The COVID-19 pandemic starkly revealed the impacts a global infectious disease can have on all aspects of society, let alone the cascading effects on public safety, health, and accessibility to healthcare (Daughton, 2020). Tracking the transmission, dispersion, and evolution of COVID-19 at population-wide scales is challenging, especially given the urgent need to evaluate and estimate the distribution of the virus in a timely manner. Detecting SARS-CoV-2 in wastewater is not only a non-invasive tool, but also provides an earlier snapshot of active infections than those in clinics due to virus shedding in stool which may precede the onset of symptoms (Bibby et al., 2021). Various studies have shown that increased SARS-CoV-2 concentrations in wastewater were found to precede spikes in clinical cases (Peccia et al., 2020; Saguti et al., 2021; Wu et al., 2022c; Karthikeyan et al., 2022). SARS-CoV-2 was detected in wastewater from a nursing home in Spain before an outbreak was declared, with the lag time between wastewater data and cases ranging from 5 to 19 days (Bonanno Ferraro et al., 2021). A wastewater surveillance program implemented at the University of California-San Diego used an automatic wastewater notification system to alert residents in buildings of positive wastewater samples (Karthikeyan et al., 2021). The system successfully led to early diagnosis of nearly 85% of all COVID-19 cases on the University campus, and increased testing rates by 1.9 to 13×. However, wastewater data may not serve as a leading indicator when clinical testing capacity is adequate. A study in Massachusetts determined that wastewater data had a lead time before reported cases during the first wave of the pandemic (March-August 2020), but not during the second wave (after August 2020), as testing capacity increased (Xiao et al., 2022).

The emergence of SARS-CoV-2 variants, especially variants of concern (VOC), necessitates the need for variant-targeted wastewater surveillance, such as through next-generation sequencing (NGS) or VOC-specific polymerase chain reaction (PCR)-based assays (Table 1) (Bar-Or et al., 2021; Izquierdo-Lara et al., 2021; Karthikeyan et al., 2021). Genomic sequencing of wastewater samples has detected viral lineages circulating within a population that had not yet been detected by clinical specimen sequencing (Crits-Christoph et al., 2021). A recent study sequenced wastewater samples in New York City and found an increasing frequency of novel cryptic SARS-CoV-2 lineages containing mutations belonging to the Omicron variant as well as mutations that had been rarely observed in clinical samples (Smyth et al., 2022). The SARS-CoV-2 variants, Alpha and Delta, were detected in the University wastewater samples up to 2 weeks before detection through clinical genomic surveillance (Karthikeyan et al., 2022). It is worthwhile to mention that bioinformatic pipelines customized for wastewater sequencing data processing are also important due to the ‘composite’ nature of wastewater samples. Researchers recently developed an end-to-end analysis pipeline to reconstruct the infection dynamics of different VOCs from wastewater sequencing data, and validated on multiple datasets (Schumann et al., 2022). Additionally, allele-specific PCR assays are also widely used to quantify the distribution of VOCs in the sewersheds (Heijnen et al., 2021; Lee et al., 2021; Lee et al., 2022). Thus, wastewater surveillance integrated with genomic sequencing and mutation-specific technologies are critical for tracking specific viral strains and emerging variants in the population.

Monkeypox virus, recently renamed as “Mpox” by WHO, is an enveloped, double-stranded DNA virus. Mpox has been found in infected individuals’ feces, urine, skin, semen, nasal secretions, and saliva (Peiró-Mestres et al., 2022). Current efforts to use PCR-based tools to detect Mpox DNA in wastewater has been successful (Tiwari et al., 2023). Mpox monitoring in wastewater has the potential to detect trends before the onset of symptoms and laboratory confirmation of cases, even given the limiting factors associated with viral shedding length times, environmental persistence, and sensitivity of analytical methods (Chen and Bibby, 2022). Eight out of nine publicly operated WWTFs (with a total of 287 samples collected) in California had positive detection for Mpox DNA using the CDC’s G2R_G (targeting the OPG002 gene for all Mpox) and G2R_WA (targeting the OPG002 gene for the West Africa clade) assays (Wolfe et al., 2022). A study in Amsterdam qualitatively detected Mpox in 31% of samples from five districts, 56% samples from two WWTFs, and 26% of samples from the Schiphol airport using both assays (de Jonge et al., 2022). Primer and probe mismatches have also been identified in the CDC Mpox generic real time PCR assay and seven other Mpox and orthopoxvirus diagnostic assays (Wu et al., 2022b), suggesting the need for careful evaluation of those assays before testing. Further investigation is warranted to optimize analytical methods in Mpox detection, improve clinical/biological understanding of viral shedding dynamics, evaluate the overall effectiveness of Mpox vaccines, and quantify viral recovery efficiency in wastewater, which are crucial for using WBE data for epidemiological inference.

