Wastewater Collection and Sequencing as a Proactive Approach to Utilizing Threat Agnostic Biological Defense

Introduction

Humans in the 21st century live in an unprecedentedly connected and dense world. ¹ Modern air travel infrastructure allows people and animals to traverse the globe in a manner of hours. Climate change has put tremendous pressure on ecosystems; as such, humans are encountering new animals and environments with increased frequency. These changes to human living and travel have considerably increased the risk of infectious disease spillover and global pandemics. ² Never before has the ability to rapidly detect and characterize any pathogen, be it known or novel, been so important to protecting global health. Pathogen agnostic tools, such as next-generation sequencing (NGS), enhance global biological defense and public health by addressing this exact need, but they must be deployed in a proactive manner to reach their full benefit. Using pathogen agnostic tools only during an outbreak is too retroactive and does not allow time for containment, particularly of a novel pathogen. In this commentary, we propose the development of national guidelines for a proactive, pathogen agnostic wastewater surveillance system in the United States with a focus on localities identified for risk of zoonoses and international ports of entry.

Risks of Exclusive Reliance on Event-Based Surveillance

Novel pathogens are particularly harmful to global health because of an abundance of immunonaive and susceptible human hosts and the limited availability, if any, of effective medical countermeasures.^3,4 Public health authorities today primarily rely on event-based surveillance, which is too slow to prevent and contain sustained spread of highly transmissible novel infectious diseases. Event-based surveillance “looks at reports, stories, rumors, and other information about health events that could be a serious risk to public health.” ⁵ For instance, health authorities read case reports from clinicians and look for indications that patients are sick with novel or otherwise high-consequence pathogens. ⁶ Event-based surveillance is effective at quickly detecting outbreaks of diseases that have extremely high individual pathogenicity because it relies on individuals seeking healthcare for their illness. ⁶ Yet, infectious diseases that are highly transmissible need not have extraordinarily high individual severity to cause significant harm to public health. ⁷ The 1918 H1N1 influenza was the modern world's deadliest pandemic, yet the infection fatality rate was only 2.5%, far lower than for other diseases, such as Ebola Zaire or Middle East respiratory syndrome. ⁸ Additionally, a disease without catastrophically high individual severity may be more likely to evade detection via event-based surveillance. When a disease with a high infection hospitalization rate emerges, a cluster of hospitalizations will appear in a quick manner. This alerts authorities early and allows the public health infrastructure to respond. When a grouping of hospitalizations occurs with no known cause, authorities will run a package of diagnostic tests and most often sequence samples to characterize the agent. Yet, a disease with a lower infection hospitalization rate will take more infections to cause a cluster of hospitalizations to emerge. This makes detection of novel pathogens especially difficult, as moderately ill individuals can visit a clinician, receive a host of negative molecular diagnostic tests, and assume they have a common cold.

Event-based surveillance by its nature only detects instances where a disease is signaled through patients seeking healthcare. This type of surveillance fails to account for infections that, for whatever reasons, do not receive care. For instance, a disease with a 5% infection hospitalization rate will average 400 infections to 20 individuals hospitalized. Thus, it would take possibly hundreds of cases before health authorities have a clear signal that a pathogen is spreading. Rather than relying exclusively on event-based surveillance, the impetus must instead be on developing and implementing biological surveillance that is proactive.

Proactive Biological Surveillance for Emerging Infectious Diseases

Proactive biological surveillance aims to collect information about emerging hazards to population health without waiting for a large-scale health event to occur. Wastewater surveillance—through collection and testing—represents an excellent method of proactive surveillance because it does not rely on any individuals seeking healthcare. Rather, it uses existing wastewater infrastructure to obtain pooled, anonymized samples of biological material. ⁹ The utility of wastewater collection and testing to determine when individuals in a population are ill with a pathogen has been proven in a number of real-world settings.^10,11 Wastewater testing can detect and quantify a range of enteric and nonenteric viruses. A study conducted in 2020 found that viruses from the Adenoviridae, Astroviridae, Caliciviridae, Coronaviridae, Flaviviridae, Hepeviridae, Herpesviridae, Matonaviridae, Papillomaviridae, Parvoviridae, Picornaviridae, Poxviridae, Retroviridae, and Togaviridae viral families could be detected in wastewater. ¹² Additional studies have proven that wastewater testing can determine the presence of viruses in the Orthomyxoviridae family as well as the Pneumoviridae family.^13,14 This illustrates the utility of wastewater testing to proactively detect a diverse array of viruses, including primarily respiratory pathogens of public health importance. Coupling wastewater sampling with NGS testing in a proactive manner would allow public health authorities to detect and characterize emerging and novel pathogens, as well as assist in warning when known high-consequence pathogens are detected.

