medRxiv May 2023 Background The origin of divergent SARS-CoV-2 spike sequences found in wastewater, but not in clinical surveillance, remains unclear. These “cryptic” wastewater sequences have harbored many of the same mutations that later emerged in Omicron lineages. We first detected a cryptic lineage in municipal wastewater in Wisconsin in January 2022. Named the “Wisconsin Lineage”, we sought to determine this virus’s geographic origin and characterize its persistence and evolution over time. Methods We systematically sampled maintenance holes to trace the Wisconsin Lineage’s origin. We sequenced spike RBD domains, and where possible, whole viral genomes, to characterize the evolution of this lineage over the 13 consecutive months that it was detectable. Findings The persistence of the Wisconsin Lineage signal allowed us to trace it from a central wastewater plant to a single facility, with a high concentration of viral RNA. The viral sequences contained a combination of fixed nucleotide substitutions characteristic of Pango lineage B.1.234, which circulated in Wisconsin at low levels from October 2020 to February 2021, while mutations in the spike gene resembled those subsequently found in Omicron variants. Interpretation We propose that prolonged detection of the Wisconsin Lineage in wastewater represents persistent shedding of SARS-CoV-2 from an infected individual, with ongoing within-host viral evolution leading to an ancestral B.1.234 virus accumulating “Omicron-like” mutations.

medRxiv, May 2023 "Human norovirus (HuNoV) GII RNA in wastewater solids at 145 United States wastewater treatment plants: Comparison to positivity rates of clinical specimens and modeled estimates of HuNoV GII shedders" Background: Human norovirus (HuNoV) is a leading cause of disease globally, yet actual incidence is unknown. HuNoV infections are not reportable in the United States, and surveillance is limited to tracking severe illnesses or outbreaks. Wastewater monitoring for HuNoV has been done previously and results indicate it is present in wastewater influent and concentrations are associated with HuNoV infections in the communities contributing to wastewater. However, work has mostly been limited to monthly samples of liquid wastewater at one or a few wastewater treatment plants (WWTPs). Methods: We measured HuNoV GII RNA in matched wastewater solids and liquid influent in 7 samples from a WWTP. We also applied the HuNoV GII assay to measure viral RNA in over 6000 wastewater solids samples from 145 WWTPs from across the United States daily to three times per week for up to five months. Measurements were made using digital droplet RT-PCR. Results: HuNoV GII RNA preferentially adsorbs to wastewater solids where it is present at 1000 times the concentration in influent. Concentrations of HuNoV GII RNA correlate positively with clinical HuNoV positivity rates. Model output of the fraction of individuals shedding HuNoV is variable and uncertain, but consistent with i Interpretation: These findings support the utility of wastewater solids as a matrix for community infectious disease surveillance.

Poster: COVID-19 highlighted the use of wastewater testing as a complementary method to monitor emerging outbreaks of infectious pathogens, including those harboring antimicrobial resistance (AMR) genes. Antibiotic resistant bacteria are identified as a primary public health concern that threatens to eliminate the effective use of antibiotics. As a strategy to complement population testing, wastewater samples can be evaluated to identify increases in the presence of AMR genes in communities. However, the use of molecular testing methods is challenging because many genetic mechanisms contribute to AMR, with the most common being β-Lactamases. To be an effective surveillance strategy, these tests must be designed to identify a broad range of targets associated with AMR, and the methods must be sensitive given the larger wastewater sampling volumes used for analysis. The data described here demonstrate methods for improved sample processing and a head-to-head comparison of commercially available real-time PCR-based assays to assess AMR burden in wastewater samples.

Quantifying Respiratory Viruses in Wastewater Using GT Molecular's Digital PCR Assays coupled with Ceres Nanosciences' Nanotrap® Particle Technology, the ThermoFisher MagMAX™ Microbiome Ultra Nucleic Acid Isolation Kit, and the KingFisher™ Apex Extraction Workflow™ Download full application note here Key Advantages High-throughput multiplex detection coupling GT Molecular Digital PCR Assays with the Nanotrap Partice Capture and Concentration Methods Ability to co-monitor and co-quantify multiple pathogens Assays available for the QIAGEN QIAcuity® Respiratory virus detection including Influenza A, Influenza B, and RSV in addition to SARS-CoV-2 Introduction Monitoring the spread of SARS-CoV-2, the virus that causes COVID-19, as well as other pathogens such as RSV and Influenza continues to provide vital information for interrupting chains of transmission and preventing or dampening the spread of new cases. Wastewater testing continues to gain traction as a viable means to monitor pathogens and provide timely information about the changing prevalence within individual communities. Wastewater-based epidemiology (WBE) utility has already been successfully described to track infectious diseases like hepatitis, norovirus, and polio. Viral shedding detected in wastewater often precedes case upticks in healthcare settings, providing valuable and rapid information about infection within a community. Multiple countries have implemented wastewater-based monitoring of SARS-CoV-2, and the COVID-19 case rates highly correlate with SARS-CoV-2 RNA found in wastewater. However, these upper-respiratory viruses are often found at low concentrations within wastewater, thereby necessitating a concentration step. Nanotrap Particles, coupled with GT Molecular kits, provide end users with a high-throughput and sensitive means to monitor pathogens such as SARS-CoV-2, Influenza, and RSV. APPLICATION NOTE SKU 10XXX SKU 44XXX

