5

Potential Target Expansion for National Endemic Disease Surveillance

In this chapter, the committee discusses the potential expansion of endemic targets for national wastewater surveillance. Over the past 12–18 months, the Centers for Disease Control and Prevention (CDC) has been actively exploring options for expanding endemic targets in the National Wastewater Surveillance System (NWSS) beyond COVID-19. The NWSS added Mpox monitoring (see Figure 4-7) and is piloting analysis of two other respiratory viruses targets—respiratory syncytial virus (RSV) and influenza A/B. If added to the NWSS core panel, all sites that use the national laboratory contract for analysis (i.e., approximately 9 percent of NWSS sites) would have their samples analyzed for these pathogens. Sites that conduct their own laboratory analyses could choose whether to analyze for the new pathogen targets. WastewaterSCAN,¹ which represents approximately 12 percent of NWSS sites, is currently analyzing 10 pathogen targets in addition to SARS-CoV-2: influenza, RSV, human metapneumovirus, parainfluenza, norovirus GIII, rotavirus, enterovirus D68, Mpox, Candida auris, and hepatitis A. The data from WastewaterSCAN surveillance will be extremely helpful in decision making and prioritizing additional targets for the NWSS in terms of analytical feasibility, correlation with population disease incidence, and public health action. This chapter discusses the potential value of specific targets under consideration for inclusion in the NWSS at a national scale as well as research needs for broad implementation.

In general, the additional endemic pathogens under consideration within the NWSS for national-scale surveillance include common respiratory

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¹ See https://www.wastewaterscan.org/en/pathogens.

pathogens, enteric pathogens, and antimicrobial resistance genes. While respiratory and enteric infections may be seasonal or confined to localized outbreaks, their prevalence is sufficiently common in the United States to be considered endemic. Emerging pathogens with epidemic or pandemic potential (e.g., H5N1 avian influenza) and those causing sporadic, localized, or regional outbreaks (e.g., dengue virus, Mpox) are addressed in Chapter 6.

The committee’s first report (NASEM, 2023) identified three criteria by which new targets should be evaluated: (1) the public health significance of the target, (2) analytical feasibility, including whether pathogen detection can be calibrated to public health outcomes of interest, and (3) the usefulness of community-level surveillance data to inform public health action (see Figure 5-1). Although existing wastewater surveillance samples could be analyzed for additional targets, the committee recognizes that the decision to implement additional targets will depend on available resources for laboratory analysis, data analysis, data visualization, and public health interpretation. It was beyond the task for the committee to conduct a formal evaluation of the value relative to the costs.

CURRENT AND POTENTIAL RESPIRATORY VIRUS TARGETS

As of early 2024, two respiratory viruses (RSV and influenza A and B [flu]) were under active consideration by the U.S. Centers for Disease Control and Prevention (CDC) for incorporation in the NWSS in addition to SARS-CoV-2. At the same time, WastewaterSCAN was monitoring for six classes of respiratory viruses nationally: SARS-CoV-2, RSV, influenza A and B, human metapneumovirus, and parainfluenza, and enterovirus D68 (EVD68). SARS-CoV-2, RSV, and influenza A and B (flu) are logical targets for ongoing wastewater-based infectious disease surveillance because they satisfy all three criteria in the committee’s first report for prioritizing pathogen targets (Figure 5-1), including that wastewater surveillance data add value for public health action relative to other existing methods of public health surveillance.

Now that routine clinical surveillance of SARS-CoV-2 has ceased, continued wastewater surveillance provides the primary ongoing epidemiological insight, beyond later-stage metrics such as hospitalizations and deaths, for a disease that continues to cause a significant public health burden. The evolution of the SARS-CoV-2 virus, however, may alter the infection and shedding dynamics within the population and necessitates ongoing recalibration of viral loads in wastewater to hospitalization or mortality data to ensure proper interpretation and public health action.

RSV and flu are major contributors to the annual respiratory disease burden and have had analytical feasibility demonstrated in several

wastewater settings (see Box 5-1). Given the seasonality of RSV and flu, wastewater surveillance can provide local data on increasing levels early in the annual cycle. Although RSV and flu are screened with respiratory panels in the clinical setting, these tests are not consistently ordered. Further, data that are collected vary by state and are not compiled for routine ongoing disease surveillance on a broad scale in the United States.

As discussed in Box 5-1, EVD68—a normally mild respiratory illness with the potential to cause a rare but severe neurological disorder leading to muscle weakness or paralysis in children—may also have promise for wastewater surveillance. However, additional information is needed to determine the value of the data for public health action.