Establishing a Workflow for Pathogen Agnostic Wastewater Surveillance

We propose the US Centers for Disease Control and Prevention (CDC) develop standardized guidelines for pathogen agnostic wastewater sample preparation and bioinformatics to ensure consistent approaches for reproducible detection and scalable data analysis across public health laboratory networks. As part of this process, it will be important to determine whether best practices developed for pathogen-specific testing can be applied in an agnostic approach. ¹⁵ Specific methodologies to be evaluated include sample storage, preparation (homogenization, clarification, nucleic extraction), and sequencing. These steps impact the concentration and purity of nucleic acids and the subsequent accuracy of sequences.^16,17

Due to its speed and ability to detect and catalog previously unknown biological agents, NGS represents the best pathogen agnostic approach available. ¹⁷ Yet, the sheer amount of data generated by NGS represents a challenge to utilizing it within the context of wastewater surveillance. For instance, approximately 15% of DNA in a wastewater sample comes from humans themselves. ¹⁸ A number of workarounds can be refined to reduce the amount of this unneeded data. One example is the use of the Scalable Nucleotide Alignment Program, which eliminates all human reads from an NGS readout. ¹⁹ Additionally, bioinformatic pipelines, such as SURPI (sequence-based ultrarapid pathogen identification), have been proven to rapidly identify and detect both known and novel pathogens, proving the utility of NGS for biological surveillance. ¹⁹ However, continued research is needed to develop a bioinformatics pipeline specifically calibrated for pathogen agnostic wastewater surveillance.

Overview of the Current National Wastewater Surveillance System

An example of the current state of wastewater surveillance is the National Wastewater Surveillance System (NWSS). Launched in September 2020 by CDC, the NWSS helps communities track outbreaks of COVID-19. ⁹ While this is a good starting point, the NWSS as currently constructed falls short of what the United States needs to rapidly detect an emerging novel pathogen. The current system collects wastewater from treatment plants and tests it for several specific pathogens, such as SARS-CoV-2, monkeypox virus, and poliovirus. This means that no other pathogen levels are determined and there is no coordinated attempt to find novel pathogens. There should be serious consideration to expanding this already operational wastewater surveillance system to conduct pathogen agnostic sequencing. This expansion would strengthen early warning capacities to activate prevention and containment measures both locally and globally.

Implementation of pathogen agnostic wastewater sequencing would be a truly proactive, threat-agnostic approach. Rather than relying on event-based surveillance to be triggered, public health officials would constantly monitor pooled samples, in the form of waste, for any novel pathogen. By using NGS, such a system would be able to identify the genome of any threat quickly, rather than only flag known threats. Given the danger that novel and emerging infectious diseases pose to global health security, this is a key capacity. Such wastewater surveillance systems should not be deployed only in major metropolitan areas. The current NWSS has the goal of testing wastewater from 30% of the US population, yet no comprehensive list of where wastewater testing occurs has been published by CDC. ²⁰ The NWSS is essentially a patchwork of local government efforts compiled by CDC, as opposed to a national, comprehensive effort with a focused aim. ²⁰ To more effectively detect the emergence of novel infectious diseases, CDC should explicitly aim to increase wastewater collection and testing capacities in settings with high levels of animal agriculture and proximity to wildlife, as well as global travel hubs, such as airports, in the United States. These recommendations align with the recent National Academies of Sciences, Engineering, and Medicine report, Wastewater-Based Disease Surveillance for Public Health Action, which calls for wastewater testing to be sustainable by considering specific localities most in need of biological surveillance. ²¹ To be pathogen agnostic, such wastewater testing should be conducted in part via NGS.