Quantifying Respiratory Viruses in Wastewater Using GT Molecular’s Digital PCR Assays coupled with Ceres Nanosciences' Nanotrap® Workflow and QIAGEN AllPrep PowerViral™ DNA/RNA Extraction Kit on the QIAcube™ APPLICATION NOTE SKU 10XXX SKU 44XXX Download full application note here Key Advantages > High-throughput multiplex detection coupling GT Molecular’s Digital PCR Assays with the Nanotrap Particle Capture and Concentration Methods > Ability to co-monitor and co-quantify multiple pathogens from a single sample > Assays available for the QIAcuity® Platform > Respiratory virus detection including: Influenza A, Influenza B and RSV in addition to SARS-CoV-2. Introduction Monitoring the spread of SARS-CoV-2, the virus that causes COVID-19, as well as other pathogens such as RSV and Influenza continues to provide vital information for interrupting chains of transmission and preventing or dampening the spread of new cases. Wastewater testing continues to gain traction as a viable means to monitor pathogens and provide timely information about the changing prevalence within individual communities. Wastewater-based epidemiology (WBE) utility has already been successfully described to track infectious diseases like hepatitis, norovirus, and polio. Viral shedding detected in wastewater often precedes case upticks in healthcare settings, providing valuable and rapid information about infection within a community. Multiple countries have implemented wastewater-based monitoring of SARS-CoV-2, and the COVID-19 case rates highly correlate with SARS-CoV-2 RNA found in wastewater. However, these upper-respiratory viruses are often found at low concentrations within wastewater, thereby necessitating a concentration step. Nanotrap Particles, coupled with GT Molecular kits, provide end users high-throughput and sensitive means to monitor pathogens such as SARS-CoV-2, Influenza, and RSV.

Nanotrap® Microbiome B Particles Enable Capture of Candida auris, Cryptosporidium parvum, and Giardia lamblia from Wastewater APPLICATION NOTE SKU 65XXX Download full application note here Key Advantages > Rapidly capture and concentrate pathogenic yeast and parasites, without filtration or centrifugation. > Single sample processing method can be used to identify multiple microbes of interest. > Workflow can be automated on a KingFisher™ System. > Methods are scalable for large volume sample processing. Introduction Wastewater-based epidemiology is used to identify and monitor pathogens in communities. It experienced a massive growth and was widely heralded during the SARS-CoV-2 pandemic. This interest has expanded to other pathogens, including eukaryotes such as parasitic organisms and multi-drug resistant yeast. The CDC has identified several of these type of pathogens as emerging threats including Candida auris and several infection-causing parasitic organisms. Nanotrap Microbiome A Particles are used in wastewater testing across the country to monitor SARS-CoV-2 in communities. More recently, the Nanotrap A Microbiome Particles and Nanotrap B Microbiome Particles have allowed detection of emerging diseases including mpox virus and Clostridioides difficile, both of which are included on the CDC emerging threats list. Using the Nanotrap Microbiome B Particles, a new, automation-friendly method has been developed to enable improved detection of eukaryotic pathogens (such as C. auris, Cryptosporidium parvum, and Giardia lamblia) in wastewater samples. In this application note, we show that Nanotrap Microbiome B Particles can: 1) capture and concentrate multiple eukaryotic organisms from wastewater samples; 2) improve detection of those organisms as compared to an alternative method; and 3) are compatible with multiplex qPCR assays on the Bio-Rad CFX96.

Poster: Nanotrap Microbiome Particles and Nanotrap Enhancement Reagents enable rapid and simple methods for microbe capture and concentration from wastewater samples at environmentally relevant concentrations Using Nanotrap Microbiome A Particles and Nanotrap Microbiome B Particles in a combined method delivers improved recovery of multiple viruses and bacteria, while maintaining workflow simplicity and throughput This study shows the Nanotrap Microbiome Particles can be used in a fast, effective, and simple wastewater processing method for multiple pathogen detection.

Morbidity and Mortality Weekly Report, February 2023 Notes from the Field: Aircraft Wastewater Surveillance for Early Detection of SARS-CoV-2 Variants — John F. Kennedy International Airport, New York City, August–September 2022 As SARS-CoV-2 testing declines worldwide, surveillance of international travelers for SARS-CoV-2 enables detection of emerging variants and fills gaps in global genomic surveillance (1). Because SARS-CoV-2 can be detected in feces and urine of some infected persons (2), wastewater surveillance in airports and on aircraft has been proposed by the global public health community† as a low-cost mechanism to monitor SARS-CoV-2 variants entering the United States. Sampling wastewater directly from aircraft can be used to link SARS-CoV-2 lineage data with flight origin countries without active engagement of travelers (3).

