Standards to Support an Enduring Capability in Wastewater Surveillance for Public Health Workshop
Standards for Wastewater Surveillance Workshop Organizing Committee (NIST & DHS)
Wastewater surveillance is a promising approach to monitor biological and chemical contaminants on a community level, and has recently been shown effective for the early detection of SARS-CoV-2 outbreaks. While much has been learned in the past year, much work remains to establish an enduring capability for wastewater surveillance poised to address new targets as they emerge. The goal of this workshop is to identify and prioritize standards needs and technology/measurement gaps and propose a potential path forward to develop standards that enable a robust, comparable wastewater surveillance capability. Topics to be discussed include lessons learned during the COVID-19 pandemic response, the state of the art in measurement science and technology for the entire wastewater surveillance workflow, and challenges in achieving comparable wastewater surveillance results across locations. Stakeholder input will inform future standards development activities, including consensus-based documentary standards, reference methods and protocols, and reference materials, and ensure that efforts are fit for purpose and aligned with the needs of the community. Ultimately, these standards will help to provide a foundation for communities and organizations to have increased confidence in wastewater surveillance results to better inform public health and safety decisions across the nation. This workshop is recommended for participants involved in all aspects of wastewater surveillance from sampling to results/reporting, including those from government, public health, testing and manufacturing, academia, and non-profit organizations.
More info: https://www.nist.gov/news-events/events/2021/06/dhsnist-workshop-standards-support-enduring-capability-wastewater
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▼ Data Reporting and Analytics Back to top
Using municipal data to develop geographically-precise sampling frame standards
R. Yeager, R. H. Holm, K. Saurabh, D. Talley, A. Bhatnagar, T. Smith
EzCOVID19: A Cloud-based Bioinformatics Platform for Rapid Detection, Characterization and Epidemiological Sub-typing of SARS-CoV-2
Nur Hasan
Method: EzCOVID19 generates consensus genome assembles, locates genetic variations from the SARS-CoV-2 Wuhan-Hu-1 reference (NC_045512.2), utilizes a novel SNV-based classification system for accurate identification, and produces parsimony trees based on public GISAID genomes.
Results: This user-friendly tool provides a platform for scientists to submit samples for rapid detection of SARS-CoV-2, download assembled genomes and profile tables, identify genetic variations, and compare with related genomes in public databases. EzCOVID19 also provides in-depth analyses and visualizations of the data with parsimony and maximum likelihood trees based on novel SNV sites. Its cloud-based system and online support provide immediate results from regularly updated public databases, including monitoring of variants of concern. To analyze your samples, access EzCOVID19 here https://www.ezbiocloud.net/tools/sc2.
Conclusion: Many scientists and public health workers from various research backgrounds have been required to adapt viral genomics into their research as a means of understanding SARS-CoV-2 without prior knowledge of virology or genomics. EzCOVID19 is a cloud-based, user-friendly, robust platform to assist scientists from different disciplines to research SARS-CoV-2. Regardless of method or sequencing instrument used, EzCOVID19 is able to integrate raw metagenomics or isolate sequence data for standardized bioinformatic analyses. Processed data are presented with detailed visuals and relevant information related to concurrently circulated publicly available SARS-CoV-2 genomes.
Systematic review of correlations for time-lagged COVID-19 cases and viral load in wastewater
Michael Austin
PiGx SARS-CoV-2 wastewater: A pipeline for reproducible wastewater sequencing analysis providing comprehensible geo-tagged time series reports
Vic-Fabienne Schumann, Ricardo Wurmus, Miriam Faxel, Jan Dohmen, Rafael Cuadrat, Bora Uyar, Vedran Franke, Alexander Blume, and Altuna Akalin
Wastewater SARS Public Health Environmental Response (W-SPHERE) Global Data Center
Krystin Kadonsky (1), Colleen C. Naughton (1), Clara Medina (1), Gertjan Medema (2), Panagis Katsivelis (3), Vajra Allan (4), Joan B. Rose (5)
Key words: Dashboard, COVID-19, Geographic Information System (GIS), Wastewater Based Epidemiology (WBE)
Standards and control materials used: Various standards and control methods used since data from many sources are aggregated for the data center.