Enteric viruses are also frequently detected in wastewater. Enteric pathogenic viruses are shed in high concentrations (10⁵–10¹² viral particles per gram of fecal matter) (Gerba, 2000) and tend to have low infectious doses (e.g., rotavirus and norovirus can be infectious at doses of 10–100 virus particles) (Yezli and Otter, 2011; Santiago-Rodriguez, 2022). Wastewater surveillance, as part of the “Global Polio Eradication Initiative,” was implemented in multiple countries to monitor poliovirus in the population before 2000 (Hovi et al., 2012). However, poliovirus has re-emerged as an infectious disease of concern in the United States after an unvaccinated adult in Rockland County, New York suffering from paralytic poliomyelitis tested positive for the vaccine-derived poliovirus type 2 (VDPV2) in July 2022 (Ryerson, 2022). Wastewater samples from 48 sewersheds in Rockland and surrounding counties were then tested for poliovirus from 9 March to 11 October 2022, with 8.3% of the 1,076 samples (from 10 sewersheds) having detectable concentrations of the virus (Ryerson, 2022). Routine wastewater surveillance (April to July 2022) in Jerusalem, Israel detected and identified an increase of vaccine-originated poliovirus type 2 even in the absence of oral polio vaccine (Zuckerman et al., 2022).

Besides poliovirus, other enteroviruses have also been detected in wastewater. A targeted population study in Clermon-Ferrand, France examined wastewater, using a two-step procedure of polyethylene glycol precipitation and tangential flow ultrafiltration, to detect an array of viruses including norovirus, adenovirus, rotavirus, enterovirus (EV), hepatitis A and E viruses (Bisseux et al., 2018). All 54 samples analyzed were positive for at least one virus; and of the samples EV-positive, it was determined that EV-D68 quietly circulated throughout the community in September 2015 (Bisseux et al., 2018). A study in Naples, Italy identified detectable concentrations of human enteroviruses in treated effluent samples at three WWTFs (Pennino et al., 2018), posing a concern for environmental contamination.

Virus detection in wastewater or water sources is critical given the potential transmission risks and severe health outcomes associated with the exposure of waterborne pathogens, especially for the elderly, children, and immunocompromised individuals (Teunis et al., 2010; Bogler et al., 2020). Quantitative viral tracking data in wastewater further helps identify or confirm “ongoing,” either silent or recognized, outbreaks and reflect infection patterns in the sewershed. Such data can be further used in dynamic models for epidemiological inference and prediction (McMahan et al., 2021; Phan et al., 2023). On the other hand, deep sequencing of wastewater samples could further provide genetic information, such as single nucleotide polymorphisms to understand viral evolution at the population level. Quantitative wastewater data provide a complementary perspective to understand the transmission of infectious diseases and inform public health decision-making for epidemic and pandemic responses. Thus, we believe that successful public health monitoring of viral pathogens requires a multi-prong approach of both wastewater and clinical surveillance, not only to detect emerging infectious diseases, but to enhance surveillance of seasonal diseases. And integration of wastewater and clinical surveillance will be also cost-effective for mass surveillance (Wu et al., 2022a), disease management, and interventions.

