comment

Wastewater surveillance of pathogens can

inform public health responses
Wastewater monitoring has been used to identify SARS-CoV-2 outbreaks and track new variants. This sentinel
system should be expanded to monitor other pathogens and boost public health preparedness.

Megan B. Diamond, Aparna Keshaviah, Ana I. Bento, Otakuye Conroy-Ben, Erin M. Driver,
Katherine B. Ensor, Rolf U. Halden, Loren P. Hopkins, Katrin G. Kuhn, Christine L. Moe, Eric C. Rouchka,
Ted Smith, Bradley S. Stevenson, Zachary Susswein, Jason R. Vogel, Marlene K. Wolfe,
Lauren B. Stadler and Samuel V. Scarpino

S
ince the late 1930s, scientists have
known that infectious poliovirus
was present in the sewage of cities
experiencing outbreaks1. As a result,
wastewater surveillance has played an
important part in the polio-eradication
campaign2. Building from the work on Pathogens shed Sewage collection Wastewater samples collected
poliovirus, data from wastewater have in human excreta site and transported to laboratory
complemented clinical surveillance during
the COVID-19 pandemic by offering a
comprehensive view of infection burden
and transmission — both symptomatic
Amplification of pathogen genetic Pathogen and variant Public health
and asymptomatic — and information material in laboratory detection reporting
on which SARS-CoV-2 variants are
circulating in a community3,4 (including
so-called cryptic variants that have never
been detected in clinical samples5). Yet
despite the established value of wastewater
surveillance for monitoring poliovirus
and its emerging importance during the
COVID-19 pandemic, most global public Fig. 1 | Wastewater-monitoring pathway. After SARS-CoV-2 is shed in human feces, the sewage may
health surveillance systems still rely heavily travel to centralized locations, such as wastewater treatment plants. Depending on the goals and
on medically attended case data6. This needs resources for wastewater monitoring, a variety of methods can be used to collect samples of the sewage,
to change to enable effective pandemic which is routinely done between twice weekly and once monthly. The samples are first processed in a
preparedness and response. lab to concentrate and amplify genetic evidence (RNA or DNA, depending on the pathogen). Genetic
material is then tested to detect and quantify pathogens of interest, with PCR-based methods or NGS
Sewage science used for variant detection. Results are then reported to public health departments to improve situational
Wastewater monitoring involves the awareness and inform action.
collection of pooled samples from
community- or institution-level sewage
systems. For community-level surveillance,
the sewers are a catch-all, aggregating surveillance systems and lead to widespread Among the wide range of biomarkers
human waste into centralized locations. poor health outcomes should be prioritized and chemicals that can be measured in
However, sampling can be done at much in wastewater-monitoring programs. sewage to assess health and well-being8,
more localized scales and in communities These include diseases with nonspecific one of the most promising has been
that lack centralized sewage collection symptoms, such as polio, influenza and viruses. Additionally, a growing body
and treatment systems. Sampling from viral gastroenteritis, or those for which of evidence indicates that wastewater
centralized locations can provide rich patients do not seek care until symptoms may be an ideal environment for the
insight into pathogenic viruses, bacteria are serious. There is also a strong use case detection of antimicrobial-resistant
and protozoa present in the community7 for conducting wastewater monitoring for pathogens — a rising global public
(Fig. 1). Of these, pathogens that are stable diseases for which current diagnostic tests health threat9. Other applications include
in wastewater and/or are consistently shed in are inadequate, have limited accessibility assessing community use of illicit
fecal material or urine are strong candidates and/or are expensive and for which the and therapeutic drugs10; monitoring
for wastewater monitoring. data could help guide vaccine campaigns consumption of everyday compounds,
From a public health perspective, diseases or other interventions that can improve including caffeine, nicotine and alcohol11;
that are under-reported by traditional health outcomes. and measuring exposure to environmental
1992 Nature Medicine | VOL 28 | October 2022 | 1992–1995 | www.nature.com/naturemedicine
 comment

