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All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Wastewater Surveillance for Poliovirus in Selected Jurisdictions, United States, 2022–2023

Wastewater testing can inform public health action as a component of polio outbreak response. During 2022-2023, a total of 7 US jurisdictions (5 states and 2 cities) participated in prospective or retrospective testing of wastewater for poliovirus after a paralytic polio case was identified in New York state. Two distinct vaccine-derived poliovirus type 2 viruses were detected in wastewater from New York state and New York City during 2022, representing 2 separate importation events. Of those viruses, 1 resulted in persistent community transmission in multiple New York counties and 1 paralytic case. No poliovirus was detected in the other participating jurisdictions (Connecticut, New Jersey, Michigan, and Illinois and Chicago, IL). The value of routine wastewater surveillance for poliovirus apart from an outbreak is unclear. However, these results highlight the ongoing risk for poliovirus importations into the United States and the need to identify undervaccinated communities and increase vaccination coverage to prevent paralytic polio.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

Air, surface, and wastewater surveillance of SARS-CoV-2; a multimodal evaluation of COVID-19 detection in a built environment

Environmental surveillance of infectious organisms holds tremendous promise to reduce human-to-human transmission in indoor spaces through early detection. In this study we determined the applicability and limitations of wastewater, indoor high-touch surfaces, in-room air, and rooftop exhaust air sampling methods for detecting SARS-CoV-2 in a real world building occupied by residents recently diagnosed with COVID-19. We concurrently examined the results of three 24-hour environmental surveillance techniques, indoor surface sampling, exhaust air sampling and wastewater surveillance, to the known daily census fluctuations in a COVID-19 isolation dormitory. Additionally, we assessed the ability of aerosol samplers placed in the large volume lobby to detect SARS-CoV-2 multiple times per day. Our research reveals an increase in the number of individuals confirmed positive with COVID-19 as well as their estimated human viral load to be associated with statistically significant increases in viral loads detected in rooftop exhaust aerosol samples (p = 0.0413), wastewater samples (p = 0.0323,), and indoor high-touch surfaces (p < 0.001)). We also report that the viral load detected in lobby aerosol samples was statistically higher in samples collected during presence of occupants whose COVID-19 diagnostic tests were confirmed positive via qPCR compared to periods when the lobby was occupied by either contact-traced (suspected positive) individuals or during unoccupied periods (p = 0.0314 and <2e–16). We conclude that each daily (24h) surveillance method, rooftop exhaust air, indoor high-touch surfaces, and wastewater, provide useful detection signals for building owner/operator(s). Furthermore, we demonstrate that exhaust air sampling can provide spatially resolved signals based upon ventilation exhaust zones. Additionally, we find that indoor lobby air sampling can provide temporally resolved signals useful during short duration sampling periods (e.g., 2-4 hours) even with intermittent occupancy by occupants diagnosed with COVID-19.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

SARS-CoV-2 raw wastewater surveillance from student residences on an urban university campus

The COVID-19 pandemic brought about an urgent need to monitor the community prevalence of infection and detect the presence of SARS-CoV-2. Testing individual people is the most reliable method to measure the spread of the virus in any given community, but it is also the most expensive and time-consuming. Wastewater-based epidemiology (WBE) has been used since the 1960s when scientists implemented monitoring to measure the effectiveness of the Polio vaccine. Since then, WBE has been used to monitor populations for various pathogens, drugs, and pollutants. In August 2020, the University of Tennessee-Knoxville implemented a SARS-CoV-2 surveillance program that began with raw wastewater surveillance of the student residence buildings on campus, the results of which were shared with another lab group on campus that oversaw the pooled saliva testing of students. Sample collection began at 8 am, and the final RT-qPCR results were obtained by midnight. The previous day’s results were presented to the campus administrators and the Student Health Center at 8 am the following morning. The buildings surveyed included all campus dormitories, fraternities, and sororities, 46 buildings in all representing an on-campus community of over 8,000 students. The WBE surveillance relied upon early morning “grab” samples and 24-h composite sampling. Because we only had three Hach AS950 Portable Peristaltic Sampler units, we reserved 24-h composite sampling for the dormitories with the highest population of students. Samples were pasteurized, and heavy sediment was centrifuged and filtered out, followed by a virus concentration step before RNA extraction. Each sample was tested by RT-qPCR for the presence of SARS-CoV-2, using the CDC primers for N Capsid targets N1 and N3. The subsequent pooled saliva tests from sections of each building allowed lower costs and minimized the total number of individual verification tests that needed to be analyzed by the Student Health Center. Our WBE results matched the trend of the on-campus cases reported by the student health center. The highest concentration of genomic copies detected in one sample was 5.06 × 10 7 copies/L. Raw wastewater-based epidemiology is an efficient, economical, fast, and non-invasive method to monitor a large community for a single pathogen or multiple pathogen targets.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

