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THE PAST, PRESENT, Wastewater’s

AND FUTURE OF
Predictive Power

WASTEWATER-BASED
Page 3
EPIDEMIOLOGY The COVID Revolution

Page 4
Wastewater’s Potential to
Protect Public Health

Sponsored By

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Tracking Pathogens
in Wastewater with
ddPCR technology
 WASTEWATER’S
PREDICTIVE POWER

W
astewater seems a pathogen’s spread in near real time based on the number of people who
like a surprising comprehensively. Since the 1980s, are served by the sewage system at
re a gent for a countries around the world gradually the sampling point. As such, WBE
d i a g no s t ic lab incorporated WBE, and the technology provides population-wide substance
t e s t . Howe ver, has helped identify silent polio virus abuse information with high spatial and
wastewater samples contain a trove of circulation and curb outbreaks in temporal resolution.
health-related information about the Finland, Israel, and the Netherlands.1,2
individuals that are served by the sewage
system. Over the years, scientists have (Antibiotic) Resistance is Futile
developed sensitive wastewater-based Water Science to Apart from pathogens and substance
detection technologies to monitor and Monitor Drug Use misuse, scientists use wastewater to
protect public health. In the early 2000s, scientists modified study antimicrobial resistance (AR)
WBE technology to expand available evolution. The rise of AR is a global
methods that evaluate illegal drug, public health threat that increasingly
The Hidden Value alcohol, and tobacco consumption at reduces treatment options for deadly
of Wastewater the population level. Traditional drug infections.4 Antibiotic resistance genes
Scientists first turned to wastewater surveillance systems include population (ARGs) arise in pathogenic and non-
as an environmental surveillance tool surveys, patient drug tests, and police pathogenic bacterial strains and
to detect polio virus circulation among seizures. These methods are tedious, can be exchanged among strains via
communities. Polio virus is a human difficult to scale, and offer data at only horizontal gene transfer.4 The breadth
RNA enterovirus that infects the gut a single timepoint. In addition, drug of circulating microorganisms makes
and nervous system and can cause misuse is a stigmatized behavior that it difficult for scientists to identify
temporary or permanent paralysis. can have legal consequences, so a self- and track ARG evolution. Here,
While the virus is nearly eradicated, the reporting bias further complicates wastewater samples allow scientists
disease remains endemic in Afghanistan surveillance efforts. Finally, people to collect, culture, and profile large
and Pakistan, and occasional outbreaks who suffer from drug addictions often amounts of diverse microorganisms to
occur around the globe. have limited access to healthcare and determine the extent of AR evolution
Only about one percent of people are typically underrepresented in within a population.4 These samples
infected with polio virus develop public health data. also represent invaluable resources for
paralysis, so a clinical polio diagnosis Because a city’s sewage system serves studying a microorganism’s therapy-
typically indicates significant community all community members and collects evading mechanisms and developing
spread of the virus. To facilitate early all the metabolites present in human strategies to overcome them.
virus detection, scientists searched excreta, wastewater-based detection With the right tools and technologies,
for new ways to improve circulating technologies offer several advantages wastewater can be a powerful early
virus surveillance and protect public that complement traditional drug warning system for monitoring and
health. Polio virus RNA can be present surveillance methods. 3 In addition, preventing a pathogen’s spread through
in sewage because the virus is excreted scientists can apply this method to a community. At the moment, WBE
in the feces of infected individuals and surveil an entire city’s population workf lows often rely on RT-qPCR to
remains relatively stable in aqueous or target subpopulations such as detect viruses, bacteria, and other
solutions at ambient temperatures. gyms, music festivals, or prisons. microorganisms. 5 However, wastewater
Researchers optimized RNA extraction Combined with sensitive detection samples are complex and contain PCR
and reverse transcription quantitative methods and knowledge of a drug’s inhibitors such as lipids and proteins.
PCR (RT-qPCR)-based assays and metabolism, excretion, and in-sewer To increase WBE sensitivity, scientists
found that they could detect the virus transformation rates, scientists now increasingly incorporate Droplet Digital
in wastewater.1 Because sewage samples deduce a community’s daily drug PCR (ddPCR) technology, which is less
contain data from entire neighborhoods, consumption rates with wastewater sensitive to PCR inhibitors.
w a s t e w at er -ba se d epidem iolog y samples.3 Researchers can then calculate
(WBE) allows researchers to monitor the extent of drug use per inhabitant See references on page 6