Table 1 | Comparison of RT-PCR versus NGS approaches for wastewater monitoring

Method Region measured Quantitative Tolerance to Novel Turnaround Target-specific Cost Interpretation Limitations
mutations variant time development
discovery time
RT-PCR Small amplicons Yes Depends on No Day Weeks Low Easy Provides only
(hundreds of location presumptive evidence,
bases) as it relies on mutation
presence or absence
as a proxy for variant
presence or absence;
inconsistent performance
over time, especially if
novel mutations emerge
in regions targeted by
assay primers
NGS Whole genome No Yes Yes Days to None High Difficult Dominant variants may
(30,000 bases) weeks overpower signal from
emerging variants;
requires initial enrichment
step using viral probes
and targeted amplicon,
which increases costs and
turnaround times

contaminants such as pesticides how and if the data should be standardized, SARS-CoV-2 was identified in South Africa,
and plasticizers12. such as using human fecal biomarkers the Wastewater Action Group mobilized
Numerous studies over the past 30 years like the pepper mild mottle virus, which to determine how wastewater monitoring
have shown that wastewater testing is common but diet dependent. Although could be combined with rapid identification
provides an accurate population-level view wastewater data have been recognized as of different genetic variants to characterize
of disease13, and recent research has shown a valuable complementary data source to the changing nature of the pandemic.
that trends in wastewater concentrations clinical case counts during the COVID- Two complementary methods
of SARS-CoV-2 are strongly predictive 19 pandemic, confusion exists about how for tracking novel variants, reverse
of clinical case counts14,15. Although the to integrate these data with existing data transcription–polymerase chain reaction
lead time may diminish as the pandemic sources, such as hospital bed capacity or (RT-PCR) and next-generation genomic
progresses16, the value of wastewater as an local sales of in-home diagnostic tests, sequencing (NGS), can be integrated into
early indicator has increased as diagnostics to provide public health officials with wastewater-monitoring programs to inform
have shifted from the health system to a more holistic measure of risk in their pandemic responses. Each approach has
at-home testing. As the rate of at-home community. More work is also needed to advantages and limitations (Table 1).
diagnostic testing increases, the reliability expand wastewater-surveillance capacity in From the original Wuhan strain of
of clinical testing data for public health rural areas and unsewered settings. Thus, SARS-CoV-2 through each new variant,
surveillance may further diminish17. complexities remain to be solved as health researchers around the globe have quickly
A warning of increased cases from departments around the world rapidly made full genome sequences publicly
wastewater surveillance can provide health adopt this tool19. A recent survey in the available, enabled by inexpensive and
departments with critical lead time for United States found a gap between public relatively fast RT-PCR assays21. This
making decisions about resource allocation health officials’ recognition of the value of sharing of viral genomes opened the
and preventive measures18. Furthermore, wastewater monitoring and their use of it for door to tracking SARS-CoV-2 variants
unlike testing of individual people, pandemic management20. not only in clinical samples but also in
wastewater testing provides insights into community wastewater.
the entire population within a catchment Variant detection Although RT-PCR has been used
area, which enables communities without The genetic code of viruses, including in clinical testing for more than three
robust syndromic surveillance systems SARS-CoV-2, continually mutates, giving decades22, its use in wastewater testing is
to have a cost-effective, efficient tool for rise to novel variants that can have different less common, although some prior work has
monitoring population health. transmissibility, severity and other disease been done to characterize bacterial variants
Current wastewater-monitoring practices characteristics. The role of variant detection in wastewater, including Escherichia coli
are not a panacea. Given the vast quantity within wastewater monitoring remains variants23 and antibiotic-resistant strains24.
of human and animal microbes that end largely underexplored scientifically and During the COVID-19 pandemic, RT-PCR
up in wastewater, the data are inherently underutilized in public health and clinical has become an essential tool for detecting
noisy, and more work is needed to reliably diagnostics. In fall 2021, The Rockefeller SARS-CoV-2 variants in wastewater. The
distinguish signal from noise in biomarker Foundation launched the Wastewater method is highly sensitive, has a quick
concentrations measured in wastewater Action Group to study various approaches turnaround time25 and is cost-effective,
samples. There is also a lack of consensus to translating wastewater data into public since one wastewater sample can yield
on wastewater-monitoring techniques and health action. When the Omicron variant of information on thousands of viral genomes

