APPLICATION NOTE SKU 10XXX SKU 65XXX Download full application note here Key Advantages > Nanotrap® Microbiome A Particles and Nanotrap Microbiome B Particles can be used in a single method to rapidly capture and concentrate bacterial and viral targets, including: Campylobacter jejuni, Escherichia coli, Listeria monocytogenes, Clostridium difficile, Salmonella enterica subsp. enterica, SARS-CoV-2, norovirus GII virus, influenza A virus, influenza B virus, and respiratory syncytial virus (RSV) from wastewater without the need of filtration and centrifugation. > The combined workflow can be automated on a Kingfisher Apex System. > Wastewater samples processed using Nanotrap Microbiome A Particles and Nanotrap Microbiome B Particles are compatible with digital PCR (dPCR) systems like the QIAcuity One 5plex. Introduction Wastewater-based epidemiology has become widely recognized as a powerful tool to identify and monitor emerg-ing pathogens in a community. Many of the efforts to date have been focused on developing wastewater testing methods that can enable monitoring of SARS-CoV-2 using RT-qPCR, but there is growing interest in expanding to other microbes and to other detection methods, like dPCR. Nanotrap Microbiome Particles enable rapid concentration of microbes from raw sewage, requiring no filtration or centrifugation and is compatible with RT-qPCR, RT-ddPCR, RT-dPCR, and sequencing based analysis methods. Labs around the world have used Nanotrap Microbiome A Particles to process many tens of thousands of wastewater samples for detection of SARS-CoV-2. Moreover, it was recently demonstrated that Nanotrap particle processing of wastewater testing can enable detection of emerging variants of concern up to 14 days earlier than clinical genomic surveillance. Nanotrap particle methods have also enabled detection of monkeypox and hepatitis A virus in wastewater. Here, we introduce a new method from Ceres Nanosciences: combining Nanotrap Microbiome B Particles with Nanotrap Microbiome A Particles in a single workflow to improve and expand microbe detection in wastewater samples. This simple and sensitive method is compatible with multiple nucleic acid extraction kits. In this application note, we show that Nanotrap Microbiome A Particles and Nanotrap Microbiome B Particles can be combined in a single workflow: 1) to capture and concentrate multiple pathogenic bacteria and viruses from wastewater samples; 2) to improve detection of those bacteria and viruses relative to using Nanotrap Microbiome A Particles alone; and 3) that is compatible with dPCR assays run on the QIAcuity One 5plex.

Science of the Total Environment, February 2023 ABSTRACT: Wastewater-based epidemiology (WBE) has been deployed broadly as an early warning tool for emerging COVID-19 outbreaks. WBE can inform targeted interventions and identify communities with high transmission, enabling quick and effective responses. As the wastewater (WW) becomes an increasingly important indicator for COVID-19 transmission, more robust methods and metrics are needed to guide public health decision-making. This research aimed to develop and implement a mathematical framework to infer incident cases of COVID-19 from SARS-CoV-2 levels measured in WW. We propose a classification scheme to assess the adequacy of model training periods based on clinical testing rates and assess the sensitivity of model predictions to training periods. A testing period is classified as adequate when the rate of change in testing is greater than the rate of change in cases. We present a Bayesian deconvolution and linear regression model to estimate COVID-19 cases from WW data. The effective reproductive number is estimated from reconstructed cases using WW. The proposed modeling framework was applied to three Northern California communities served by distinct WW treatment plants. The results showed that training periods with adequate testing are essential to provide accurate projections of COVID-19 incidence.