▼ Interpretation and Use of Data Back to top
Targeted communications using wastewater monitoring at the sub-sewershed scale in Davis, CA
Heather N. Bischel, Hannah R. Safford, Rogelio Zuniga-Montanez, Minji Kim, Lezlie Rueda, C. Winston Bess, Michelle Dunigan, Randi Pechacek, Karen Shapiro, Tod Stoltz
HDT formed a Wastewater Action Committee (WAC) to coordinate communications of all wastewater results between HDT scientists and City leadership on a weekly basis and to develop and implement responses to wastewater results. A press release invited community members to opt into the pre-existing Yolo Alert messaging system to receive notifications when elevations are detected.
Virus levels in wastewater that exceed pre-defined action thresholds are reviewed by the WAC with three primary response strategies, together intended to encourage participation in HDT’s widely available, free asymptomatic COVID-19 testing program. First, HDT posts results on its website, broken down by sampling region to keep the public informed each week. The slope of a two-week moving average is evaluated to denote on the HDT website if levels in each sampling region are increasing, staying about the same, or decreasing. Second, the highest absolute normalized values of detection are used by HDT and the WAC to identify priority regions within the city and to cross-check participation in asymptomatic individual testing programs within different regions. Third and finally, geo-targeted text, email, and social media messages can be sent to a sub-sewershed region following a sustained elevation of normalized virus concentrations above the limit of detection. Sub-sewershed samples represent smaller populations (hundreds to low thousands of people) and are expected to frequently yield nondetects; an increase in levels above the detection limit represents a significant and easily identifiable change. The city used its opt-in text message alert system as well as Nextdoor to notify residents within the associated sub-sewershed region based on sewer system data collected in March 2021.
WBE Data Sharing, Ethics, and Community Outreach
Lauren Anderson1,2, Alexis Ecarma1,2, and Ted Smith1,2
Using high-resolution sampling in Davis, CA to understand how to best analyze and act on WBE data
H. Safford, R. Zuniga-Montanez, M. Kim, L. Rueda, R. Pechacek, R. Guerrero, M. Clauzel, X. Liu, L. Wei, M. Daza, M. Nuno, J. Sharpnack, K. Shapiro, H. Bischel
The Healthy Davis Together (HDT) program in Davis, CA applies WBE at all three of the above scales. Since Fall 2020, HDT has been collecting and analyzing levels of SARS-CoV-2 in wastewater collected from the following sites: • Building/neighborhood outflow [collected 1–3x/week]: o 22 UC Davis residential buildings or complexes o 6 Davis neighborhoods o 1 apartment complex o 1 elementary school • Sewershed sub-regions [3x/week]: o 15 sub-regions of the Davis sewershed • WWTP influent [7x/week]: o City of Davis WWTP o UC Davis WWTP
We are leveraging the high temporal and spatial resolution of HDT wastewater analysis to understand how to best analyze and act on WBE data. We are pleased to present and discuss early insights related to the following key topics and questions: • Treatment of non-detects. Relatively low concentrations of SARS-CoV-2 in environmental matrices like wastewater mean that qPCR technical replicates of the same sample frequently yield a mix of positive (“detect”) and negative (“non-detect”) results. Researchers commonly substitute a single constant value (e.g., zero or half the detection limit) for non-detects during data analysis. We are (i) illustrating how this crude approach biases results, and (ii) exploring multiple-imputation methods for more sophisticated handling of non-detects. • Sub-regional comparison of wastewater and clinical data. Many groups have demonstrated strong correlation between trends in wastewater SARS-CoV-2 concentrations and clinically confirmed COVID-19 cases at the city or college level. We are exploring whether similar relationships exist at the sub-regional level.
