The Evolution of Wastewater-Based Epidemiology

The Evolution of Wastewater-Based Epidemiology Wastewater monitoring is a rapid, cost-effective and non-invasive method of tracking public health threats. Wastewater is a rich source of information, allowing health agencies to easily monitor whole communities, regardless of income or access to healthcare. As the scope for wastewater-based epidemiology (WBE) increases, versatile and accurate methods of detecting even low levels of pathogens are needed to predict outbreaks and limit spread. Father of epidemiology, Dr Jon Snow first identifies cholera as a waterborne disease. The handle of the Broad Street water pump in London is removed, and the outbreak abates. 1854 Researchers find a correlation between the incidence of paralytic polio in the community and high levels of poliovirus in sewage samples. 1 1940s Dot blot hybridization methods are developed to detect hepatitis A virus in sewage-polluted waters. 2 1985 PCR enables more rapid, sensitive and efficient detection of wastewater pathogens, and becomes the new gold standard for WBE. 3 1990s WBE technology is used to evaluate illegal drug, alcohol and tobacco consumption at a population level. 4 2000s Early in the COVID-19 pandemic, SARS-CoV-2 RNA is discovered in untreated wastewater. WBE is widely used to monitor the spread of the virus at a community level and predict outbreaks. 5 2020 The widespread use of WBE during the COVID-19 pandemic has brought these methods to the forefront of public health surveillance. Appropriate infrastructure has been widely established and other emerging or continued public health threats can now be addressed e.g., Today Antimicrobial resistance surveillance Predicting and preventing epidemics Tracking chemical exposures Data down the drain Wastewater data can be used to inform and implement public health measures. Samples are collected from wastewater processing plants and processed to detect and quantify microbial populations. Wastewater collection Viral particle concentration RNA extraction RNA detection WBE application + Surrogate controls Community monitoring Prevalence estimation Muddying the waters: the challenges of WBE While quantitative PCR (qPCR) remains an important analytical tool for pathogen RNA detection in wastewater, sewage samples pose multiple challenges for achieving accurate and efficient results. A more accurate approach Droplet DigitalTM PCR (ddPCRTM) technology uses a water-emulsion droplet system to separate nucleic acid samples and perform PCR amplification within each droplet. It enables absolute quantification of a target in a single-step process with multiple advantages over standard qPCR. Advantages of ddPCR qPCR can lack sensitivity for low viral copy numbers. Sewage samples contain high levels of lipids and proteins that can interfere with qPCR amplification steps. Expert-designed solutions Optimized ddPCR assays and kits are available to detect a growing list of infectious pathogens in wastewater samples, along with predesigned assays to detect variants of concern: 1 Prepare ddPCR reaction mix 2 Generate droplets 3 PCR amplification 4 Read droplets and analyze results References 1. Metcalf TG, Melnick JL, Estes MK. Environmental virology: from detection of virus in sewage and water by isolation to identification by molecular biology—a trip of over 50 years. Annu Rev Microbiol. 1995;49(1):461-487. doi:10.1146/annurev.mi.49.100195.002333 2. Jiang X, Estes MK, Metcalf TG. Detection of hepatitis A virus by hybridization with single-stranded RNA probes. Appl Environ Microbiol. 1987;53(10):2487-2495. doi:10.1128/aem.53.10.2487-2495.1987 3. Toze S. PCR and the detection of microbial pathogens in water and wastewater. Water Research. 1999;33(17):3545-3556. doi:10.1016/s0043-1354(99)00071-8 4. Huizer M, ter Laak TL, de Voogt P, van Wezel AP. Wastewater-based epidemiology for illicit drugs: A critical review on global data. Water Research. 