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The virus behind the COVID-19 pandemic, SARS-CoV-2, can be detected in the feces of patients infected with COVID-19, both symptomatic and asymptomatic.  Thus, wastewater becomes a reservoir of valuable information related to the status of the pandemic within a community.  Through monitoring of wastewater at several locations in a community-wide “sewershed”, wastewater-based epidemiology (WBE) has the potential to act as a leading indicator in tracking the spread of COVID-19, where the “hidden” infections can be accounted for.  Government agencies and municipal utilities have begun a nationwide surveillance of wastewater for SARS-CoV-2 as a supplement to clinical data, though the most effective way to detect the virus is still unclear.

The initial step in the quantification of the virus in wastewater is a viral concentration step designed to reduce the volume of wastewater that contains the viral material into a much smaller volume (1-2 mL) while still retaining the viral signal present in the larger volume.  The methods to concentrate the virus are still not standardized around the world, or even across the country, with many different groups using different methods that have been optimized for an enveloped virus like SARS-CoV-2. 

The goal of this project is to compare several different common concentration methods by concentrating aliquots of the same sample of wastewater treatment plant influent and determining the recovery of SARS-CoV-2 RNA relative to the other methods.  The chosen methods were electronegative membrane filtration, ultrafiltration with centrifugal devices, and polyethylene glycol (PEG) precipitation.  For each chosen method, there were several different implementations of the method run to represent different ways the method could be implemented in a laboratory.  For centrifugal ultrafiltration and PEG precipitation, the solids were either removed beforehand or included in the concentration step.  This helped evaluate another research question that asks much of the virus partitioned to solids, since groups currently conducting sewershed surveillance have differed on their choice to remove or keep large wastewater solids in the sample for the concentration. 

The different implementations that have been/will be run are:

1. Electronegative membrane filtration
   1. Acid and MgCl2 added
   2. Acid added
   3. MgCl2 added
   4. Nothing added (unamended wastewater)
2. Centrifugal ultrafiltration
   1. Large wastewater solids removed through centrifugation before ultrafiltration
   2. Large wastewater solids added to centrifugal ultrafiltration device
3. PEG precipitation
   1. Large wastewater solids removed by filtration and discarded before precipitation
   2. Separate virus extraction step performed for large solids and included in PEG precipitation

This comparison will inform, and has already informed, the concentration work being done at Clean Water Services to lower method detection limit for SARS-CoV-2.  In the future, this work may be compared to similar studies that use spike tests of common surrogate viruses like murine hepatitis virus and the bacteriophage Pseudomonas ϕ6 to simulate the concentration of SARS-CoV-2. 

This project presenter is available for live video chat on Sept. 1, 2020 from 10:15 a.m. - 12:00 p.m. PDT.