POTENTIAL ENTERIC PATHOGEN TARGETS

The four enteric pathogens that have been discussed publicly by CDC for potential inclusion in the NWSS at a national scale are adenovirus types F40 and 41, Shiga-toxin-producing E. coli (STEC), Campylobacter, and norovirus. WastewaterSCAN is currently surveilling adenovirus group F (including both F40 and F41 types), norovirus, and rotavirus. Detection of these three viral targets were associated with clinical positivity data (Ammerman et al., 2024; Boehm et al., 2024; Kazmer et al., 2024). Both the viral targets (adenovirus group F, norovirus, rotavirus) and the bacterial targets (STEC and Campylobacter) generally meet the first two National

Academies’ (NASEM, 2023) prioritization criteria—public health significance and analytical feasibility (see Figure 5-1 and Box 5-2). All cause significant health effects, and most have little other consistent clinical surveillance data. As enteric pathogens, each of these is shed readily into wastewater at relatively high levels (Kosulin et al., 2016; Lenaker et al., 2024; Teunis et al., 2014), and analytical quantification of adenoviruses, noroviruses, rotavirus, and Campylobacter in wastewater has been shown to be feasible. Additional research on the specificity of polymerase chain reaction (PCR)-based detection of STEC may be needed because the targeted Shiga-toxin genes may not always indicate presence in pathogenic E. coli. Shiga-toxin genes may be detected that are not expressed or are not

functional (cryptic copies) or are carried in bacteriophage rather than E. coli, and therefore would require confirmation by selective culture.

Prior to national implementation, additional testing would be informative to demonstrate the extent to which these data can inform meaningful health actions or interventions, given the timing of each illness and the nature of disease spread relative to the timing of wastewater surveillance. The committee supports the rollout of these agents at a limited number of NWSS sites or analysis of public health response from the WastewaterSCAN data, supported by the NWSS Centers of Excellence, to further explore the value of national data collection on enteric pathogens for public health action. A limited rollout could assess whether wastewater surveillance of

these targets can provide sufficiently timely information such that actions could be implemented to alter community-level pathogen dynamics (e.g., advising increased handwashing and increased cleaning at schools). These pilots could also inform the development of guidance for local public health jurisdictions on how to interpret these new data (see also Chapter 4). Even if the surveillance data prove to be not timely enough to mitigate localized outbreaks, the pilot could determine whether the data can be useful to understand the community-scale disease trends over time to inform future public health actions or increase knowledge of disease dynamics in the community to a degree that justifies the investment. This approach with support from academic partners can minimize unnecessary burden on public health agency staff to interpret the data until the value for public health action is determined and guidance and messaging plans are available.

An additional enteric target candidate with potential is Salmonella, which has great public health significance. It is a highly prevalent foodborne pathogen, second to norovirus as the most confirmed etiology for foodborne infections in the United States (and above both STEC and Campylobacter). If technical challenges could be resolved allowing detection of disease-causing strains (see Box 5-2), wastewater surveillance of Salmonella may allow local health care jurisdictions to better communicate foodborne disease risks in their communities.

ANTIBIOTIC RESISTANCE GENE TARGETS

Four classes of antimicrobial resistance genes have been considered for potential inclusion in the NWSS: carbapenamases, extended spectrum β-lactamases (ESBLs), colistin resistance, and vancomycin resistance (see Box 5-3). Each of these targets has public health significance and maps to the National Action Plan for Combatting Antibiotic Resistant Bacteria 2020–2025 (Federal Task Force for Combating Antibiotic-Resistant Bacteria, 2020).

Although these antibiotic resistance genes can be detected in municipal wastewater, the interpretation of the data with respect to public health is by no means straightforward. Antibiotic resistance can be naturally occurring (intrinsic) in certain bacterial strains (notably with colistin resistance), acquired by chromosomal changes in genes (e.g., mutations), or acquired through mobile genetic elements. In fact, antibiotic resistance genes are frequently found on plasmids or other mobile elements that enhance their spread and can be detected in organisms in community wastewater (Hackenberger et al., 2024). Because there is no single antibiotic resistance gene target for major classes of resistance, detection of the full range of antibiotic resistance genes will require using multiple assays.