Drugs and their metabolites in wastewater tend to be in low concentration due to dilution, therefore requiring highly sensitive and specific detection methods. High-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) has become the gold standard tool to detect and quantify illicit drug compounds in wastewater (Table 1). LC-MS/MS is regarded as the definitive approach to determine specific drugs because of its high sensitivity, selectivity, and reproductivity (Harper et al., 2017; Liu et al., 2018). However, it also requires sample extraction and preparation, well-trained personnel, and advanced facilities (Harper et al., 2017), making this a challenging method to adopt for high-throughput WBE programs. Other detection techniques include gas chromatography–mass spectrometry (GC-MS), surface-enhanced Raman spectroscopy (SERS), lateral flow immunoassay, and carbon nanotube electrode, which efficiently detected illicit drugs in complex matrices (Angelini et al., 2019; De Rycke et al., 2020; Dragan et al., 2021; Azimi and Docoslis, 2022; Makanye et al., 2022). Recently, advances in sensor technology, specifically with aptamer sensors (aptasensors, Table 1) have shown promise as a new analytical tool that is selective, stable, automatic, and easy to use with low cost (Mao et al., 2020; Kumar et al., 2022). Aptasensor is generally composed of an artificial single stranded nucleic acid for specific binding of the targeted drug compound and a reporter system to signal the binding. The aptasensor-based assay can be further integrated with portable devices for point-of-care detection such as for cocaine and norfentanyl (Mao et al., 2017; Kumar et al., 2022). However, the performance of aptasensors may be impaired by the low concentration and non-specific binding of drug compounds in wastewater, which requires further tests on the assay’s specificity and robustness.

Detection and quantification of drugs in wastewater sheds light on the illicit drug usage and spatiotemporal consumption patterns at the population level. A multi-city study in Croatia analyzed wastewater samples and identified that most illicit drugs consumed included cannabis, heroin and cocaine (Krizman et al., 2016). Drug consumption patterns identified in wastewater data indicated regional and seasonal variations between coastal and continental cities, with associations with summer tourist visits. Such regional and temporal differences of illicit drugs and pharmaceuticals in wastewater were also widely observed in other countries (Zhang et al., 2019; González-Mariño et al., 2020; Guzel, 2022). Oxycodone and tramadol, as opposed to illicit drugs such as heroin and fentanyl, were the most commonly measured opioids within an American Indian Reservation (Driver et al., 2022), suggesting that prescribed opioids may be a greater health concern as opposed to illicit drugs in some areas. Furthermore, the flexibility in sampling offered by wastewater analysis permits specific research scopes for illicit drug use to be pursued, such as detecting trends during weekends, holidays, and social events (Gerrity et al., 2011; Ort et al., 2014; Krizman et al., 2016).

Quantitative wastewater data can be further used to estimate the number of illicit drug consumption rate and overdoses. For example, the target compound’s concentration in wastewater (e.g., ng/mL) can be converted to the total load (e.g., ng/day) through multiplying the influent flow, and then utilized to estimate the consumption rates (mg/day/1,000 people) (Gushgari et al., 2019; Endo et al., 2020). The consumption rate can then be used to estimate drug-overdose deaths, which aligned well with the number of reported opioid deaths or prevalence estimates (Zuccato et al., 2008; Gushgari et al., 2019). However, those back-calculation estimates are impacted by multiple factors including loss rate in sewer lines, degradation of drug and corresponding metabolites in wastewater, total flows, population size in the sewershed, and the purity of street products, as discussed elsewhere (European Monitoring Centre for Drugs and Drug Addiction., 2016; Gracia-Lor et al., 2016; Margetts et al., 2020). Thus, further studies in characterizing those factors (e.g., quantification of compounds’ stability and standardization of calculation methods) will improve the inference from wastewater data.

Due to challenges in gathering data regarding actual opioid and illicit drug usage, wastewater analysis offers a complementing approach to gauge the consumption rates at the population level. These WBE data can further inform new policies and programs to mitigate drug abuse, track the long-term impact of interventions on community health, and perform educational outreach to specific populations (Gushgari et al., 2019; Driver et al., 2022). Accurate detection of which drugs and their concentrations are being used in a specific community may address knowledge gaps and limitations associated with survey health assessments. Longitudinal sampling at upstream locations (e.g., manholes) or WWTFs can further indicate the temporal characteristics of illicit drug and opioid use in the sewershed. Geographical sampling could help identify communities or subgroups with higher consumptions and suggest a more targeted public health intervention. Finally, the WBE data can be further combined with clinical data such as overdose-related hospitalizations and deaths to understand the pattern and burden of opioid and illicit drug use in the sewershed.