"Comparative analysis of adsorption-extraction (AE) and Nanotrap® magnetic virus particles (NMVP) workflows for the recovery of endogenous enveloped and non-enveloped viruses in wastewater" Science of The Total Environment, November 2022 ABSTRACT: In this study, two virus concentration methods, namely Adsorption-extraction (AE) and Nanotrap® Magnetic Virus Particles (NMVP) along with commercially available extraction kits were used quantify endogenous pepper mild mottle virus (PMMoV) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in nucleic acid extracted from 48 wastewater samples collected over six events from eight wastewater treatment plants (WWTPs). The main aim was to determine which workflow (i.e., concentration and extraction methods) produces greater concentrations of PMMoV and SARS-CoV-2 gene copies (GC) in comparison with each other. Turbidity and total suspended solids (TSS) of wastewater samples within and among the eight WWTPs were highly variable (41–385 NTU and 77–668 mg/L TSS). In 58 % of individual wastewater samples the log10 GC concentrations of PMMoV were greater by NMVP workflow compared to AE workflow. Paired measurements of PMMoV GC/10 mL from AE and NMVP across all 48 wastewater samples were weakly correlated (r = 0.455, p = 0.001) and demonstrated a poor linear relationship (r2 = 0.207). The log10 GC concentrations of SARS-CoV-2 in 69 % of individual samples were greater by AE workflow compared to NMVP workflow. In contrast to PMMoV, the AE and NMVP derived SARS-CoV-2 GC counts were strongly correlated (r = 0.859, p < 0.001) and demonstrated a strong linear relationship (r2 = 0.738). In general, the PMMoV GC achieved by the NMVP workflow decreased with increasing turbidity, but the PMMoV GC by the AE workflow did not appear to be sensitive to either turbidity or TSS levels. These findings suggest that suspended solids concentration, and the intended target for analysis should be considered when validating an optimal workflow for wastewater surveillance.

APPLICATION NOTE SKU 10XXX SKU 65XXX Download full application note here Key Advantages > Nanotrap® Microbiome B Particles rapidly capture and concentrate Campylobacter jejuni, Escherichia coli O157:H7, Listeria monocytogenes, Clostridium difficile, Salmonella enterica subsp. Enterica, and crAssphage from wastewater without the need of filtration, bead-beating, or centrifugation. > Nanotrap Microbiome B Particles enable a simple and rapid automated or manual method for detecting pathogens, including bacteria, in wastewater samples. > Nanotrap Microbiome B Particles are compatible with several commercially available nucleic acid extraction kits. Introduction Recently, wastewater surveillance has become widely recognized as a powerful tool to identify and monitor emerging pathogens in a community. Many of the investments made to date have been focused on developing wastewater surveillance methods that can enable rapid and reliable monitoring of SARS-CoV-2, but there is growing interest in expanding the utility of these methods to other microbes. The Nanotrap particle technology enables rapid concentration of microbes from raw sewage, requiring no filtration or centrifugation and is compatible with RT-qPCR, RT-ddPCR, RT-dPCR, and sequencing based analysis methods. Labs around the world have used Nanotrap Microbiome A Particles to process many tens of thousands of wastewater samples for detection of SARS-CoV-2. Moreover, it was recently demonstrated that Nanotrap particle processing of wastewater testing can enable detection of emerging variants of concern up to 14 days earlier than clinical genomic Surveillance. Nanotrap particle methods have also enabled detection of monkeypox and hepatitis A virus in wastewater. Here, we introduce two new products from Ceres Nanosciences: Nanotrap Microbiome B Particles and Nanotrap Enhancement Reagent 3 (ER3), along with a sensitive, rapid, and easy-to-use concentration method for wastewater-based epidemiology testing for bacterial targets. This simple and sensitive method is compatible with several magnetic bead DNA extraction kits including standard “off the shelf” kits provided by MACHEREY-NAGEL and Thermo Fisher Scientific and requires no bead-beating steps. We show that Nanotrap Microbiome B Particles 1) capture and concentrate multiple pathogenic bacteria from wastewater samples resulting in equivalent or better detection as compared to labor-intensive manual HA Filter wastewater concentration methods, 2) are compatible with multiple automation-friendly nucleic acid extraction kits, and 3) enable detection of a wastewater control organism that is commonly utilized to normalize detection results across unique wastewater samples.

APPLICATION NOTE SKU 10XXX SKU 44XXX Download full application note here Key Advantages > A high-throughput method to capture and concentrate multiple pathogens including Monkeypox virus > Scalable and automatable method with increased sensitivity using large volume samples > Compatible with a variety of extraction kits and downstream methods > Utilizes the same Nanotrap® particle-enabled work-flow as SARS-CoV-2 Introduction Monkeypox was discovered in humans in 1970 in the Democratic Republic of the Congo. Since then, it has become endemic in western and central Africa, and most cases outside that region have been linked to travel or imported animals. In May 2022, cases began appearing in Europe and around the world with no known link to western or central Africa. To date, there have been over 21,000 monkeypox cases in the US and over 55,000 in the world as a result of this ongoing outbreak. Interest in wastewater-based epidemiology has increased dramatically in recent years as it has been used to track the SARS-CoV-2 virus in communities. There is growing interest in tracking the monkeypox virus through wastewater as well. Ceres Nanotrap Microbiome A Particles capture and concentrate a wide range of viral and bacterial pathogens present in wastewater. In this study, we demonstrate that Nanotrap Microbiome A Particles bind and concentrate monkeypox virus from wastewater using both manual and automated workflows.