A modeling pipeline to relate municipal wastewater surveillance and regional public health data

As COVID-19 becomes endemic, public health departments require improved passive indicators, which are independent of voluntary testing data, to estimate the prevalence of COVID-19 in local communities. Quantification of SARS-CoV-2 RNA from wastewater has the potential to be a powerful passive indicator. However, connecting measured SARS-CoV-2 RNA to community prevalence is challenging. We have developed a generalized methodology to improve the predictive power of wastewater measurements and applied it to data collected from treatment plants in the Chicago area. We built and compared a set of multi-linear regression models, which incorporate pepper mild mottle virus (PMMoV) as a population biomarker, Bovine coronavirus (BCoV) as a recovery control, and wastewater system flow rate into a corrected estimate for SARS-CoV-2 RNA concentration. For our data, models with BCoV performed better than those with PMMoV, but all model estimates of prevalence significantly improved correlation compared to doing no correction. We also investigate the utility of RNA measurements in wastewater as a leading indicator of COVID-19 trends. We do this in a rolling manner for corrected wastewater data and for other prevalence indicators, and statistically compare the temporal relationship between new increases in the wastewater data and those in other prevalence indicators. We find that wastewater trends often lead other COVID-19 indicators in predicting new surges.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

SARS-CoV-2 wastewater variant surveillance: pandemic response leveraging FDA’s GenomeTrakr network

ABSTRACT Wastewater surveillance has emerged as a crucial public health tool for population-level pathogen surveillance. Supported by funding from the American Rescue Plan Act of 2021, the FDA‘s genomic epidemiology program, GenomeTrakr, was leveraged to sequence SARS-CoV-2 from wastewater sites across the United States. This initiative required the evaluation, optimization, development, and publication of new methods and analytical tools spanning sample collection through variant analyses. Version-controlled protocols for each step of the process were developed and published on protocols.io. A custom data analysis tool and a publicly accessible dashboard were built to facilitate real-time visualization of the collected data, focusing on the relative abundance of SARS-CoV-2 variants and sub-lineages across different samples and sites throughout the project. From September 2021 through June 2023, a total of 3,389 wastewater samples were collected, with 2,517 undergoing sequencing and submission to NCBI under the umbrella BioProject,PRJNA757291. Sequence data were released with explicit quality control (QC) tags on all sequence records, communicating our confidence in the quality of data. Variant analysis revealed wide circulation of Delta in the fall of 2021 and captured the sweep of Omicron and subsequent diversification of this lineage through the end of the sampling period. This project successfully achieved two important goals for the FDA’s GenomeTrakr program: first, contributing timely genomic data for the SARS-CoV-2 pandemic response, and second, establishing both capacity and best practices for culture-independent, population-level environmental surveillance for other pathogens of interest to the FDA. IMPORTANCE This paper serves two primary objectives. First, it summarizes the genomic and contextual data collected during a Covid-19 pandemic response project, which utilized the FDA’s laboratory network, traditionally employed for sequencing foodborne pathogens, for sequencing SARS-CoV-2 from wastewater samples. Second, it outlines best practices for gathering and organizing population-level next generation sequencing (NGS) data collected for culture-free, surveillance of pathogens sourced from environmental samples.

Microbiology↗
DOE OSTI2025

Benchmarking Concentration and Extraction Methods for Wastewater-Based Surveillance of Eight Human Respiratory Viruses: Implications for Rapid Application to Novel Pathogens

To provide early warning and support a rapid response to a novel virus through wastewater surveillance, it would be ideal to understand in advance which concentration and extraction methods are likely to be effective for dPCR-based methods, depending on virus characteristics. In this study, we spiked raw wastewater samples with eight human respiratory viruses and processed them with four methods that concentrate and/or extract nucleic acids from both liquid and solid fractions (Promega, Nanotrap, and InnovaPrep) or only the solid fraction of wastewater (Solids). Our findings provide encouraging evidence that all four methods combined with dPCR could detect an emerging virus in wastewater, although they differed in sensitivity. The pattern of recovery efficiency for adenoviruses, coronaviruses, and influenza A viruses was consistent across methods, with Promega producing higher median recovery efficiencies, while distinct patterns were observed for coxsackieviruses. We also normalized the concentration data with two endogenous fecal indicators, PMMoV and Carjivirus (formerly crAssphage). We found that normalization could reduce method-associated differences if the indicator exhibited a recovery pattern similar to that of the target virus. These findings can guide the selection of concentration and extraction methods for wastewater monitoring based on the properties of target viruses, thus enhancing pandemic preparedness.