The presence of antibiotic resistance genes from nonhuman sources, the growth of antibiotic-resistant bacteria in biofilms within the sewage system, and the transfer of mobile genes within the environment may further confound any interpretation of wastewater antibiotic resistance gene prevalence at the community scale for public health and health system action. Consolidated wastewater streams may serve not just as a reservoir but also an amplifier of antimicrobial resistance genes, making it difficult to relate the levels observed to carriage in the population. Culture approaches have shown Aeromonas, an abundant member of the microbial community of sewage (McLellan and Roguet, 2019), has a wide range of β-lactamase genes (Piotrawska et al., 2017), highlighting the importance of considering the contribution of resident microorganisms in wastewater systems to antibiotic resistance signals. Similarly, Enterococcus containing VanA, which

confers vancomycin resistance, is also widespread in untreated wastewater (Liu et al., 2023). The committee expects wastewater surveillance to detect the already prevalent antibiotic resistance genes in nearly all community-level (i.e., wastewater treatment plant) samples. Therefore, at the community scale, wastewater surveillance data on antibiotic resistance gene occurrence cannot be reliably calibrated relative to public health outcomes of interest, and widespread monitoring at current NWSS wastewater treatment plant sampling sites is unlikely to be useful to inform public health actions.

In light of these confounding factors, as discussed in Chapter 2, facility-level antibiotic resistance gene monitoring would be more actionable than wastewater treatment plant monitoring. Facility wastewater is less likely to be confounded by nonhuman or environmental reservoirs of resistance genes, although this has yet to be tested. The detection or changes in levels of rare antibiotic resistance targets (e.g., rarer carbapenem and colistin resistance targets) in hospitals, skilled nursing facilities, and assisted living facilities is valuable given the more vulnerable population in these facilities and the higher use of antibiotics in these settings. Detection of antibiotic resistance genes using current laboratory PCR methods alone, however, is of questionable utility for public health action because antibiotic resistance can occur in both bacterial pathogens and commensal bacteria, which are carried without causing disease in most individuals. For example, several ESBL-conferring genes including bla_CTX-M are already commonly carried in human microbiomes (Woerther et al., 2013) and detected ubiquitously and in high levels in wastewater using PCR-based methods (Amos et al., 2014; Auguet et al., 2017; Rodriguez-Mozaz et al., 2015). Wastewater detection of rare antibiotic-resistant genes at a facility would likely trigger follow-up testing (e.g., selection by microbial culture, long-read genomic sequencing, proximity ligation sequencing) to determine whether the target antibiotic resistance genes are linked to pathogens. These approaches are difficult to integrate into the sample handling, laboratory analysis, and data analysis currently in place for routine national wastewater surveillance (Brown et al., 2021). Once antibiotic-resistant pathogens are confirmed at a facility, actions such as disinfection, individual clinical testing, contact prevention, and contact tracing could be implemented to limit its spread.

Periodic community-based wastewater surveillance for the rarer carbapenemases and colistin resistance genes would be useful to establish a baseline of which genes are present and could shed light on if or when organisms carrying these genes form reservoirs in wastewater systems. However, such periodic monitoring need only take place at a limited number of wastewater treatment plants across the United States to get useful data on baseline trends. Implementation at all NWSS community-based

surveillance sites is likely to provide redundant baseline data that are not useful for public health action at the local scale.

OTHER POTENTIAL ENDEMIC TARGETS

Another target that has been publicly discussed for potential inclusion in NWSS national sampling is Candida auris. C. auris is a yeast and emerging pathogen with an extreme level of antimicrobial resistance and therefore of high public health significance. Most infections with C. auris to date have originated in a hospital setting, and current clinical screening approaches are recommended only for at-risk patients or residents. Relatively little is known about the distribution, reservoirs, or behavior of C. auris in the wastewater system (e.g., persistence, growth), although potential environmental reservoirs have been identified for other Candida species (Akinbobola et al., 2023; Stone et al., 2012). Therefore, interpretability of the data still needs to be established. Community-level wastewater surveillance of C. auris was first demonstrated during an ongoing outbreak in southern Nevada (Barber et al., 2023; Rossi et al., 2022) and has since been initiated in several other locations in Florida, California, and Arizona (e.g., Babler et al., 2023). Inclusion of C. auris in a wastewater surveillance panel to understand the breadth of distribution is understandable, but the actionability of community-level surveillance data may be limited. There are public health actions (e.g. contact prevention, contact tracing, environmental disinfection) that could be taken at long-term care facilities or hospitals if C. auris is detected. Barber et al. (2023) suggested that community-level wastewater surveillance may be useful in areas with no reported C. auris to spur additional screening at hospitals and long-term care facilities if a new community-level detection occurs. Overall, considering its healthcare-associated occurrence and the current uncertainty of its behavior in larger wastewater systems, facility-level rather than community-level surveillance may provide the best information to inform public health action, but continued exploration of the value of community-level versus facility-level surveillance still needs to be assessed. The ongoing surveillance by WastewaterSCAN for C. auris is expected to help close this gap.