2021;207:117789. doi:10.1016/j.watres.2021.117789 5. Ahmed W, Angel N, Edson J, et al. First confirmed detection of SARS-COV-2 in untreated wastewater in Australia: a proof of concept for the wastewater surveillance of COVID-19 in the community. Sci Total Environ. 2020;728:138764. doi:10.1016/j.scitotenv.2020.138764 Public health guidelines implemented qPCR can only provide relative quantification based on standard curves. Surveillance of multiple pathogens or variants is limited by qPCR. Less affected by PCR inhibiting contaminants Simpler data analysis Rapid turnaorund, results in <8h Quantitative without requiring a standard curve Allows multiplexing for different pathogens or variants Smaller sample requirements, reducing cost Enrichment of rare targets, resulting in increased sensitivity Channel 1 amplitude Channel 2 amplitude Template Primer(s) Probe(s) PCR mix Mixture Get a clearer picture of public health with ddPCR technology; discover solutions for wastewater-based epidemiology PCR reagents and sample are loaded into a multichannel cartridge with droplet generation mineral oil. The droplet generator creates a vacuum with negative pressure. This divides the sample into water-in-oil droplets. The subsamples are amplified by PCR. The PCR plate is placed into a droplet reader, which analyzes each droplet individually. The story so far: for over 150 years, water has provided essential insights into infectious diseases SARS-CoV-2 Adenovirus Monkeypox Norovirus Respiratory syncytial virus Legionella pneumophila Enterococcus https://www.bio-rad.com/en-us/feature/wastewater-partner-epidemiology https://doi.org/10.1146/annurev.mi.49.100195.002333 https://doi.org/10.1128/aem.53.10.2487-2495.1987 https://www.sciencedirect.com/science/article/abs/pii/S0043135499000718?via%3Dihub https://www.sciencedirect.com/science/article/abs/pii/S0043135499000718?via%3Dihub https://doi.org/10.1016/j.watres.2021.117789 https://doi.org/10.1016/j.scitotenv.2020.138764 https://doi.org/10.1146/annurev.mi.49.100195.002333 https://doi.org/10.1146/annurev.mi.49.100195.002333 https://doi.org/10.1146/annurev.mi.49.100195.002333 https://doi.org/10.1146/annurev.mi.49.100195.002333 https://doi.org/10.1146/annurev.mi.49.100195.002333 https://doi.org/10.1146/annurev.mi.49.100195.002333 https://doi.org/10.1146/annurev.mi.49.100195.002333 https://doi.org/10.1128/aem.53.10.2487-2495.1987 https://doi.org/10.1128/aem.53.10.2487-2495.1987 https://doi.org/10.1128/aem.53.10.2487-2495.1987 https://doi.org/10.1128/aem.53.10.2487-2495.1987 https://doi.org/10.1128/aem.53.10.2487-2495.1987 https://doi.org/10.1128/aem.53.10.2487-2495.1987 https://doi.org/10.1128/aem.53.10.2487-2495.1987 https://www.sciencedirect.com/science/article/abs/pii/S0043135499000718?via%3Dihub https://www.sciencedirect.com/science/article/abs/pii/S0043135499000718?via%3Dihub https://www.sciencedirect.com/science/article/abs/pii/S0043135499000718?via%3Dihub https://www.sciencedirect.com/science/article/abs/pii/S0043135499000718?via%3Dihub https://www.sciencedirect.com/science/article/abs/pii/S0043135499000718?via%3Dihub https://www.sciencedirect.com/science/article/abs/pii/S0043135499000718?via%3Dihub https://www.sciencedirect.com/science/article/abs/pii/S0043135499000718?via%3Dihub https://www.sciencedirect.com/science/article/abs/pii/S0043135499000718?via%3Dihub https://www.sciencedirect.com/science/article/abs/pii/S0043135499000718?via%3Dihub https://doi.org/10.1016/j.watres.2021.117789 https://doi.org/10.1016/j.watres.2021.117789 https://doi.org/10.1016/j.watres.2021.117789 https://doi.org/10.1016/j.watres.2021.117789 https://doi.org/10.1016/j.watres.2021.117789 https://doi.org/10.1016/j.watres.2021.117789 https://doi.org/10.1016/j.watres.2021.117789 https://doi.org/10.1016/j.scitotenv.2020.138764 https://doi.org/10.1016/j.scitotenv.2020.138764 https://doi.org/10.1016/j.scitotenv.2020.138764 https://doi.org/10.1016/j.scitotenv.2020.138764 https://doi.org/10.1016/j.scitotenv.2020.138764 https://doi.org/10.1016/j.scitotenv.2020.138764 https://doi.org/10.1016/j.scitotenv.2020.138764