Biological and medical sciences↗
DOE OSTI2026

Citywide indoor air sampling mirrors wastewater and clinical for environmental surveillance of respiratory viruses

Wastewater surveillance of respiratory pathogens can provide timely estimates of viral activity and disease trends in a population. Indoor air surveillance could be used similarly with some advantages but remains largely unvalidated at the community -scale. Here, an indoor air surveillance program was employed as part of public health environmental surveillance in Chicago, Illinois, USA. Ten air samplers were placed in healthcare and congregate living settings across the city. Weekly air samples were evaluated for influenza A, influenza B, respiratory syncytial virus, and SARS -CoV-2 over two respiratory virus seasons (2023 -2025). Citywide, aggregated air sample positivity and viral load were closely correlated with local clinical case and wastewater surveillance data across all respiratory viruses. Virus trends in air data often preceded clinical and wastewater, although this varied across pathogens and respiratory virus seasons. Further, whole -genome sequencing of SARS -CoV-2 showed close correlation of variant proportions across all datasets. At the building -scale, air samples obtained from a single sampling device provided efficient respiratory virus surveillance, with respiratory pathogen levels mirroring citywide clinical surveillance data. These data demonstrate that air surveillance can provide respiratory virus case and variant trend data at a building or community -scale, serving as an alternative or complementary tool for public health environmental surveillance.

Wilton, Rosemarie↗
DOE OSTI2023

Intensity of sample processing methods impacts wastewater SARS-CoV-2 whole genome amplicon sequencing outcomes

Wastewater SARS-CoV-2 surveillance has been deployed since the beginning of the COVID-19 pandemic to monitor the dynamics in virus burden in local communities. Genomic surveillance of SARS-CoV-2 in wastewater, particularly efforts aimed at whole genome sequencing for variant tracking and identification, are still challenging due to low target concentration, complex microbial and chemical background, and lack of robust nucleic acid recovery experimental procedures. The intrinsic sample limitations are inherent to wastewater and are thus unavoidable. Here, we use a statistical approach that couples correlation analyses to a random forest-based machine learning algorithm to evaluate potentially important factors associated with wastewater SARS-CoV-2 whole genome amplicon sequencing outcomes, with a specific focus on the breadth of genome coverage. We collected 182 composite and grab wastewater samples from the Chicago area between November 2020 to October 2021. Samples were processed using a mixture of processing methods reflecting different homogenization intensities (HA + Zymo beads, HA + glass beads, and Nanotrap), and were sequenced using one of the two library preparation kits (the Illumina COVIDseq kit and the QIAseq DIRECT kit). Technical factors evaluated using statistical and machine learning approaches include sample types, certain sample intrinsic features, and processing and sequencing methods. The results suggested that sample processing methods could be a predominant factor affecting sequencing outcomes, and library preparation kits was considered a minor factor. Finally, a synthetic SARS-CoV-2 RNA spike-in experiment was performed to validate the impact from processing methods and suggested that the intensity of the processing methods could lead to different RNA fragmentation

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Performance of methods for SARS-CoV-2 variant detection and abundance estimation within mixed population samples