THE PAST, PRESENT, AND FUTURE OF WASTEWATER-BASED EPIDEMIOLOGY 2

 THE COVID
REVOLUTION

D
espite early city-w ide limited.4 In parallel, researchers applied the technology analyzes bulk cDNA in
quarantines and nation- WBE and detected viral circulation large volumes, it relies on a standard
wide lockdowns, SARS- in several countries weeks to months curve to provide relative quantifications,
CoV-2 rapidly spread before reports of the first clinical has poor performance when detecting
across the globe. In the two cases. 3,5-7 Combined with other reports low-abundance targets, and is sensitive
years since the coronavirus’ emergence, of successful SARS-CoV-2 detection to PCR inhibitors. The concentration
scientists estimate that it has caused in treated or untreated wastewater of PCR inhibitors, including proteins
more than 5 million deaths worldwide, samples, these studies confirmed WBE’s and lipids, determines a reaction’s
and that its economic burden in the US sensitivity and revealed its value as an amplification efficiency, so scientists
alone will surpass $17 trillion by 2030.1 early warning system to signal SARS- must properly dilute all samples prior to
Public health professionals have CoV-2 presence in a population. analysis to reliably detect and estimate a
struggled to contain SARS-CoV-2 sample’s viral load.11,12
circulation with interventions such In contrast, ddPCR technology
as clinical testing, social distancing, The Struggle for Reproducibility partitions sample and reagents in tens
and contact tracing. This is partially and Sensitivity of thousands of droplets. The technology
due to the virus’ asymptomatic and Communities around the world have performs end-point PCRs that quantify
pre-symptomatic spread. A recent now incorporated WBE for SARS- cDNA molecules in individual droplets,
study showed that SARS-CoV-2 CoV-2 surveillance, and scientists which increases the assay’s sensitivity
mainly spreads through individuals estimate that they can detect the compared to RT-qPCR and allows for
who are unaware that they carry the virus in a wastewater sample if only 2 absolute target quantitation without
virus. 2 Clearly, identif ication and people are infected out of the 10,000 the need for a reference standard.11,12
isolation of symptomatic patients served by the sewer system. 8 Viral In addition, due to the assay’s small
alone will not suffice to curb the quantification from sewage samples volumes, PCR inhibitors are present in
pandemic, and there is a need for more is a complex process that involves much smaller amounts that typically
untargeted, sensitive, and scalable wastewater sampling and molecular- don’t affect target amplif ication
surveillance methods. based methods. To date, researchers efficiency. These characteristics make
have used a variety of methods for the technology well-suited for WBE-
sample collection, viral concentration, based SARS-CoV-2 surveillance, and
Testing the Waters extraction, and detection, with no ddPCR is now a recommended method
While SARS-CoV-2 primarily affects clear consensus on the most effective for wastewater-based SARS-CoV-2
the respiratory system, the virus infects approach.9 A recent study found that RNA detection by the Center for Disease
many other tissues, including the gut. the type of concentration method does Control and Prevention (CDC).13
When researchers discovered viral RNA not affect viral recovery efficiency.10 In Several research teams compared
in COVID-19 patients’ stool and urine contrast, wastewater characteristics such viral RT-qPCR and ddPCR detection
samples, this opened the door to develop as turbidity, surfactant and detergent and found greater sensitivity and
wastewater-based epidemiology (WBE) load, and its storage temperature do reproducibilit y for SA R S - CoV-2
technologies that monitor viral spread at inf luence experimental sensitivity quantification with ddPCR.12,14,15 In
the community level. 3 and reproducibility.10 addition, one team used optimized
As patients shed virus throughout ddPCR primer sets to discern between
the course of an infection, viral material wildtype virus or the delta variant
enters wastewater systems, allowing ddPCR Technology for the Win in wastewater.16 Therefore, ddPCR
scientists to sample and analyze it. In Once RNA is extracted, scientists technolog y permits scientists to
a proof-of-concept study, researchers choose between reverse transcription multiplex primer sets and simultaneously
showed that they could detect viral quantitative PCR (RT-qPCR) or Droplet assess the circulation of various SARS-
RNA in untreated wastewater samples Digital PCR (ddPCR) to quantify SARS- CoV-2 variants within a community.
collected during the first few months CoV-2. RT-qPCR has several limitations
of the pandemic when viral spread was for wastewater-based detection. Because See references on page 6