An organism-centric approach to performance metrics for metagenomic NGS-based diagnostics
Jason G. Kralj, Stephanie L. Servetas, Samuel P. Forry, Scott A. Jackson
Early Warnings of COVID-19 Second Wave in Detroit MI
Brijen Miyani, Liang Zhao, Maddie Spooner, Zachary Gentry, Irene Xagoraraki
▼ Other Back to top
Wastewater Surveillance’s Role as Part of a Whole-of-University Response in Campus Protection
Jeffrey W. Bethel(1), Benjamin Dalziel(2,3), Roy Haggerty(4), Kathryn A. Higley(5), Katherine R. McLaughlin(6), F. Javier Nieto(7), Justin L. Sanders(8), Brett M. Tyler(9,10), and Tyler S. Radniecki(11).
(1) https://www.forbes.com/sites/jemimamcevoy/2020/09/11/19-of-the-25-worst-us-coronavirus-outbreaks-are-in-college-towns/?sh=13d0f4491df7 (2) https://trace.oregonstate.edu/
▼ Processing/Testing Methods Back to top
First detection of SARS-CoV-2 RNA in wastewater in North America: A study in Louisiana, USA
Dr. Samendra sherchan
First detection of SARS-CoV-2 proteins in wastewater samples by mass spectrometry
Lara-Jacobo Linda R, Islam Golam, Gedge Ashley, Desaulniers Jean-Paul, Kirkwood Andrea E and Simmons Denina BD
Parameters influencing the methods of detection for SARS-CoV-2 wastewater surveillance using RT-PCR
*Golam Islam, Ashley Gedge, Linda Lara-Jacobo, Denina Simmons, Jean-Paul Desaulniers, Andrea Kirkwood
Targeted NGS for Sensitive Detection of SARS-CoV-2 Genomes
Jordan RoseFigura, Evan Hughes, Karl Spork, Laurie Kurihara
1 Spurbeck et al. Science of the Total Environment. 789 (2021) 147829 3 Fontenele el al. https://doi.org/10.1101/2021.01.22.21250320
A Step-By-Step Approach to Evaluating SARS-CoV-2 Methods: Adaptability for Future Use with Other Microorganisms
Brienna L Anderson-Coughlin and Kalmia E. Kniel
Importance of Validation for Wastewater Surveillance
Brian Swalla
SARS-CoV-2 Wastewater Surveillance in a remote municipality in the Aleutian Islands of Alaska
Karie Holtermann
Betancourt WQ, Schmitz BW, Innes GK, et al. COVID-19 containment on a college campus via wastewater-based epidemiology, targeted clinical testing and an intervention. The Science of the Total Environment. 2021 Jul;779:146408. DOI: 10.1016/j.scitotenv.2021.146408.
Medema G, Heijnen L, Elsinga G, Italiaander R, Brouwer A. Presence of SARS-Coronavirus-2 RNA in Sewage and Correlation with Reported COVID-19 Prevalence in the Early Stage of the Epidemic in The Netherlands. Environ Sci Technol Lett. 2020;acs.estlett.0c00357. Published 2020 May 20. doi:10.1021/acs.estlett.0c00357
Fuqing Wu, Amy Xiao, Jianbo Zhang, Katya Moniz, Noriko Endo, Federica Armas, Richard Bonneau, Megan A Brown, Mary Bushman, Peter R Chai, Claire Duvallet, Timothy B Erickson, Katelyn Foppe, Newsha Ghaeli, Xiaoqiong Gu, William P Hanage, Katherine H Huang, Wei Lin Lee, Mariana Matus, Kyle A McElroy, Jonathan Nagler, Steven F Rhode, Mauricio Santillana, Joshua A Tucker, Stefan Wuertz, Shijie Zhao, Janelle Thompson, and Eric J Alm. 2020. “SARS-CoV-2 titers in wastewater foreshadow dynamics and clinical presentation of new COVID-19 cases.” medRxiv.