"A secretory form of Parkin-independent mitophagy contributes to the repertoire of extracellular vesicles released into the tumour interstitial fluid in vivo" Journal of Extracellular Vesicles, June 2022 ABSTRACT: We characterized the in vivo interstitial fluid (IF) content of extracellular vesicles (EVs) using the GFP-4T1 syngeneic murine cancer model to study EVs in-transit to the draining lymph node. GFP labelling confirmed the IF EV tumour cell origin. Molecular analysis revealed an abundance of IF EV-associated proteins specifically involved in mitophagy and secretory autophagy. A set of proteins required for sequential steps of fission-induced mitophagy preferentially populated the CD81+/PD-L1+ IF EVs; PINK1, TOM20, and ARIH1 E3 ubiquitin ligase (required for Parkin-independent mitophagy), DRP1 and FIS1 (mitochondrial peripheral fission), VDAC-1 (ubiquitination state triggers mitophagy away from apoptosis), VPS35, SEC22b, and Rab33b (vacuolar sorting). Comparing in vivo IF EVs to in vitro EVs revealed 40% concordance, with an elevation of mitophagy proteins in the CD81+ EVs for both murine and human cell lines subjected to metabolic stress. The export of cellular mitochondria proteins to CD81+ EVs was confirmed by density gradient isolation from the bulk EV isolate followed by anti-CD81 immunoprecipitation, molecular sieve chromatography, and MitoTracker export into CD81+ EVs. We propose the 4T1 in vivo model as a versatile tool to functionally characterize IF EVs. IF EV export of fission mitophagy proteins has broad implications for mitochondrial function and cellular immunology.

"Metagenomics of Wastewater Influent from Wastewater Treatment Facilities across Ontario in the Era of Emerging SARS-CoV-2 Variants of Concern" Environmental Microbiology May 2022 ABSTRACT: We report metagenomic sequencing analyses of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in composite wastewater influent from 10 regions in Ontario, Canada, during the transition between Delta and Omicron variants of concern. The Delta and Omicron BA.1/BA.1.1 and BA.2-defining mutations occurring in various frequencies were reported in the consensus and subconsensus sequences of the composite samples.

"Wastewater-Based Epidemiology for COVID-19: Handling qPCR Nondetects and Comparing Spatially Granular Wastewater and Clinical Data Trends" ACS EST Water July, 2022 ABSTRACT: Wastewater-based epidemiology (WBE) is a useful complement to clinical testing for managing COVID-19. While community-scale wastewater and clinical data frequently correlate, less is known about subcommunity relationships between the two data types. Moreover, nondetects in qPCR wastewater data are typically handled through methods known to bias results, overlooking perhaps better alternatives. We address these knowledge gaps using data collected from September 2020–June 2021 in Davis, California (USA). We hypothesize that coupling the expectation maximization (EM) algorithm with the Markov Chain Monte Carlo (MCMC) method could improve estimation of “missing” values in wastewater qPCR data. We test this hypothesis by applying EM-MCMC to city wastewater treatment plant data and comparing output to more conventional nondetect handling methods. Dissimilarities in results (i) underscore the importance of specifying nondetect handling method in reporting and (ii) suggest that using EM-MCMC may yield better agreement between community-scale clinical and wastewater data. We also present a novel framework for spatially aligning clinical data with wastewater data collected upstream of a treatment plant (i.e., distributed across a sewershed). Applying the framework to data from Davis reveals reasonable agreement between wastewater and clinical data at highly granular spatial scales─further underscoring the public-health value of WBE.