Lancet Regional Health, Americas February 2023 Safer at school early alert: an observational study of wastewater and surface monitoring to detect COVID-19 in elementary schools Background: Schools are high-risk settings for SARS-CoV-2 transmission, but necessary for children's educational and social-emotional wellbeing. Previous research suggests that wastewater monitoring can detect SARS-CoV-2 infections in controlled residential settings with high levels of accuracy. However, its effective accuracy, cost, and feasibility in non-residential community settings is unknown. Methods: The objective of this study was to determine the effectiveness and accuracy of community-based passive wastewater and surface (environmental) surveillance to detect SARS-CoV-2 infection in neighborhood schools compared to weekly diagnostic (PCR) testing. We implemented an environmental surveillance system in nine elementary schools with 1700 regularly present staff and students in southern California. The system was validated from November 2020 to March 2021. Findings: In 447 data collection days across the nine sites 89 individuals tested positive for COVID-19, and SARS-CoV-2 was detected in 374 surface samples and 133 wastewater samples. Ninety-three percent of identified cases were associated with an environmental sample (95% CI: 88%-98%); 67% were associated with a positive wastewater sample (95% CI: 57%-77%), and 40% were associated with a positive surface sample (95% CI: 29%-52%). The techniques we utilized allowed for near-complete genomic sequencing of wastewater and surface samples. Interpretation: Passive environmental surveillance can detect the presence of COVID-19 cases in non-residential community school settings with a high degree of accuracy.

Nature, Scientific Reports, Feb. 2023 ABSTRACT: Presented here is a magnetic hydrogel particle enabled workflow for capturing and concentrating SARS-CoV-2 from diagnostic remnant swab samples that significantly improves sequencing results using the Oxford Nanopore Technologies MinION sequencing platform. Our approach utilizes a novel affinity-based magnetic hydrogel particle, circumventing low input sample volumes and allowing for both rapid manual and automated high throughput workflows that are compatible with Nanopore sequencing. This approach enhances standard RNA extraction protocols, providing up to 40 × improvements in viral mapped reads, and improves sequencing coverage by 20–80% from lower titer diagnostic remnant samples. Furthermore, we demonstrate that this approach works for contrived influenza virus and respiratory syncytial virus samples, suggesting that it can be used to identify and improve sequencing results of multiple viruses in VTM samples. These methods can be performed manually or on a KingFisher automation platform.

Water Research, February 2023 ABSTRACT: Monitoring SARS-CoV-2 in wastewater is a valuable approach to track COVID-19 transmission. Designing wastewater surveillance (WWS) with representative sampling sites and quantifiable results requires knowledge of the sewerage system and virus fate and transport. We developed a multi-level WWS system to track COVID-19 in Atlanta using an adaptive nested sampling strategy. From March 2021 to April 2022, 868 wastewater samples were collected from influent lines to wastewater treatment facilities and upstream community manholes. Variations in SARS-CoV-2 concentrations in influent line samples preceded similar variations in numbers of reported COVID-19 cases in the corresponding catchment areas. Community sites under nested sampling represented mutually-exclusive catchment areas. Community sites with high SARS-CoV-2 detection rates in wastewater covered high COVID-19 incidence areas, and adaptive sampling enabled identification and tracing of COVID-19 hotspots. This study demonstrates how a well-designed WWS provides actionable information including early warning of surges in cases and identification of disease hotspots.

"Evaluation of wastewater surveillance for SARS-CoV-2 in Massachusetts correctional facilities, 2020–2022" Frontiers in Water, 2023 Introduction: Correctional facilities have environmental, resource, and organizational factors that facilitate SARS-CoV-2 transmission and challenge clinical testing of staff and residents. In Massachusetts, multiple state prisons implemented wastewater surveillance for strategic testing of individuals and isolation of COVID-19 cases early in the course of infection, as recommended by the Centers for Disease Control and Prevention (CDC). Our objective was to quantify the correlation of COVID-19 cases with facility-level wastewater surveillance compared to standard case surveillance in towns in closest geographic proximity to participating correctional facilities. Materials and methods: Available data included number of reported COVID-19 cases in residents from each of eight participating facilities (labeled A-H for anonymity), wastewater viral concentrations at each facility, and COVID-19 cases reported to routine surveillance in towns geographically nearest each facility. We selected data from December 2020-February 2022. Spearman's rank correlation was calculated at each facility to assess agreement between town cases and facility resident cases, and between wastewater concentrations and facility resident cases. We considered a correlation of ≤0.3 as weak and ≥0.6 as strong. Results: Facilities housed a mean of 502 individuals (range 54–1,184) with mean staffing of 341 (range 53–547). In 7/8 facilities, the town/resident cases correlation coefficients (ρ) were statistically significant (range 0.22–0.65); in all facilities, the wastewater/facility resident cases correlations were statistically significant (range 0.57–0.82). Consistently, ρ values were higher for facility-specific wastewater/resident cases than for town/resident cases: A (0.65, 0.80), B (0.59, 0.81), C (0.55, 0.70), D (0.61, 0.82), E (0.46, 0.62), F (0.51, 0.70), and H (0.22, 0.57). Conclusion: We conclude that wastewater surveillance for SARS-CoV-2 can provide an additional signal to objectively supplement existing COVID-19 clinical surveillance for the early detection of cases and infection control efforts at correctional facilities.