Assessment of qPCR- targets and protocols for quantifying anthropogenic impacts of antibiotic resistance to the water environment
Ishi Keenum, Jeanette Calarco, Benjamin C. Davis, Krista Liguori, Erin Milligan, Valerie J. Harwood, Amy Pruden
However, there are thousands of known ARGs that could be targeted, each varying in their relevance to human health and their overall contribution to dissemination of antibiotic resistance. Further, there are various methodological aspects, such as sample concentration, extraction, and PCR inhibition that need to be evaluated to ensure that measurements are representative and comparable across studies. Here we conducted a critical review to identify ARGs and assays that are most commonly measured by qPCR in wastewater, recycled water, and surface water, specifically: sul1, intI1, vanA, blaCTX-M, and tetA. We identified 117 peer-reviewed studies meeting the search criteria and systematically assessed the corresponding workflows reported, including sample collection and concentration, DNA extraction, amplification conditions, amplicon length, and level of validation. Resulting concentrations reported for various water matrices were compared across the studies. Based on this evaluation, recommend assays, a standardized workflow, and reporting guidelines for the five genes of interest. Implications for emerging qPCR approaches, such as droplet digital qPCR and high-throughput qPCR, are also discussed and a path forward for standardization is proposed.
Comparison of Sampling Frequency and Concentration Methodology for the Detection and Quantification of SARS-CoV-2 in Wastewater
Andrea D. George, Devrim Kaya, Blythe A. Layton, Kestrel Bailey, Christine Kelly, Kenneth J. Williamson, Tyler S. Radniecki
Nucleic Acid Extraction with Microbubbles from Wastewater Produces Simple and High-Yield Workflows
M Maloney1, C Livi1, J Roussey1, N Petlakh-Co1, V Kelchner1, B Macintyre1, E Grimley1, A Pierri2, C Wegner1
Sources Of Variability In Methods For Processing, Storing, And Concentrating SARS-CoV-2 In Influent From Urban Wastewater Treatment Plants
Joshua A. Steele, Amy G. Zimmer-Faust, John F. Griffith, Steven B. Weisberg
Rapid Capture of SARS-CoV-2 Using Nanotrap® Magnetic Virus Particles
Alex Barclay,1 Smruthi Karthikeyan,2 Robbie Barbero,1 Patrick Andersen,1 Daniel Goldfarb,1 Kevin Kolb,1 Joshna Seelam,1 Tara Jones-Roe,1 Rob Knight,2 Ben Lepene1
Review of culture methods for monitoring antibiotic resistant Acinetobacter, Aeromonas, and Pseudomonas in wastewater, recycled and receiving water
Erin Milligan, Jeanette Calarco, Ben Davis, Ishi Keenum, Krista Liguori, Amy Pruden, Valerie J. Harwood
Single Particle ICP-MS: A Powerful Tool for Characterization of Nanomaterials in Wastewater Surveillance
Monique E. Johnson, Karen E. Murphy, Antonio R. Montoro Bustos, Michael Winchester
Analytical methods, critical for the detection and characterization of nanomaterials in wastewater, can address current gaps in knowledge. In particular, single particle inductively coupled plasma mass spectrometry (spICP-MS) is an emerging technique that enables measurements of size distributions and number concentrations of nanoparticles suspended in liquids. Special characteristics of spICP-MS that make it attractive for wastewater surveillance include extremely good nanoparticle detection capability down to truly environmentally relevant levels and extremely rapid analysis. The use of spICP-MS for wastewater surveillance has been outlined in recent reviews [2,3] and demonstrated in publications describing the detection and sizing of TiO2 nanoparticles in municipal sewage treatment plants [4] and the size characterization of silver nanoparticles in wastewater [5,6]. However, the technique presents challenges and limitations that researchers have highlighted and are striving to overcome, particularly with respect to the lack of reference materials and standardization of spICP-MS methodologies. Provision of suitable reference materials and standard methods would enable widespread effective and efficient application of spICP-MS for wastewater surveillance to ensure that human health is not affected detrimentally by nanomaterials.