Sexually transmitted infections (STIs) represent another potential opportunity for endemic wastewater surveillance. Additional information from wastewater on the occurrence of STIs, for which clinical case data are poor and slow to respond to outbreaks, could inform the allocation of resources to public health messaging campaigns and better inform clinicians. Although the analytical feasibility of wastewater surveillance has not been demonstrated for key targets like Treponema palladium, Neisseria gonorrhea, and Chlamydia trachomatis, molecular assays for their detection in clinical samples are readily available (Aboud et al., 2021; Tayoun et al., 2015).

CONSIDERATIONS WHEN REFINING NWSS ENDEMIC TARGETS

Tradeoffs are involved when expanding wastewater surveillance targets in NWSS beyond a pilot scale. There are clear opportunities to increase the value of information from existing NWSS samples. However, there are additional analytical costs, the significance of which would depend on whether the new targets require different analytical concentration and extraction methods. Additional cost increases will be associated with data reporting, visualization, and analysis. In addition to needing to adapt existing local and national data visualization and analysis systems to include new targets, state and local public health authorities will need to manage, understand, and communicate the significance of the new target data in their communities. Public health jurisdictions have spent a considerable amount of time bringing wastewater surveillance online for SARS-CoV-2, including data analysis and public communication activities. While some states and localities actively use wastewater surveillance data to take public health action, others are still working to understand and communicate its value for decision making (see Chapter 4). Expansion of targets without a correlative increase in personnel time, training, and support tools may compound the burden on local public health. Another potential unintended consequence is loss of interest by some state and local public health jurisdictions due to a lack of understanding of why new targets were chosen and not finding them relevant to their local public health practice.

In light of budget uncertainty for the NWSS and potentially severe pending budget constraints (see Chapter 1), the recommendations for improving comparability and reducing variability in sampling and analysis and strengthening data analysis in the existing system (see Chapters 2, 3, and 4) are a higher priority than significantly expanding targets across NWSS sampling sites. By addressing these recommendations, the NWSS can ensure that public health agencies at local, state, and national scales can get the most information out of NWSS investments.

If budgets allow, an evidence-based, data-driven process that also considers local public health agency perspectives is needed prior to requiring additional targets in NWSS throughout the United States. Targets should only be added at all NWSS sites when specific uses for near-term or long-term public health action can be identified. Each of the targets discussed in this chapter meets one or more of the three criteria for new targets identified in the committee’s Phase 1 report (Figure 5-1; NASEM, 2023), which suggests they are reasonable targets for consideration for nationwide surveillance. If funds are available, the committee supports the near-term addition of RSV and influenza, which meet the three NASEM (2023) criteria and can be processed using the same analytical methods as for SARS-CoV-2,

reducing the additional analytical costs. For the other potential endemic targets discussed in this chapter, not all meet all three criteria, and more research or pilot testing may be needed to determine the feasibility and value of wastewater surveillance.

Prior to the inclusion of additional endemic targets at all NWSS sites, research should demonstrate that wastewater surveillance data can be reliably calibrated to public health outcomes of interest (NASEM, 2023). In some cases, this may be challenging as clinical data can be incomplete or nonexistent. Although one study has been conducted for correlations between adenovirus F40/41 and clinical sample positivity rates (Boehm et al., 2024), more studies are needed to understand the reliability of these trends to reflect infection in the community. Further, the bacterial enteric pathogens (STEC and Campylobacter) and antibiotic resistance gene targets do not yet have wastewater surveillance data that have been shown to reflect case burden or other health outcomes.

Approaches to address these research needs include both mechanistic and correlative approaches. Both have been applied to SARS-CoV-2 data and should not be considered mutually exclusive. For example, fecal shedding studies of SARS-CoV-2 during infection coupled with physical models of sewersheds demonstrated mechanistically the link between cases in a community and levels in wastewater (Prasek et al., 2022). Alternatively, many studies have identified strong correlations between COVID-19 cases in a community and wastewater concentrations (X. Li et al., 2023). For many of the expansion targets, good epidemiological data to relate to the wastewater surveillance data may be lacking, at least initially. Likewise, fecal shedding studies of most pathogens are sparse, limiting the mechanistic approach. Researchers have taken advantage of local outbreaks, such as with influenza A (Wolfe et al., 2022) and adenovirus 4 (Kazmer et al., 2024), to demonstrate correlations with wastewater. These outbreak periods resulted in higher clinical illness surveillance than during normal periods and thus provided a unique opportunity to study correlations between wastewater levels and cases. Prospective epidemiology studies could be used to validate measurements of community-wide burdens for comparison to wastewater data. Such studies would also begin to move the field forward toward using wastewater surveillance to estimate disease prevalence in addition to measuring trends.