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comment

Table 2 | Case studies for wastewater surveillance

Location Method Approach Benefits
Oklahoma RT-PCR Since October 2020, several municipalities across Testing revealed distinct patterns across different
Oklahoma have implemented wastewater monitoring racial or ethnic groups29, provided an early warning
for SARS-CoV-2 to provide public health officials of Oklahoma’s transition from Delta to Omicron,
with an early warning of increasing SARS-CoV-2 and signaled to local physicians and hospitals that a
prevalence in the community. The results are surge in COVID-19 cases was imminent. As Omicron
communicated weekly to Tulsa’s public health leaders. infections can be resistant to the antibody treatments
When Omicron was first detected in the state, Tulsa used for Delta infections, the variant information from
adapted its wastewater testing to assess the relative wastewater monitoring also enabled local physicians to
contributions of the Delta and Omicron variants, target antibody treatments to patients on the basis of
using allele-specific RT-PCR. the relative abundance of Omicron in their community’s
wastewater.
Houston NGS In May 2020, the Houston Health Department Researchers were able to detect Omicron in the
collaborated with Rice University to establish a wastewater days before the first clinical case was
city-wide wastewater epidemiology program to reported in Houston. The health department quickly
monitor SARS-CoV-2 among the 2.3 million Houston incorporated wastewater detection of Omicron into
residents. The system provides weekly measurements ZIP-code-level metrics that enabled them to prioritize
and targeted amplicon sequencing of the SARS-CoV-2 resources. The city’s public health authority also
genome at 39 centralized wastewater treatment provided local infectious-disease doctors and hospital
plants. By the time the first Omicron case was leaders with wastewater-based data on Omicron
identified in the Houston area in December 2021, spread, which they used to inform staffing and personal
the Houston Health Department had already protective equipment needs.
implemented a rapid wastewater variant-screening
pipeline, collecting wastewater samples each Tuesday
and summarizing the variant sequencing and other
wastewater results in reports generated each Friday.
Southwestern Variant detection without The 5.4 million American Indians and Alaska Natives This approach not only circumvents concerns associated
tribal nations sequencing in the United States encompass 574 distinct tribes with genetic sequencing but also overcomes a key
with more than 130 languages. Cultural preferences challenge with wastewater sequencing: degradation of
and, in some cases, past ethical breaches in research viral RNA in the sewer system. Wastewater-borne viral
on tribal communities have led some tribal nations to proteins may be more stable than viral RNA in the sewer
prohibit genetic sequencing of wastewater samples system. As a result, sequencing of high-copy-number
during the COVID-19 pandemic. When two or more proteins and peptides in wastewater may be another
tribes use a single wastewater-collection system, promising alternative for detection of a persistent signal
genetic sequencing cannot be ethically performed of the public health threat.
if any one of their communities or villages voices
objections. In such situations, an alternative approach
is to analyze the gene products, such as virus-specific
proteins and peptides, using mass spectrometry.

in a population, which has made it the quantification of known variants) variant-surveillance approach. First,
workhorse of public health response. is therefore the best approach. Ideally, clinical sequences are often needed for
NGS of viral genetic material in these techniques can be used at different the identification of novel variants and for
wastewater, by contrast, can be used to stages, with NGS being used infrequently guiding the development of primers for assays
indicate the relative abundance of viral at sentinel sites to scan for novel variants used to identify those variants in wastewater.
mutations in wastewater, providing a and PCR being used for more routine, Although this dependence can help hone
broader view of genetic diversity than the cost-effective testing. focus on clinically and epidemiologically
limited presence-or-absence information The emergence of the Omicron variant relevant variants, it also makes wastewater
that PCR methods provide. Because of SARS-CoV-2 was a defining moment for sequencing vulnerable to shifts in capacity for
wastewater samples are pooled from an the role of wastewater monitoring in public timely sequence information about current
entire sewer-service population, the use of health, as it revealed the practical value and emerging clinical strains.
NGS with wastewater testing can provide of this approach for detecting emerging In addition, many of the academic
information on viral mutations across many variants in a community in real time26, labs that have been the backbone of
different infections. Routine sequencing of which were then used to inform decision wastewater-sequencing efforts will pivot
wastewater samples offers a foundation for making in Texas, Oklahoma, Kentucky and back to their usual research, which will
studying how individual mutations change Arizona (Table 2). compromise retainment of their expertise.
over time, and how variants of interest and A successful transition into long-term
variants of concern emerge and propagate. Building acceptance surveillance for public health will require
Combining NGS (for a broad view of the Considerable hurdles must be overcome continued cooperation in sharing of the
full range of variants present) and targeted at each phase of wastewater monitoring research labs’ deep expertise with their
RT-PCR assays (for rapid and sensitive before it reaches its full potential as a public and commercial partners.