The goal of our presentation is to highlight a few examples of the work that has been done within the NIST Inorganic Chemical Metrology Group (646.01) toward the advancement of spICP-MS as a mature technique. This work has provided a foundation for developing the standards and reference materials that are necessary to enable routine wastewater surveillance for nanoparticles. In the past five years, our group has engaged in several activities to build a robust nanomaterial spICP-MS measurement infrastructure, including: • validation of spICP-MS for routine characterization of gold nanoparticles of different sizes, coatings, and surface charge at environmentally relevant concentrations in water; • analysis of silver and titanium dioxide nanoparticles in water; • characterization of silicon dioxide food additive materials in aqueous media; • the characterization of the uptake of gold nanoparticles in Caenorhabditis elegans; and • the potential application of spICP-MS for the characterization of nanoplastics suspended in water.
Minimizing uncertainties of COVID-19 prevalence by establishing standards for wastewater surveillance
Guangming Jiang, Xuan Li, Shuxin Zhang, Jiahua Shi, Stephen P. Luby
The Development of a Sensitive and Reliable qRT-PCR-Based SARS-CoV-2 Wastewater Analysis Protocol
Agustin Pierri and Douglas Sieglaff
SARS-CoV-2 improved primers and probes and RT-qPCR detection assay for variants of concern B.1.1.7, B.1.351, P.1 and B.1.617
Karin Yaniv, Eden Ozer, Marilou Shagan, Satish Lakkakula, Noam Plotkin, Nikhil Suresh Bhandarkar and Ariel Kushmaro
A Direct Nucleic Acid Capture Method for Purification and Detection of SARS-CoV-2 Genetic Material from Wastewater
Nathan Feirer and Subhanjan Mondal
In parallel, we formulated RT-qPCR enzyme mixes that demonstrate resistance to PCR inhibitors commonly found in wastewater. RT-qPCR assays were developed to detect N1, N2 (nucleocapsid) and E (envelope) gene fragments of SARS-CoV-2 as part of multiplexed assays. Pepper Mild Mottle Virus (PMMoV), a fecal indicator RNA virus present in wastewater, and an exogenous inhibition control were included in all PCR reactions for quality control. The workflow has a limit of detection of 1.6 GC/ ml with 40 ml of raw wastewater sample. Using this workflow, we monitored wastewater samples from three WWTP in Dane County, Wisconsin, serving a combined population of over 300,000 people, for four months covering the peak of community prevalence. Our analysis was compared to the COVID-19 cases declared by the municipalities, demonstrating strong correlation between the SARS-CoV-2 viral load present in wastewater and clinical cases.
Challenges with Standardizing the Measurement of SARS-CoV-2 Recovery Efficiency in Wastewater
Kara L. Nelson, Rose S. Kantor, Hannah D. Greenwald, Lauren C. Kennedy
▼ Sampling Back to top
Equity in standardized collection of pooled community stool for pathogen detection: Moving from pit latrines to sewered systems globally
Rochelle H. Holm, and Ted Smith
Optimizing Sampling Strategies for Large Rural Regions
Anuj Tiwari, Aaron Packman, Charles Williams, Wilnise Jasmin, Rachel Poretsky, Wayne Duffus, Sarah Patrick, James Wendt, Leslie Wise, Charlie Catlett
The State of Illinois is a primarily rural region comprising 58k square miles with a population of roughly 16M, over half of whom live within 50 miles of Chicago. Outside of the Chicago area, Illinois has roughly 140 cities with populations greater than 10k and nearly 1,000 with populations under 10k. To understand and track infectious disease such as COVID-19 across these communities, a comprehensive WBE sampling plan would involve over 1,000 WWTPs and countless private septic systems.