THE PAST, PRESENT, AND FUTURE OF WASTEWATER-BASED EPIDEMIOLOGY 3

 WASTEWATER’S POTENTIAL
TO PROTECT PUBLIC HEALTH

T
he SARS-CoV-2 pandemic leadership to take timely action before multiple pathogens requires method
w i l l on ly end when individual infections initiated a optimization and standardization.6
communities around the widespread outbreak.1 Based on WBE’s Nex t generat ion sequencing
world c a n implement success at various universities, several (NGS) is an attractive technology for
measures that prevent pre- cities and states, including Houston comprehensive WBE programs because
symptomatic and asymptomatic viral and North Carolina, 2,3 implemented it provides an untargeted approach
spread. Unfortunately, most public cit y- or state-w ide W BE-based to identify all pathogens in a sewage
health offices are unable to regularly SARS-Cov-2 surveillance programs. sample.7 While powerful, untargeted
test all community members for reasons These programs include interactive WBE has a slow turnaround time
related to cost, laboratory logistics, and maps that inform public health because researchers rely on algorithms
individual compliance. SARS-CoV-2 officials and individuals about their to align short sequencing reads with
surveillance with wastewater-based community’s COVID-19 burden. known pathogenic genomes.7 Compared
epidemiology (WBE) overcomes these Initial measures to reduce SARS- to targeted pathogen detection,
limitations because it detects SARS- C oV-2 t ra n sm ission, i nclud i ng current NGS-based WBE-workf lows
CoV-2 before an individual becomes lockdowns and social distancing, cannot generate data rapidly enough
symptomatic and does not depend on significantly affected the travel sector. To to alert public health officials to
patient compliance. Therefore, WBE can resume all aspects of the pre-pandemic an emerging problem.
provide near real-time information about lifestyle, scientists are searching for ways For targeted WBE, scientists employ
a population’s COVID-19 outbreak risk. to minimize transportation-related viral RT-qPCR or ddPCR technology for
spread. Researchers recently applied pathogen detection. Based on its superior
WBE-based surveillance methods sensitivity and reproducibility compared
Resuming “Life as We Know It” to airline and cruise ship sanitation to RT-qPCR-based methods, 8 ddPCR
Early in the pandemic, most schools systems and showed the technology’s technology is the preferred technology
transitioned to virtual learning to potentia l to pr ior itize clinica l for comprehensive, multi-pathogen
protect their students’ health. While testing and contact tracing among WBE. To facilitate standardized
virtual platforms quickly become disembarking passengers.4,5 Provided methodology implementation, Bio-
more accessible and inclusive, online the implementation of rapid on-site Rad offers optimized ddPCR assays
learners still lack the social aspects of analysis stations in airports and harbors, to detect a plethora of viruses and
in-person learning. Several universities WBE can likely support a safe return to bacteria, including SARS-CoV-2,
implemented local WBE-based viral long-distance travel. Norovirus, Adenovirus, Legionella, and
monitoring to stave off COVID-19 Enterococcus. In addition, Bio-Rad has
outbreaks and safely resume on-campus predesigned assays to detect variants
education. For example, researchers at Looking Beyond COVID-19 of concern so researchers can access
Colorado State University incorporated While researchers adapt available WBE and implement them as quickly as
an autosampler to collect composite technologies to monitor SARS-CoV-2 possible.9 Because multiplexed ddPCR
samples: wastewater collected over circulation surveillance nation-wide, analysis is less prone to artifacts and
a 24 -hour period at 15-minute public health officials are also interested has simpler data analysis compared
intervals.1 Because the team samples 17 in expanding current workf lows for to other technologies,10 ddPCR-based
different sites across campus, they can simultaneous surveillance of multiple WBE should allow researchers to
also extract spatial information about pathogens. Wastewater sample supply is accurately analyze the circulation of
local transmission hot spots within the near infinite and scientists can already multiple pathogens, including variants
university. The scientists incorporated detect a wide variety of individual viruses of concern, in societies and inform public
Droplet Digital PCR (ddPCR) technology and bacteria in wastewater samples. health officials in a timely manner to
into their WBE workf lows to provide However, translating these experimental prevent future outbreaks.
accurate daily updates on viral methods into scalable, multiplexed public
circulation, which allowed university health tools that simultaneously detect See references on page 6

THE PAST, PRESENT, AND FUTURE OF WASTEWATER-BASED EPIDEMIOLOGY 4

Tracking Pathogens in
Wastewater with ddPCR technology
Many pathogens are excreted into the Small Residential City University
Village Area Center Campus
sewer system through bodily fluids,
where scientists detect and quantify
them with reverse transcriptase
quantitative PCR (RT-qPCR) or
Droplet Digital PCR (ddPCR).