Due to the limited scope and time delay of phenotyping pathogens from human clinical infections (Hendriksen et al., 2019), efforts to characterize AMR from wastewater has drastically increased. Metagenomic sequencing techniques, utilizing both long and short reads, facilitate ARGs profiling in the sample (Table 1), compared to culture-based methodologies. Short-read next-generation sequencing data has the ability to identify and quantify thousands of ARGs and associated bacterial taxa, virulence genes, and pathogens (Hendriksen et al., 2019). However, these short reads are susceptible to assembly errors and make it challenging to identify the original organism with specific ARGs (Arango-Argoty et al., 2018; Garner et al., 2021). Long-read sequencing with a typical read length 10–30 kilobases (Ardui et al., 2018) provides a greater insight regarding ARGs genetics such as genome assembly, mapping certainty, and locations (plasmid or chromosome) (Amarasinghe et al., 2020; Dai et al., 2022). Other techniques for evaluating ARGs in wastewater include primarily culture independent methods: single-cell fusion PCR, digital PCR, high-throughput quantitative PCR, 16S rRNA amplicon sequencing and correlation analysis, fluorescence-activated cell sorting and sequencing, and genomic cross-linking (Nguyen et al., 2021). Details on the advantages and disadvantages of each method are discussed elsewhere (Ishii, 2020; Nguyen et al., 2021).

ARG diversity and abundance has been found to vary between different locations and even within WWTF biotreatment compartments. A significant difference in AMR gene abundance was identified between high-income countries (Europe/North and America/Oceania) and low-income countries (Africa/Asia/South America) (Hendriksen et al., 2019). For example, Brazil, Vietnam, and India were identified to have the most divergent distribution of AMR genes, suggesting that these countries could be hot spots for the emergence of new ARGs. AMR gene abundance was found to be associated with several parameters regarding health sanitation, including female child mortality rates, infection and malnutrition, and number of physicians, among others. Another study determined that within the same WWTF, the microbial communities and ARGs were found to be significantly different in both liquid phase effluent and recycled activated sludge (Quintela-Baluja et al., 2019). The recycled activated sludge had a lower ARG richness than the liquid effluent and downstream receiving waters. Spatial differences in ARG abundance and diversity in WWTF effluent have also been reported in China and Hong Kong (Zhang et al., 2016; Yin et al., 2022). The results indicated the spatiotemporal dynamics of ARGs in wastewater and its relevance with socioeconomic, health, and environmental factors.

WWTFs are considered a critical component in AMR monitoring and mitigation, due to their roles as reservoirs for ARGs and ARG exchange, as well as a barrier by limiting the environmental release of ARGs through treatment processes (Nguyen et al., 2021). The discharge of treated wastewater into the environment also poses an increased risk of exposure for humans via multiple routes such as contaminated food (e.g., irrigated produce) and biosolid reuse in agriculture. The transfer of ARGs into human pathogens is also an emerging public health concern.

While ARG detection and monitoring is critical both nationally and globally for addressing the global burden and transmission of AMR, how to interpret the wastewater data for antibiotic stewardship and infection prevention should be considered. Stewardship can be directed through the ARG data measured in wastewater. If AMR is increasing in certain urban areas, for example, genes associated with extended-spectrum beta-lactamases, physicians might limit or modify their prescriptions accordingly. Additionally, it will alert healthcare systems to be on guard because these infections may be challenging to treat. One example of this importance is the potential detection of an antimicrobial resistance gene of public health importance upon importation into a new geographic area. The early detection of this resistance gene can influence and inform clinical surveillance and may enable public health intervention and mitigation before the wide spread of resistance gene in the sewershed. The rise of novel variants of AMR bacteria are extremely concerning and may be identified in wastewater long before they appear clinically. Analysis of ARG in wastewater used for urban agriculture can be also used to understand the AMR transmission risk among humans and animals (Bougnom et al., 2019), particularly in locations where ARG leaks into densely populated areas. In addition, identifying exposure thresholds, such as through quantitative microbial risk assessment (Pruden et al., 2018), by which ARGs and ARB may adversely impact human health need to be quantified. Public health efforts for managing AMR via wastewater is primarily focused on characterization, but future work incorporating quantitative AMR and ARG data into mathematical modeling could further inform and implement mitigation strategies. While there is plenty work left to do in ARG surveillance in wastewater, it holds great promise in increasing our knowledge of the dissemination and asymptomatic carriage of resistant pathogens.