The accurate identification of SARS-CoV-2 (SC2) variants and estimation of their abundance in mixed population samples (e.g., air or wastewater) is imperative for successful surveillance of community level trends. Assessing the performance of SC2 variant composition estimators (VCEs) should improve our confidence in public health decision making. Here, we introduce a linear regression based VCE and compare its performance to four other VCEs: two re-purposed DNA sequence read classifiers (Kallisto and Kraken2), a maximum-likelihood based method (Lineage deComposition for Sars-Cov-2 pooled samples (LCS)), and a regression based method (Freyja). We simulated DNA sequence datasets of known variant composition from both Illumina and Oxford Nanopore Technologies (ONT) platforms and assessed the performance of each VCE. We also evaluated VCEs performance using publicly available empirical wastewater samples collected for SC2 surveillance efforts. Bioinformatic analyses were performed with a custom NextFlow workflow (C-WAP, CFSAN Wastewater Analysis Pipeline). Relative root mean squared error (RRMSE) was used as a measure of performance with respect to the known abundance and concordance correlation coefficient (CCC) was used to measure agreement between pairs of estimators. Based on our results from simulated data, Kallisto was the most accurate estimator as it had the lowest RRMSE, followed by Freyja. Kallisto and Freyja had the most similar predictions, reflected by the highest CCC metrics. We also found that accuracy was platform and amplicon panel dependent. For example, the accuracy of Freyja was significantly higher with Illumina data compared to ONT data; performance of Kallisto was best with ARTICv4. However, when analyzing empirical data there was poor agreement among methods and variations in the number of variants detected (e.g., Freyja ARTICv4 had a mean of 2.2 variants while Kallisto ARTICv4 had a mean of 10.1 variants). This work provides an understanding of the differences in performance of a number of VCEs and how accurate they are in capturing the relative abundance of SC2 variants within a mixed sample (e.g., wastewater). Such information should help officials gauge the confidence they can have in such data for informing public health decisions.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

SARS-CoV-2 Monitoring in Wastewater Reveals Novel Variants and Biomarkers of Infection

Wastewater-based epidemiology (WBE) is a popular tool for the early indication of community spread of infectious diseases. WBE emerged as an effective tool during the COVID-19 pandemic and has provided meaningful information to minimize the spread of infection. Here, we present a combination of analyses using the correlation of viral gene copies with clinical cases, sequencing of wastewater-derived RNA for the viral mutants, and correlative analyses of the viral gene copies with the bacterial biomarkers. Our study provides a unique platform for potentially using the WBE-derived results to predict the spread of COVID-19 and the emergence of new variants of concern. Further, we observed a strong correlation between the presence of SARS-CoV-2 and changes in the microbial community of wastewater, particularly the significant changes in bacterial genera belonging to the families of Lachnospiraceae and Actinomycetaceae . Our study shows that microbial biomarkers could be utilized as prediction tools for future infectious disease surveillance and outbreak responses. Overall, our comprehensive analyses of viral spread, variants, and novel bacterial biomarkers will add significantly to the growing body of literature on WBE and COVID-19.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Evaluation of the Impact of Concentration and Extraction Methods on the Targeted Sequencing of Human Viruses from Wastewater

Sequencing human viruses in wastewater is challenging due to their low abundance compared to the total microbial background. This study compared the impact of four virus concentration/extraction methods (Innovaprep, Nanotrap, Promega, and Solids extraction) on probe-capture enrichment for human viruses followed by sequencing. Different concentration/extraction methods yielded distinct virus profiles. Innovaprep ultrafiltration (following solids removal) had the highest sequencing sensitivity and richness, resulting in the successful assembly of several near-complete human virus genomes. However, it was less sensitive in detecting SARS-CoV-2 by digital polymerase chain reaction (dPCR) compared to Promega and Nanotrap. Across all preparation methods, astroviruses and polyomaviruses were the most highly abundant human viruses, and SARS-CoV-2 was rare. These findings suggest that sequencing success can be increased using methods that reduce nontarget nucleic acids in the extract, though the absolute concentration of total extracted nucleic acid, as indicated by Qubit, and targeted viruses, as indicated by dPCR, may not be directly related to targeted sequencing performance. Further, using broadly targeted sequencing panels may capture viral diversity but risks losing signals for specific low-abundance viruses. Overall, this study highlights the importance of aligning wet lab and bioinformatic methods with specific goals when employing probe-capture enrichment for human virus sequencing from wastewater.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

2022 Annual Site Environmental Report for Sandia National Laboratories, Kaua'i Test Facility, Hawai'i

Sandia National Laboratories is a multimission laboratory managed and operated by National Technology & Engineering Solutions of Sandia, LLC, a wholly owned subsidiary of Honeywell International Inc., for the. U.S. Department of Energy’s National Nuclear Security Administration. The National Nuclear Security Administration’s Sandia Field Office administers the contract and oversees contractor operations at the Sandia National Laboratories Kaua'i Test Facility in Hawai'i. Activities at the site are conducted in support of U.S. Department of Energy weapons programs., and the site has operated as a rocket preparation launching and tracking facility since 1962. The U.S. Department of Energy and its management and operating contractor are committed to safeguarding the environment, assessing sustainability practices, and ensuring the validity and accuracy of the monitoring data presented in this annual site environmental report. This report summarizes the environmental protection, restoration, and monitoring programs in place at Sandia National Laboratories, Kaua'i Test Facility, during calendar year 2022. Environmental topics include cultural resource management, chemical management, air quality, meteorology, ecology, oil storage, site sustainability, terrestrial surveillance, waste management, water quality, wastewater discharge, and implementation of the National Environmental Policy Act. This report is prepared in accordance with and as required by DOE O 231.1B, Admin Change 1, Environment, Safety and Health Reporting and has been approved for public distribution.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2023

