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How to Automate Your SARS-CoV-2 Wastewater Testing Workflows: 4 Key Questions

By 10.07.2021

The COVID-19 pandemic has spawned a year of innovation never before seen in the biotech and pharma industries. From the rapid development and manufacturing of novel mRNA vaccines on a global scale to the creation of myriad diagnostic tests, our scientific community has helped protect against the current and continued threat of the SARS-CoV-2 virus.

The discovery that SARS-CoV-2 viral particles and fragments are shed in the feces of COVID-19 patients, led to the inventive use of wastewater testing to measure infection dynamics within small to large communities.1 This epidemiological approach has been used to track infectious diseases for centuries and this continues with COVID-19 monitoring. There has been widespread adoption and implementation of testing programs worldwide and wastewater-based epidemiology research projects have expanded over the past year from dozens to more than two hundred.

And it’s easy to see why: The method has helped anticipate future infection burden and has several advantages over traditional diagnostics, including the non-invasive, unbiased identification of asymptomatic carriers.1 It’s also a cost-effective and time-saving alternative to individualized testing, which requires readily available access to healthcare personnel and facilities.

Maximizing the Potential of Wastewater Surveillance Workflows

As SARS-CoV-2 viral spread continues internationally, wastewater testing continues to be a critical part of assessing the infection dynamics of communities, the appearance of new variants, and overall vaccine efficacy. However, the use of wastewater samples (and other related sample types) for community testing presents distinct challenges in establishing accurate, efficient, and high-throughput workflows, that can accommodate school, county, or city-wide scales.

Depending on testing protocols, wastewater testing workflows can require immense amounts of hands-on time, including time for the collection of samples, extraction of viral RNA, and detection using methodologies such as quantitative real-time PCR (qRT-PCR), digital droplet PCR (ddPCR), isothermal amplification, or next-generation sequencing (NGS). Implementing the proper workflow that suits the needs of your monitoring program and community requires you to consider some key details at each step of the process.

If you’re currently in the planning stages for wastewater-based epidemiology program, we have 4 key questions to reflect on so that your workflows operate as accurately and efficiently as possible.

The Dynabeads can efficiently isolate SARS-CoV-2 from cell culture media, virus transport media and wastewater samples. They precipitate the virus the solution by tying up water molecules, which results in less-soluble components (i.e., virus) being forced out of solution. Dynabeads Intact Virus Enrichment beads are used on KingFisher purification systems for processing up to 96 virus-containing samples in ~20 minutes.

What type of samples will you be processing?

Several publications have reported successful detection of SARS-CoV-2 from a variety of sample types, including primary sludge, municipal sewage, and untreated wastewater. The CDC and other research groups recommend collecting and using either untreated wastewater, which includes human fecal waste from a variety of sources, or sludge, made up of sedimented solids that settle out of wastewater before treatment.

While there is no scientific consensus on the most reliable sample type, there are distinct advantages and disadvantages to using either sludge or untreated wastewater. Sludge typically has higher concentrations of SARS-CoV-2 RNA, requires lower collection volumes, and can help identify rare cases in large communities. Because this sample type is concentrated, however, it may also have higher concentrations of inhibitors that can complicate nucleic acid extraction and/or downstream analysis.

Using untreated wastewater can get around these problems, especially if a wastewater treatment facility adds disinfectant before sedimentation. The drawback of using untreated wastewater is that it can require the collection of higher volumes, sometimes up to 1 L, that then need to be concentrated. Depending on the method used, sample concentration can be time-consuming and limit your throughput.

More targeted sampling can also be done of schools, universities, workplaces, or other facilities with the added benefit of closer and more tailored monitoring and surveillance. While a more targeted approach can be a time- and cost-effective way of monitoring high-risk populations, concentrations of SARS-CoV-2 can be highly variable and may require individual testing to validate workflows.

What sample volume will you be collecting? 

Several publications report the collection of 10 to 250 mL of untreated wastewater, but larger volumes have been collected and processed for analysis.4,7,9 Sludge and untreated wastewater samples will have different concentrations and will therefore require different sample volumes for detecting SARS-CoV-2 RNA. It has been reported that for certain concentration methods (read the section below for more information), larger volumes (50 mL to 1 L) can reduce the efficiency of virus recovery or result in the inhibition of downstream assays.

To get a better understanding of the limit of pathogen detection for your workflow, you can answer the questions we’ve posed in this blog by performing pilot experiments with spiked-in, inactive SARS-CoV-2 viral particles.

Read about our robust workflows

What virus concentration method will you use? 

Several virus concentration methods for untreated wastewater have been reported and validated, including ultracentrifugation, filtration with 0.45 µm filter, precipitation with polyethylene glycol (PEG) 8000, and more. These methods can be time-consuming, require a lot of operational hands-on time, and may not be amenable to the development of high-throughput workflows.