Sewage samples pose several

challenges for pathogen detection;
they are rich in lipids and proteins
that can inhibit PCR-mediated
amplification and can contain low
or high pathogen concentrations.
Successful translation of wastewater-
based epidemiology (WBE) into tools Sample 1 Sample 2 Sample 3 Sample 4
that inform public health decisions PCR-Inhibitors Low Low High Medium
requires sensitive, robust methods Viral Load Low Medium Medium High
with a wide dynamic range. Variant of Concern: Absent Absent Absent Present

Deciding between RT-qPCR and

ddPCR technology for WBE
Because ddPCR technology Reverse Transcription Droplet Digital PCR (ddPCR)
partitions a sample into small Quantitative PCR (RT-qPCR)
droplets and performs end-
1 2
point PCR in each individual 4 Variant 1
droplet, the method has 2
several advantages for WBE-
based pathogen detection 3 4
compared to RT-qPCR. Detection
4 Variant 2
Limit 3
1

Assay Sensitivity When infection rates are low, RT-qPCR fails to detect the virus whereas
(Sample 1, brown) ddPCR accurately quantifies individual viral molecules in each droplet.

Assay Accuracy When viral loads are medium or high, RT-qPCR and ddPCR both perform
(Sample 2, orange) well. RT-qPCR has a wider dynamic range than ddPCR.

Sensitivity to PCR High lipids and protein concentrations inhibit RT-qPCR-mediated

inhibitors amplification. ddPCR is less sensitive to PCR inhibitors because it partitions
(Sample 3, green) the sample.