2022 Annual Site Environmental Report for Sandia National Laboratories, Tonopah Test Range, Nevada

Sandia National Laboratories is a multimission laboratory managed and operated by National Technology & Engineering Solutions of Sandia, LLC, a wholly owned subsidiary of Honeywell International Inc., for the. U.S. Department of Energy’s National Nuclear Security Administration. The National Nuclear Security Administration’s Sandia Field Office administers the contract and oversees contractor operations at Sandia National Laboratories, Tonopah Test Range. Activities at the site are conducted in support of U.S. Department of Energy weapons programs and have operated at the site since 1957. The U.S. Department of Energy and its management and operating contractor are committed to safeguarding file environment, assessing sustainability practices, and ensuring the validity and accuracy of the monitoring data presented in this annual site environmental report. This report summarizes the environmental protection, restoration, and monitoring programs in place at Sandia National Laboratories, Tonopah Test Range during calendar year 2022. Environmental topics include cultural resource management, chemical management, air quality, ecology, environmental restoration, oil storage, site sustainability, terrestrial surveillance, waste management, water quality, wastewater discharge, and implementation of the National Environmental Policy Act. This report is prepared in accordance with and as required by DOE 0 231.IB, Admin Change 1, Environment, Safety and Health Reporting and has been approved for public distribution.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2024

2023 Annual Site Environmental Report for Sandia National Laboratories, Tonopah Test Range, Nevada

Sandia National Laboratories is a multimission laboratory managed and operated by National Technology & Engineering Solutions of Sandia, LLC, a wholly owned subsidiary of Honeywell International Inc., for the U.S. Department of Energy’s National Nuclear Security Administration. The National Nuclear Security Administration’s Sandia Field Office administers the contract and oversees contractor operations at Sandia National Laboratories, Tonopah Test Range. Activities at the site are conducted in support of U.S. Department of Energy weapons programs and have operated at the site since 1957. The U.S. Department of Energy and its management and operating contractor are committed to safeguarding the environment, assessing sustainability practices, and ensuring the validity and accuracy of the monitoring data presented in this annual site environmental report. This report summarizes the environmental protection, restoration, and monitoring programs in place at Sandia National Laboratories, Tonopah Test Range during calendar year 2023. Environmental topics include cultural resource management, chemical management, air quality, ecology, environmental restoration, oil storage, site sustainability, terrestrial surveillance, waste management, water quality, wastewater discharge, and implementation of the National Environmental Policy Act. This report is prepared in accordance with and as required by DOE O 231.1B, Admin Change 1, Environment, Safety and Health Reporting, and has been approved for public distribution.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2024

2023 Annual Site Environmental Report for Sandia National Laboratories, Kaua‘i Test Facility, Hawai‘i

Sandia National Laboratories is a multimission laboratory managed and operated by National Technology & Engineering Solutions of Sandia, LLC, a wholly owned subsidiary of Honeywell International Inc., for the U.S. Department of Energy’s National Nuclear Security Administration. The National Nuclear Security Administration’s Sandia Field Office administers the contract and oversees contractor operations at the Sandia National Laboratories Kaua‘i Test Facility in Hawai‘i. Activities at the site are conducted in support of U.S. Department of Energy weapons programs, and the site has operated as a rocket preparation launching and tracking facility since 1962. The U.S. Department of Energy and its management and operating contractor are committed to safeguarding the environment, assessing sustainability practices, and ensuring the validity and accuracy of the monitoring data presented in this annual site environmental report. This report summarizes the environmental protection, restoration, and monitoring programs in place at Sandia National Laboratories, Kaua‘i Test Facility, during calendar year 2023. Environmental topics include cultural resource management, chemical management, air quality, meteorology, ecology, oil storage, site sustainability, terrestrial surveillance, waste management, water quality, wastewater discharge, and implementation of the National Environmental Policy Act. This report is prepared in accordance with and as required by DOE O 231.1B, Admin Change 1, Environment, Safety and Health Reporting, and has been approved for public distribution.

54 ENVIRONMENTAL SCIENCES↗