A critical open question is to what degree wastewater surveillance of some of the individual targets would be able to inform public health action that reduces or prevents further spread and what is the value of this information for public health. Data from WastewaterSCAN may provide guidance on the value of additional targets for public health action.

The addition of targets may also have value for long-term public health action from the standpoint of understanding a disease, even when the

short-term value of the data is limited due to the timing of wastewater surveillance data relative to disease dynamics. For example, a retrospective analysis of the spatial and temporal distribution of a target measured in wastewater in relation to limited annual case reports or outbreaks may improve the understanding of disease spread and inform future public health action. Such targets could be deemed worth adding at a pilot or national scale, particularly if they are added with minimal additional cost or sampling burden. However, such additions should be clearly communicated to states and localities as data that will be analyzed by researchers so that the burden of data analysis and interpretation is not perceived as resting on local public health agencies.

Much will continue to be learned about the value of wastewater surveillance beyond COVID-19 in the years ahead as new targets are incorporated and public health practitioners become more comfortable with the data. CDC should conduct a systematic review of any expanded targets 3–5 years after implementation to evaluate the value of the information provided for public health. This information can be used to further refine the wastewater surveillance targets for optimal public health benefit.

CONCLUSIONS AND RECOMMENDATIONS

The expansion of wastewater surveillance to include influenza and RSV in addition to SARS-CoV-2 is expected to provide useful public health information for minimal additional analytical costs, but more information is needed for several other targets prior to a decision to implement broadly across NWSS sites. The committee evaluated additional targets in active consideration by NWSS against the three criteria proposed in the Phase 1 report (NASEM, 2023): (1) the public health significance of the target, (2) analytical feasibility, and (3) the usefulness of community-level surveillance data to inform public health action. RSV, influenza, and SARS-CoV-2 clearly meet the three criteria and all of the proposed targets had public health significance. However, for other targets, data are not yet available to demonstrate that community-level wastewater surveillance data are correlated with health outcomes of interest or that the information is useful to inform near-term public health actions that can alter community infection dynamics. Ongoing data collection for several targets by WastewaterSCAN may fill this gap. Additional research or limited implementation supported by the NWSS Centers of Excellence can be used to resolve remaining uncertainties for proposed targets. Each pathogen target added has an accompanying cost to local and state public health agencies that collect that data associated with data analysis and interpretation. Therefore, the public health value of each new target should be clearly understood and communicated prior to nationwide implementation. Added targets should be reviewed

after 3–5 years to determine the value and usefulness of the data for public health action. Under highly constrained budgets, the committee judges that improvements to data quality and comparability to be a higher near-term priority than target expansion.

The usefulness of routine wastewater surveillance for antibiotic resistance genes at the community (wastewater treatment plant) level is questionable because of limited ability to correlate the data with antibiotic resistance in clinically relevant pathogens. High priority antibiotic resistance phenotypes (such as carbapenem and vancomycin resistance) are encoded by multiple clusters of related genes and are present in wastewater in host-associated bacterial pathogens, a wide range of nonpathogenic bacteria found in humans, and other environmental reservoirs. Consequently, the laboratory methods currently used for wastewater surveillance are not always useful for detection of bacterial pathogens containing antibiotic resistance genes of interest. Approaches to link target antibiotic resistance genes to pathogens are difficult to integrate into the sample handling, laboratory analysis, and data analysis currently in place for routine national surveillance. Furthermore, the presence of antibiotic resistance genes from nonhuman sources, potential growth of organisms in biofilms within the wastewater systems, and the transfer and persistence of mobile resistance genes outside of a host confounds any interpretation at the community scale for public health action. The committee recommends that screening for antibiotic resistance genes focus on facility-level monitoring (e.g., hospitals, nursing homes) with wastewater treatment plant monitoring limited to specific needs, such as testing whether a high-priority antibiotic resistance gene is widely present in the community. Additional culture-based analytical approaches would still be necessary at the facility level to confirm that the antibiotic resistance is associated with a pathogenic organism.

The following research and development needs should be addressed in the area of target identification:

• Explore the potential value of community-level wastewater surveillance data for adenovirus group F and norovirus for public health action.
• Determine the analytical feasibility and potential value of wastewater surveillance for STEC, Campylobacter, Salmonella, EVD68, and STIs, with an emphasis on determining the correlation between wastewater concentrations and health outcomes of interest and the extent to which these data are useful to support public health action.
• Understand the environmental reservoirs of antibiotic resistance genes to better gauge the potential value of wastewater surveillance at the community level.

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