Pathogen Agnostic Wastewater Surveillance in High-Risk Settings

The US National Biodefense Strategy and Implementation Plan calls for government agencies to “increase domestic surveillance and sampling of potential sources of zoonotic spillover of existing and emerging diseases.” ²² Conducting pathogen agnostic wastewater surveillance in these locations represents an optimal method, allowing for surveillance of geographic sources of potential spillover without invasive testing of individuals or animals. This method would support rapid detection and identification of priority zoonoses as well as capture spillover events that evade traditional mechanisms of surveillance for early warning and response.

Key to its success is an interagency effort to determine areas with the highest risk of a zoonotic event. Comprehensive modeling can be used to identify target geographic locations; models will need to account for the presence of susceptible animals, the interactions between animals and humans in a particular environment, and impacts of climate on the geographic distribution and migration of humans and/or animals. ²³ Other focus areas may be driven more by human occupation and travel. Occupational exposure has been a repeat issue in many emerging and reemerging disease outbreaks. Recent surveillance efforts of biological workers indicate that animal agriculture workers are regularly exposed to viruses circulating in livestock under their care, even in high-resource settings.^24-26 While these studies do not point to imminent public health threats, they do illustrate that even in the United States, animal agriculture workers are regularly exposed to potentially zoonotic viruses. That no major spillover has happened may be more indicative of random luck in which pathogens workers are being exposed to rather than strong occupational health programs in the animal agriculture industry.

Another location for government officials to target for pathogen agnostic wastewater surveillance is high-volume ports of entry. International travel, particularly air travel, played a key role in the initial global spread of SARS-CoV-2 and likely will do so in the event of a future aerosol- or close-contact-transmitted pandemic. ²⁷ Routine sampling and sequencing of wastewater from flights coming from designated high-risk areas could support early detection and identification of underlying pathogen spread between regions or continents. Testing wastewater from airplanes would represent a less invasive method for disease surveillance than mandatory sampling of individual travelers from high-risk or endemic areas. ²⁷ A recent CDC study highlights that wastewater collection procedures added only 3 minutes to the standard maintenance time of the airplane, illustrating that this intervention would add minimal burden to travelers or airlines and should be considered as a method of proactive surveillance. ²⁸

While these sources and locations are crucial in gathering data to strengthen early warning and response mechanisms, a critical gap remains. We do not have normal signals or values for expected or anticipated pathogens under routine wastewater sampling. As pathogen agnostic wastewater testing has not occurred at scale, no baseline exists to evaluate and determine deviations that would trigger a response. Routine data collection is crucial for further deployment and for full use of agnostic wastewater surveillance as a public health tool.

Triggering a Public Health Response

To make a pathogen agnostic wastewater surveillance system actionable, rigorous data sharing must occur between local and federal officials. Access to data in real time will support appropriate assessments and the allocation and authorization of a public health response. Current challenges to the agnostic approach are the evaluation and determination of public health action should a novel pathogen be detected. Yet, advances in computational biology allow for a more nuanced and thorough understanding of a biological agent's potential public health impacts simply based on a sequence. ²⁹ As such, the first step upon detection of a novel agent via wastewater surveillance could be characterization via computational biology tools, such as the Pathogenicity Prediction for Bacterial Genomes (PaPrBaG). ³⁰ Other tools allow for pathogenicity predictions for sequences of viruses. ²⁹ If an agent were flagged as a potential risk to public health, infectious disease experts and epidemiologists could trigger additional early warning systems while scientists adapt or develop appropriate diagnostic tests and conduct informed-risk analysis for public health impacts. Additionally, research could be conducted to characterize the pathogen to inform timely public risk communications on prevention and containment, and if necessary, begin the process of developing medical countermeasures.

Conclusion

Pathogen agnostic tools represent a new path forward for biological defense and global health security. Their use allows for an efficient method of detection and characterization of any pathogen, be it novel or known. While their utility in an outbreak is clear, authorities should consider how to deploy them proactively in settings with a high risk of disease emergence. The strides made in wastewater surveillance must be capitalized on using emerging pathogen agnostic tools. The US government must prioritize wastewater surveillance in places at the highest risk of zoonosis and high-traffic international ports of entry. While this approach is not possible everywhere, as technology progresses and sequencing costs decrease, the ability to use a pathogen agnostic approach for wastewater surveillance will become available in more and more settings. ³¹ Shifting the paradigm from event-based biological threat detection to proactive threat detection will greatly benefit global health and promote biological defense.