation’s strategy to manage infectious disease outbreaks, including continued surveillance of SARS-CoV-2 variants, resurgences of known pathogens, and newly emergent pathogens. The emergency establishment of wastewater surveillance has proven its value, and the efforts at local and national scales to establish the NWSS provide a solid basis for expanded applications. Infectious diseases, whether endemic, seasonal, newly emergent, or re-emergent, are dynamic and never fully predictable. The high likelihood that SARS-CoV-2 variants will continue to emerge and circulate is alone a strong rationale to maintain and strengthen a national wastewater surveillance system. The recent use of wastewater surveillance for poliovirus and mpox in mid-2022 illustrates the advantages of a maintained national system for detecting re-emerging pathogens and pathogens recently introduced into the United States.

To achieve its goals, a national wastewater surveillance system should be flexible, equitable, integrated, actionable, and sustainable. Flexibility includes the ability to track multiple pathogens simultaneously and pivot quickly to new threats. A national wastewater surveillance system should be as equitable as possible across population demographics, with efforts to engage underrepresented communities and extrapolate findings, where feasible, to unsewered communities. Integration, including coordination and collaboration across multiple partners (e.g., utilities, laboratories, and public health agencies) and analysis of data from different disease surveillance systems, ensures effective data interpretation in support of public health decision making. For the information to be actionable and inform decisions about clinical and public health resource allocations as well as policy decisions, it must also be timely, available, reliable, representative, and interpretable. Finally, the system needs to be fiscally and operationally sustainable. Although the NWSS supports both local and national public health decision making, a sustainable national wastewater surveillance program may not serve every locality’s objectives but should allow for locally funded initiatives, such as pilot surveillance of a pathogen of emerging regional concern.

When evaluating potential targets for future wastewater surveillance, CDC should consider three criteria: (1) public health significance of the threat, (2) analytical feasibility for wastewater surveillance, and (3) usefulness of community-level wastewater surveillance data to inform public health action. Applying these criteria to known and emergent/re-emergent pathogens of concern can guide strategic allocation of effort and resources. Assessment of the public health significance of a microbial threat is important to develop and maintain a system that is responsive to current public

health needs. Assessment of the feasibility to detect a specific pathogen in wastewater for disease surveillance is necessary to determine technical readiness and can also drive research or technology development for microbial threats that meet the other criteria. Finally, it is critical that the value of wastewater surveillance information for a given pathogen be considered in the context of the broader universe of surveillance approaches so as to maximize the use of resources to inform public health action (e.g., allocation of clinical or public health resources). Candidate pathogens will need to be re-evaluated periodically as scientific knowledge, technology, and infectious disease risks evolve.

Temporal and spatial resolution of the NWSS sampling program should be subject to intentional design, informed by rigorous and iterative analysis of data for prioritized pathogens. Collaborative and frequent analysis of incoming NWSS data is essential to determine the spatial and temporal scales of sampling and analysis needed, both for effective COVID-19 monitoring as well as detection of emerging pathogens. Temporal and spatial resolution should be regularly re-evaluated to ensure the system is capable of detecting meaningful change with sufficient lead time needed to inform public health action. CDC should also give careful attention to the need for more representative sampling for prioritized use cases. Currently, the system consists of localities, tribes, and states that were willing and able to participate during a pandemic emergency, and this current distribution of sampling sites might not be representative of the range of demographic and geographic characteristics desired in a national network nor equitable, optimally actionable, or sustainable. Because 16 percent of the U.S. population resides in unsewered communities, wastewater surveillance in and of itself cannot be fully representative of the population but should be viewed as one key component of a national infectious disease surveillance system.

CDC should take additional steps to bring the benefits of wastewater surveillance to critical areas not addressed by the NWSS. The committee identified three steps that CDC could take to ensure that resources expended on wastewater surveillance systems are not distributed inequitably. First, CDC should create a comprehensive outreach program to provide information to selected public health officials and utility personnel in localities that are not currently using wastewater surveillance about the potential benefits of joining the national system. Second, CDC should reduce financial and staff capacity barriers to joining the system. CDC could reduce barriers by providing continued and expanded funding to state, tribal, local, and territorial health departments and utilities and by creating an easily operable data management and analysis system wherein local wastewater surveillance programs can easily transmit their samples and data for centralized analysis and data visualization (see Chapter 4). Finally, because some areas that are

important to understanding national infectious disease transmission will remain outside the wastewater surveillance system even with these resources in place (e.g., in unsewered areas), CDC should assess whether tools can be used to extrapolate data from monitored regions to estimate disease burden in areas without wastewater surveillance. CDC and local health departments should also maintain robust infectious disease surveillance programs using other sources of data on disease trends and provide public education about how to interpret wastewater data alongside other indicators.

As part of a national wastewater surveillance system, strategic incorporation of sentinel sites is recommended as a mechanism for early detection. Sentinel sites should be intentionally selected to monitor for specific emerging pathogens at their points of entry into human communities. Sites that can directly inform community wastewater-based surveillance, especially as related to emerging pathogens, will provide important and distinct benefits in the context of a national surveillance network. Such sentinel sites could include wastewater surveillance at major international airports with a large number of global travelers to detect emerging pathogens and antimicrobial resistance genes. Sentinel monitoring at ports of entry could allow early detection of emerging pathogens entering the country that otherwise may be too dilute to detect at the community scale. Wastewater treatment plants with zoos or major livestock farms that contribute to a sewer system could also serve as valuable sentinel sites to detect the emergence and transmission of zoonotic pathogens. Developing useful sentinel sites will require careful planning and thoughtful experimentation with site selection, program design, and data interpretation based on the pathogen(s) of interest. Sentinel sites are a cornerstone of any public health system, and the NWSS should seek to incorporate these sites in a way that will ensure the surveillance system is nimble and adaptive as needed to address emerging threats.