ARTICLE

A Highly Sensitive and Rapid Method for SARS-CoV-2 Detection in Wastewater

Introduction

When the COVID-19 outbreak began, Dante Labs recognized the critical need for an effective population monitoring method for the SARS-CoV-2 virus in addition to diagnostic testing. One option which was widely discussed by researchers was the utilization of wastewater-based epidemiology. This case study details Dante Labs’ development and implementation of a wastewater testing methodology which is highly sensitive, fast and cost-effective and can be used to support their customers globally.

COVID-19

What has become known as coronavirus disease 2019 (COVID-19) was first reported in December 2019 in Wuhan City, China, as “pneumonia of unknown cause.” It was soon linked to a novel coronavirus pathogen, SARS-CoV-2, that was initially spread from animals to humans, and then spread person-to-person. The SARS-CoV-2 virus can be transmitted from one person to another when they are in close contact. The primary means of virus transmission is within respiratory droplets emitted from an infected person’s nose or mouth and which then come in contact with another person’s eyes, nose, or mouth.

As of 19 January 2021, more than 94 million positive cases of SARS-CoV-2 infection have been reported globally along with over 2 million deaths due to COVID-19 disease.1 The risk of severe COVID-19 disease increases with age, smoking, and the presence of other serious medical conditions. Severe cases involve extensive lung impairment that can lead to respiratory failure, organ failure, and death. Fatality rates vary greatly across nations and within populations.2

Outbreak Prevention Strategies and Challenges

It is imperative that there be a sensitive, accurate, and scalable means of identifying subpopulations with high rates of SARS-CoV-2 infection to trend infection rates and put corrective measures in place to address and mitigate outbreaks of COVID-19 disease. The initial strategy was to conduct clinical testing of symptomatic persons followed by quarantining by those testing positive. This strategy alone, however, does nothing to prevent the spread of the virus before the onset of symptoms. A strategy of much broader scope would be to conduct large-scale clinical testing that is not limited to symptomatic persons. Such an effort would, however, be logistically difficult and expensive to conduct.

What is needed is a way to monitor human populations to identify virus hotspots and increasing trends of COVID-19 presence. Targeted diagnostic testing could then be conducted to identify infected individuals and the information used to thwart COVID-19 disease outbreaks.

Researchers have been considering potential monitoring approaches as a way to conduct group monitoring. To be logistically feasible and scalable, scientists and researchers have focused on the use of wastewater-based epidemiology (WBE). In WBE, wastewater samples are collected from sewage treatment plants and/or sewers serving facilities, where groups of people work, congregate or live, such as office buildings, apartment complexes, businesses, hospitals, college campuses and government facilities. Wastewater, prior to its treatment, is typically collected over a 24-hour period, representing a composite sample. Thus, the wastewater sample provides information on that sub-population that could be used to trend and identify virus hotspots for further action.

Dante Labs Develops Wastewater Testing Methodology

Dante Labs in L’Aquila, Italy, forged a new path for the genomics industry in 2016 with their mission to make advanced genomics accessible to everyone. Dante Labs delivers whole genome sequencing using next generation sequencing (NGS) technology and artificial intelligence-based advanced analytics platforms. For five years, Dante Labs has used their advanced technologies to provide exceptional molecular diagnostics and biomarker identification services to individuals, human and animal health organizations, and environmental industries.

When the COVID-19 outbreak began, Dante Labs recognized the critical need for an effective population monitoring method for the SARS-CoV-2 virus besides individual diagnostic testing. As well, their customers began asking them for a solution. Led by Generoso Ianniciello, Dante Labs' Innovation Manager, the team began research and development efforts in January 2020. By May 2020, Dante Labs was providing SARS-CoV-2 in wastewater testing service to customers worldwide. Mr. Ianniciello shares the story of how it came to be.

Protocol Challenges and Development

Ianniciello describes the criteria his customers presented in early discussions on protocol development. “They wanted fast results that were sensitive and accurate enough for them to use in decision-making at their facilities,” he explains. “And ideally, they wanted a comprehensive end-to-end solution that began with sample collection and ended with immediate access to results.” With these and other considerations in mind, Dante Labs began reviewing studies and guidelines for similar viruses and sample matrices as possible proxies for method development. The following guidelines were particularly helpful in guiding their protocol development:

  • Internal Dante Labs methods for other viruses
  • World Health Organization (WHO) guidelines for viruses similar to SARS-CoV-2
  • Italy’s National Institute of Health guidelines for SARS-CoV-2 testing in humans, animals, and environmental samples

The biggest challenges they encountered during protocol development were:

  • Determination of the sample volume needed to achieve high sensitivity levels
  • Standardization of sample volume and pretreatment procedures
  • Providing rapid turnaround time from sample receipt to results availability
  • Inclusion of robust quality control (QC) checks throughout the protocol

Ianniciello and his team received assistance from PerkinElmer Inc. during protocol development, including the benefits of high-performing instruments and materials, workflow design consultation, and technical support. Dante Labs’ final validated protocol consists of:

  • Customer collects a 250 ml sample and sends to Dante Labs for analysis.
  • Upon receipt, the sample is preprocessed beginning with pasteurization followed by concentration by centrifugation and aqueous polymer separation/filtration with polyethylene glycol. Sample concentration takes 14 hours to complete—the most time-consuming step of the protocol but one that is vital for downstream sensitivity and accuracy.
  • Viral RNA is then extracted from the concentrated sample using the PerkinElmer chemagic360 instrument. The chemagic 360 completes the extraction in 30 minutes.
  • The extracted RNA is quantified using PerkinElmer’s Nucleic Acid Detection Kits (real-time PCR kits).
  • The PerkinElmer JANUS® G3 workstation automates much of the sample processing and analysis

QC checks are conducted at strategic points in the protocol, some key highlights below:

  • Sample processing: Matrix recovery controls
  • Sample concentration: Confirmation of the presence of SARS-CoV-2 in the concentrated sample
  • RNA extraction: Negative controls (blanks) and sample swaps
  • RNA quantification:
    • Negative controls
    • Matrix recovery controls
    • Quantitative measurement controls
    • Human fecal normalization controls
    • Inhibition assessment controls

    In addition, a unique bar code follows the sample from collection through processing and analysis to results reporting. This provides an added layer of QC to standard chain-of-custody (COC) procedures and documentation.

    The Dante Labs protocol provides 24-hour turnaround time from sample receipt to results availability thanks to the PerkinElmer chemagic 360. The chemagic 360 reduces the extraction time from one hour to 30 minutes, a 50% improvement over other extraction technologies. The protocol also generates reliable, actionable results thanks to PerkinElmer PCR kits that have the highest sensitivity levels available. The rapid turnaround time and high sensitivity fully support the policy or decision-making responsibilities of public health officials, business owners, school chancellors, and operations managers.

    Dante Labs Customer Outcomes

    Dante Labs customers are now enjoying the highest quality testing available for SARS-CoV-2 in wastewater thanks to the expertise and commitment of their scientists and the advanced capabilities of PerkinElmer instruments and materials.

    In addition to meeting these needs, Dante Labs went further and designed a turnkey, end-to-end solution for their customers. For each sampling event, customers receive a wastewater sampling kit that includes sample containers, labels with bar codes, COC forms, ice packs, bags, wipes, prepared shipping labels, and detailed instructions for sample collection and shipping.

    Customers also get real-time updates on sample status. They receive a text message when their results are available with a link to the Dante Labs' electronic portal for results viewing and download. Again, all within 24 hours.

    As a result of these outstanding capabilities and service, Dante Labs’ customers routinely submit samples one to two times per week. They use the monitoring results to adjust controls in place to minimize viral spread and include targeted diagnostic testing, enabling them to accurately identify infected persons and prevent widespread outbreaks at their facilities. Several customers have shared how the wastewater testing program is helping them manage their operations, such as:

    • Customers that have more than 50 people at their facility find routine wastewater testing a cost-effective way to monitor the risk status at their facility.
    • Employers consider the monitoring to be another way they can contribute to the health and safety of employees and their families.
    • Risk managers use the results to limit risk of outbreaks at their facilities or businesses.
    • Routine monitoring helps ensure business continuity.
    • Update or revise public health guidance and safety protocols.

    Dante Labs Leads the Way

    Dante Labs is using their highly sensitive, fast, cost effective method to support companies and organizations worldwide in their efforts to prevent COVID-19 disease outbreaks at their facilities. PerkinElmer’s instruments, materials, and technical expertise play an important part in Dante Labs’ success.

    Ianniciello reports they currently utilize 30 chemagic 360 extraction instruments and are in the process of increasing the number of JANUS G3 workstations from 7 to 10. “I consider PerkinElmer a trusted partner for our business development and market strategy,” he explains. “Their strong technical support and ongoing assistance made them indispensable in our protocol development.”

    For research use only. Not for use in diagnostic procedures.

    References

    1. World Health Organization COVID-19 Dashboard, December 16, 2020. https://covid19.who.int/
    2. Johns Hopkins University of Medicine Mortality Analyses, December 16, 2020. https://coronavirus.jhu.edu/data/mortality