In Sep 2020, we introduced a machine learning based COVID-19 Vulnerability Index (C19VI) using CDC’s six themes: (a) socioeconomic status, (b) household composition & disability, (c) minority status & language, (d) housing type & transportation, (e) epidemiological factors, and (e) healthcare system factors . This model uses an ensemble learning approach with recursive partitioning to optimally compute non-linear relationships between input themes. We refined the model in early 2021 with additional demographic and sequencing information to evaluate WBE sampling strategies across Illinois counties to support an expansion of WBE from currently several dozen to over 150 WWTPs. The vulnerability index supported the evaluation of various sampling strategies, such as selecting the largest population centers within each of the eleven COVID-19 “Restore Illinois” regions , and eventually the current strategy of sampling from the largest population center in each of Illinois’ 102 counties. In this poster we outline the models and methods used, including various sampling strategies explored, the current strategy, and experiments planned to evaluate the sampling strategy as we expand to 150 WWTPs in 2021.
CEDAR-MC: Clinical and Environmental Dynamics of Antibiotic Resistance within Microbial Communities
George Hanna1,2, Bashir Hamidi1, Scott Curry1, Cheryl Carmack2, Alexander V. Alekseyenko1
Methods: We bring together environmental and clinical measurements of the microbial communities with evidence for emerging resistance by linking existing local clinical and environmental surveillance programs. The clinical specimens are sourced from the Medical University of South Carolina (MUSC) infection surveillance culture program that routinely samples the MUSC patient population for clinically relevant pathogens. The environmental specimens are the result of partnership with a local non-profit, Charleston Waterkeeper, that performs water quality monitoring at popular recreational spots in the area.
Results: The surplus infection surveillance specimens are made available for research via a Living µBiome Bank system that enables nuanced electronic phenotyping of patient populations for just-in-time capture of clinical microbiology specimens. We show the feasibility of sequencing these clinical specimens for microbiota composition, which in combination with resistance status is invaluable in characterizing the broader structure of resistant microbial communities. The environmental specimens collected from May 2018 through October 2020 have been banked to be assessed for evidence of antibiotic resistance of their constituent microbiota.
Discussion: We continue to expand the resources and the biobanks associated with the clinical and environmental surveillance programs with an overarching goal to control infections by understanding the dynamics of emergence and maintenance of resistance. This project demonstrates the value of collaboration between academic healthcare and community-funded environmental monitoring programs.
▼ Standards/Reference Materials Back to top
Whole Cell Reference Materials to Assess Microbial Detection in Wastewater
Sandra Da Silva1, Joy P. Dunkers1, Kirsten H. Parratt1, Guilherme Pinheiro1,2, Samuel T. Hailemichael1,3, and Nancy J. Lin1
1Hendriksen, R.S., Munk, P., Njage, P. et al. Global monitoring of antimicrobial resistance based on metagenomics analyses of urban sewage. Nat Commun 10, 1124 (2019). https://doi.org/10.1038/s41467-019-08853-3
NIST SRM 2917: A Plasmid DNA Standard for Molecular Recreational Water Quality Testing
Scott A. Jackson, Jason G. Kralj, Blaza Toman, Stephanie S. Servetas, Monique E. Hunter, Jessica R. Willis, and Orin C. Shanks
A DNA-based reference material for pathogen detection via metagenomic next-generation sequencing (mNGS)
Jason G. Kralj, Monique Hunter, Blaza Toman, Scott A. Jackson
Antimicrobial Resistance (AMR) in Water Environments: Standardizing Monitoring
Krista Liguori, Ben Davis, Ishi Keenum, Erin Milligan, Jeanette Calarco, Valerie (Jody) Harwood, Amy Pruden
Development of RNA Standards for Quantification of SARS-CoV-2 and PMMoV by RT-qPCR for Wastewater Surveillance
Subhanjan Mondal, Nathan Feirer, and Dongping Ma