Quantifying the Relationship between SARS-CoV-2 Wastewater Concentrations and Building-Level COVID-19 Prevalence at an Isolation Residence: A Passive Sampling Approach Int. J. Environ. Res. Public Health, 2022 ABSTRACT: SARS-CoV-2 RNA loads can be detected in the excreta of individuals with COVID-19 and have demonstrated positive correlations with clinical infection trends. Consequently, wastewater-based epidemiology (WBE) approaches have been implemented globally as a public health surveillance tool to monitor community-level prevalence of infections. The majority of wastewater specimens are gathered as either composite samples via automatic samplers (autosamplers) or grab samples. However, autosamplers are expensive and can be challenging to maintain in cold weather, while grab samples are particularly susceptible to temporal variation when sampling sewage directly from complex matrices outside residential buildings. Passive sampling can provide an affordable, practical, and scalable sampling system while maintaining a reproducible SARS-CoV-2 signal. In this regard, we deployed tampons as passive samplers outside of a COVID-19 isolation unit (a segregated residence hall) at a university campus from 1 February 2021–21 May 2021. Samples (n = 64) were collected 3–5 times weekly and remained within the sewer for a median duration of 24 h. SARS-CoV-2 RNA was quantified using reverse-transcription quantitative polymerase chain reaction (RT-qPCR) targeting the N1 and N2 gene fragments. We quantified the mean viral load captured per individual and the association between the daily viral load and total persons, adjusting for covariates using multivariable models to provide a baseline estimate of viral shedding. Samples were processed through two distinct laboratory pipelines on campus, yielding highly correlated N2 concentrations. Data obtained here highlight the success of passive sampling utilizing tampons to capture SARS-CoV-2 in wastewater coming from a COVID-19 isolation residence, indicating that this method can help inform building-level public health responses.

MedRxiv July 2022 ABSTRACT: Wastewater represents a composite biological sample from the entire contributing population. People infected with monkeypox excrete monkeypox virus DNA via skin lesions, saliva, feces and urine and these can enter the wastewater via toilets, sinks, and shower drains. To test whether monkeypox can be detected and monitored in wastewater during a period when publicly reported monkey cases in the region were increasing, we deployed digital PCR assays that target genomic DNA from the monkeypox virus in our routine, ongoing wastewater surveillance program in the Greater Bay Area of California, USA. We measured monkeypox virus DNA daily in settled solids samples from nine wastewater plants over the period of approximately 4 weeks. During that period, we detected monkeypox virus DNA in wastewater solids at nearly all the wastewater plants we routinely sample. Frequency of occurrence and concentrations were highest at plants serving San Francisco County. To confirm the presence of monkeypox DNA, we used two assays that target distinct sequences on the monkeypox genome on a subset of samples and results from both assays were in close agreement strongly suggesting true positives in the wastewater. Additionally, we show that concentrations of monkeypox DNA is 103 times higher in the solid fraction compared to the liquid fraction of wastewater on a mass-equivalent basis.

Nature, July 2022 ABSTRACT: As SARS-CoV-2 becomes an endemic pathogen, detecting emerging variants early is critical for public health interventions. Inferring lineage prevalence by clinical testing is infeasible at scale, especially in areas with limited resources, participation, or testing/sequencing capacity, which can also introduce biases. SARS-CoV-2 RNA concentration in wastewater successfully tracks regional infection dynamics and provides less biased abundance estimates than clinical testing. Tracking virus genomic sequences in wastewater would improve community prevalence estimates and detect emerging variants. However, two factors limit wastewater-based genomic surveillance: low-quality sequence data and inability to estimate relative lineage abundance in mixed samples. Here, we resolve these critical issues to perform a high-resolution, 295-day wastewater and clinical sequencing effort, in the controlled environment of a large university campus and the broader context of the surrounding county. We develop and deploy improved virus concentration protocols and deconvolution software that fully resolve multiple virus strains from wastewater. We detect emerging variants of concern up to 14 days earlier in wastewater samples, and identify multiple instances of virus spread not captured by clinical genomic surveillance. Our study provides a scalable solution for wastewater genomic surveillance that allows early detection of SARS-CoV-2 variants and identification of cryptic transmission.

APPLICATION NOTE SKU 10XXX SKU 44XXX Download full application note here Key Advantages > Enables detection of multiple SARS‐CoV‐2 variants > Provides absolute quantification of low copy number samples with higher precision Introduction Since the emergence of new SARS-CoV-2 variants, which have been associated with increased transmissibility and/or immune escape, there is an urgent need for methods that enable specific and timely detection and quantification of the occurrence of these variants in the community. Droplet Digital Polymerase Chain Reaction (ddPCR™) was developed to provide high-precision, absolute quantification of nucleic acid target sequences within samples through sample partitioning. This technology also mitigates the effects of target competition, making PCR amplification less sensitive to inhibition. In this application note, we show the compatibility of Nanotrap® Magnetic Virus Particles with RT-ddPCR and demonstrate the detection and absolute quantification of SARS-CoV-2 variants (i.e. Omicron, Delta) in wastewater samples.