More recently, magnetic bead-based concentration methods have been used, allowing the use of a high-throughput, automated sample preparation instrument, the Kingfisher™ Flex Purification System. Dynabeads™ Intact Virus Enrichment and Nanotrap™ beads have both been validated in wastewater testing workflows and can capture nearly 100% of virus present with 10mL of wastewater samples. However, at larger volumes enrichment efficiency goes down.

Protocols using this approach can take as little as 40 minutes to process 24 samples and is key to the execution of large-scale, automated wastewater surveillance programs.

How to do SARS-CoV-2 RNA Extraction?

There are several RNA extraction methods available and ultimately, the upstream decisions you make about sample type, sample volume, concentration methods, and throughput required may make the decision about which extraction protocol is right for your workflow. Guanidinium-phenol extraction methods, like those made possible with TRIzol reagent, have been the gold standard in RNA extraction for some time. Resultant RNA is high-quality and amenable to nearly any downstream application.

One major disadvantage of using guanidinium-phenol extraction is that protocols are low throughput and exposure to phenol can cause skin irritation or burns during processing.

Thermo Fisher’s column- or bead-based nucleic acid extraction kits offer a high-quality alternative that requires less hands-on time. Kits like our MagMAX™ Microbiome Ultra are designed for difficult sample types, such as stool, and are amenable to automation with our Kingfisher Flex instrument, allowing the processing of 96 wastewater samples in approximately 1 hour. The use of MagMAX for wastewater testing has been validated by several research groups and furthers the use of automation in these important workflows.

Continued Viral Monitoring Through Automation

The degree to which wastewater testing programs have sprung up over the past year is a testament to their utility for SARS-CoV-2 monitoring and protecting community health. As the use of these programs continues to expand, focusing on how we can save time in our workflows, while still generating actionable data is critical to our continued success and innovation.

At Thermo Fisher Scientific, we believe automation using high-quality reagents is the key to empowering your lab to continue the fight against emerging pathogens. To learn more about how we can help, learn more about automated DNA and RNA extraction and purification systems or schedule a demo.

Start from the beginning and dive into the History of Wastewater: History of Pathogen Detection in Wastewater

This article is for Research Use Only. Not for use in diagnostic procedures.

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References

  1. Sims N, Kasprzyk-Hordern B. Future perspectives of wastewater-based epidemiology: Monitoring infectious disease spread and resistance to the community level. Environ Int. 2020;139:105689.
  2. The myriad ways sewage surveillance is helping fight COVID around the world. Nature website: https://www.nature.com/articles/d41586-021-01234-1. Published May 10, 2021. Accessed June 22, 2021.
  3. How sewage could reveal true scale of coronavirus outbreak. Nature website: https://www.nature.com/articles/d41586-020-00973-x. Published April 3, 2020. Accessed June 22, 2021.
  4. Robust detection of SARS-CoV-2 in wastewater samples. Thermo Fisher website: https://assets.thermofisher.com/TFS-Assets/BID/Application-Notes/robust-detection-of-sars-cov-2-in-wastewater-samples.pdf. Published May 26, 2021. Accessed June 22, 2021.
  5. Wastewater Surveillance Testing Methods. CDC website: https://www.cdc.gov/coronavirus/2019-ncov/cases-updates/wastewater-surveillance/testing-methods.html. Published November 23, 2020. Accessed June 22, 2021.
  6. Alpaslan Kocamemi B, Kurt H, Sait A et al. SARS-CoV-2 detection in Istanbul wastewater treatment plant sludges. medRxiv 2020.2020;05.12.20099358.
  7. 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, 7, 7, 511–516. Published 2020 May 20.
  8. Karthikeyan S, Ronquillo N, Belda-Ferre P, et al. High-Throughput Wastewater SARS-CoV-2 Detection Enables Forecasting of Community Infection Dynamics in San Diego County. mSystems. 2021;6(2):e00045-21. Published 2021 Mar 2.
  9. Developing a Wastewater Surveillance Sampling Strategy. CDC website: https://www.cdc.gov/coronavirus/2019-ncov/cases-updates/wastewater-surveillance/developing-a-wastewater-surveillance-sampling-strategy.html. Published November 23, 2020. Accessed June 22, 2021.
  10. Targeted Wastewater Surveillance at Facilities, Institutions, and Workplaces. CDC website: https://www.cdc.gov/coronavirus/2019-ncov/cases-updates/wastewater-surveillance/targeted-use-case.html. Published November 23, 2020. Accessed June 22, 2021.
  11. Detection of SARS-CoV-2 in fecal samples and wastewater. Thermo Fisher website: https://assets.thermofisher.com/TFS-Assets/BID/Application-Notes/detection-sars-cov-2-fecal-wastewater-samples-app-note.pdf. Published October 23, 2020. Accessed June 23, 2021.
  12. Automated high throughput viral concentration from wastewater using the KingFisher Flex platform. Protocols.io website: https://www.protocols.io/view/automated-high-throughput-viral-concentration-from-bptemnje. Published November 19, 2020. Accessed June 25, 2021.

 

 

 

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