Using differentially labeled probes (red and green) with ddPCR technology
Multiplexing ability
more accurately quantifies different pathogens or variants of concern than
(Sample 4, purple)
RT-qPCR because of its enhanced sensitivity and accuracy.
References
and community transmission of CSU,” The Rocky Mountain Collegian,
Article 1: Wastewater’s COVID-19,” Environ Sci Pollut R, 1–12, https://collegian.com/2020/09/
Predictive Power 2021. category-news-the-science-behind-
1 ) T. Hovi et al., “Role of environmental 8 ) A.C.U. Jørgensen et al., “Eurofins the-on-campus-wastewater-testing-at-
poliovirus surveillance in global polio Covid-19 SentinelTM Wastewater Test csu/, accessed December 16, 2021.
eradication and beyond,” Epidemiol Provide Early Warning of a potential 2 ) “Houston health department launches
Infect, 140(1):1-13, 2012. COVID-19 outbreak,” Medrxiv, 2020. dashboard displaying wastewater virus
2 ) W.J. Lodder et al., “Feasibility 9 ) W. Ahmed et al., “Surveillance of levels,” https://www.houstontx.gov/
of quantitative environmental SARS-CoV-2 RNA in wastewater: health/NewsReleases/houston-health-
surveillance in poliovirus eradication methods optimisation and quality department-launches-dashboard-
strategies,” Appl Environ Microb, control are crucial for generating displaying-wastewater-virus-levels.
78(11):3800-5, 2012. reliable public health information,” html, Houston Health Department,
3 ) M. Huizer et al., “Wastewater-based Curr Opin Environ Sci Heal, 17:82–93, accessed December 16, 2021.
epidemiology for illicit drugs: a critical 2020. 3 ) “COVID-19 North Carolina
review on global data,” Water Res, 10 ) J.L. Kevill et al., “A comparison of Dashboard,” https://covid19.
207:117789, 2021. precipitation and filtration-based ncdhhs.gov/dashboard?mc_
4 ) A. Pruden et al., “Seizing the moment: SARS-CoV-2 recovery methods cid=2c5d221348&mc_eid=7773ff8fe0,
now is the time for integrated global and the influence of temperature, North Carolina Department of Health
surveillance of antimicrobial resistance turbidity, and surfactant load in and Human Services, accessed
in wastewater environments,” Curr urban wastewater,” Sci Total Environ, December 16, 2021.
Opin Microbiol, 64:91-99, 2021. 808:151916, 2022. 4 ) W. Ahmed et al., “Detection of SARS-
5 ) K. Mao et al., ”The potential of 11 ) S.C. Taylor et al., “Droplet digital CoV-2 RNA in commercial passenger
wastewater-based epidemiology as PCR versus qPCR for gene expression aircraft and cruise ship wastewater:
surveillance and early warning of analysis with low abundant targets: a surveillance tool for assessing
infectious disease outbreaks,” Curr from variable nonsense to publication the presence of COVID-19 infected
Opin Environ Sci Heal, 17:1-7, 2020. quality data,” Sci Rep, 7(1):2409, 2017. travellers,” J Travel Med, 27:taaa116,
12 ) M. Ciesielski et al., “Assessing 2020.
Article 2: The COVID Revolution sensitivity and reproducibility of 5 ) W. Ahmed et al., “Wastewater
RT-ddPCR and RT-qPCR for the surveillance demonstrates high
1 ) S. Chen et al., ”The economic burden
quantification of SARS-CoV-2 predictive value for COVID-19
of COVID-19 in the United States:
in wastewater,” J Virol Methods, infection on board repatriation flights
estimates and projections under
297:114230, 2021. to Australia,” Environ Int, 158:106938,
an infection-based herd immunity
13 ) Centers for Disease Control and 2022.
approach,” J Econ Ageing, 20:100328,
Prevention, “Wastewater surveillance 6 ) W. Ahmed et al., “Surveillance of
2021.
testing methods,” https://www.cdc. SARS-CoV-2 RNA in wastewater:
2 ) M.A. Johansson et al., “SARS-CoV-2
gov/healthywater/surveillance/ methods optimisation and quality
transmission from people without
wastewater-surveillance/ control are crucial for generating
COVID-19 symptoms,” Jama Netw
testing-methods.html?CDC_AA_ reliable public health information,”
Open, 4;4(1):e2035057, 2021.
ref Val=https%3A%2F%2Fwww. Curr Opin Environ Sci Heal, 17:82–93,
3 ) G. Medema et al., ”Presence of SARS-
cdc.gov%2Fcoronavirus%2F2019- 2020.
Coronavirus‑2 RNA in sewage and
ncov%2Fcases- 7 ) C. Barras, “Going to waste,” Nat Med,
correlation with reported COVID-19
updates%2Fwastewater- 24:14847, 2018.
prevalence in the early stage of the
surveillance%2Ftesting-methods.html, 8 ) S.C. Taylor et al., “Droplet digital
epidemic in the Netherlands,” Environ
accessed December 16, 2021. PCR versus qPCR for gene expression
Sci Tech Let, 7:511–6, 2020.
14 ) C. Park et al., “Comparison of digital analysis with low abundant targets:
4 ) W. Ahmed et al., “First confirmed
PCR and quantitative PCR with from variable nonsense to publication
detection of SARS-CoV-2 in untreated
various SARS-CoV-2 primer-Probe quality data,” Sci Rep, 7(1):2409, 2017.
wastewater in Australia: A proof of
Sets,” J Microbiol Biotechn, 31(3):358-
concept for the wastewater surveillance
67, 2021.
of COVID-19 in the community,” Sci
15 ) H.N. Vasudevan et al., “Digital droplet
Total Environ, 728:138764, 2020.
PCR accurately quantifies SARS-
5 ) G. Fongaro et al., ”The presence of
CoV-2 viral load from crude lysate
SARS-CoV-2 RNA in human sewage
without nucleic acid purification,” Sci
in Santa Catarina, Brazil, November
Rep, 11(1):780, 2021.
2019,” Sci Total Environ 778:146198,
16 ) L. Heijnen et al., “Droplet digital RT-
2021.
PCR to detect SARS-CoV-2 signature
6 ) W. Ahmed et al., “SARS-CoV-2
mutations of variants of concern
RNA monitoring in wastewater as
in wastewater,” Sci Total Environ,
a potential early warning system
799:149456, 2021.
for COVID-19 transmission in the
community: A temporal case study,” Sci
Total Environ 761:144216, 2021. Article 3: Wastewater’s Potential
7 ) H. Dutta et al., ”Wastewater-based
to Protect Public Health
epidemiology: a new frontier for
1 ) H. DeGan, “The science behind the
tracking environmental persistence
on-campus wastewater testing at

THE PAST, PRESENT, AND FUTURE OF WASTEWATER-BASED EPIDEMIOLOGY 6