1994 Nature Medicine | VOL 28 | October 2022 | 1992–1995 | www.nature.com/naturemedicine

 comment

Delays in wastewater reporting and ad of expansion should include multi-pathogen KY, USA. 14Northwestern University, Evanston, IL,
hoc communication of findings can hamper detection, to provide situational awareness USA. 15University of Oklahoma, Norman, OK, USA.
the value and early-warning potential of of changes in the transmission or burden 16
Civil and Environmental Engineering Department,
this approach. Much of the power of these of pathogens circulating in the community, Rice University, Houston, TX, USA. 17Northeastern
data comes from the ability to generate and early warning of novel or re-emerging University, Boston, MA, USA. 18University of
qualitative and quantitative situational threats. Dedicated wastewater monitoring, Vermont, Burlington, VT, USA. 19Santa Fe Institute,
awareness, through real-time data, about coupled with variant monitoring, is a Santa Fe, NM, USA. 20These authors contributed
the spread of diseases across geographies. valuable strategy for modernizing public equally: Megan B. Diamond, Aparna Keshaviah.
But wastewater data are rarely shared in real health surveillance systems. ✉e-mail: [email protected];
time, and when they are, relevant metadata, The development of successful [email protected];
such as specification of primer sequences or wastewater-monitoring programs will [email protected]
sample type, as well as source population, are require three critical components: funding to
often incomplete. This missing information support the necessary wastewater-sampling Published online: 8 September 2022
can impede the ability to detect outbreaks, equipment, lab testing supplies and https://doi.org/10.1038/s41591-022-01940-x
including new variants, especially those personnel; researcher expertise for deriving
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Building and maintaining trust requires Samuel V. Scarpino1,17,18,19 ✉ Acknowledgements
This work was funded in part by a contract with
incorporation of community interests 1
The Rockefeller Foundation, New York, NY, USA. Mathematica by The Rockefeller Foundation. All
throughout the decision-making process; 2
Mathematica, Princeton, NJ, USA. 3Indiana authors are part of the Wastewater Action Group, which
top-down implementation risks pushback by University, Bloomington, IN, USA. 4School of is composed of grantees funded by The Rockefeller
the communities monitored. In some cases, Sustainable Engineering and the Built Environment, Foundation. The views and findings are those of the
author and not those of The Rockefeller Foundation or the
communication about the mechanisms Arizona State University, Tempe, AZ, USA.
Pandemic Prevention Institute.
and goals of wastewater surveillance from 5
Biodesign Center for Environmental Health
local leaders and experts may be enough to Engineering, Arizona State University, Tempe, AZ, Author contributions
engender community buy-in. In other cases, USA. 6AquaVitas, Scottsdale, AZ, USA. 7Statistics M.B.D. and A.K. conceived of the project. M.B.D., A.K. and
as with some tribal nations in Arizona, trust Department, Rice University, Houston, TX, USA. S.V.S. coordinated the work. All authors jointly drafted and
revised the manuscript.
must be built by co-design with community 8
OneWaterOneHealth, Arizona State University
members, so that the technological approach Foundation, Tempe, AZ, USA. 9Global Futures Competing interests
and reporting can be modified to fit the Laboratory, Arizona State University, Tempe, R.U.H. and E.M.D. are founding members of AquaVitas,
needs of the community. AZ, USA. 10City of Houston Health Department, and R.U.H. is founder of the nonprofit project
Houston, TX, USA. 11University of Oklahoma Health OneWaterOneHealth of the Arizona State University
Foundation, with both entities being involved in the same
Maximizing value Sciences Center, Oklahoma City, OK, USA. 12Rollins intellectual space. C.L.M. receives research support for
Given the successes of wastewater School of Public Health, Emory University, Atlanta, wastewater surveillance from Ceres Nanosciences. All
monitoring for SARS-CoV-2, the next phase GA, USA. 13University of Louisville, Louisville, other authors declare no competing interests.

Nature Medicine | VOL 28 | October 2022 | 1992–1995 | www.nature.com/naturemedicine 1995