The APHC LS Method Development team has developed two different methods and workflows for quantification of SARS-CoV-2 viral load in environmental wastewater from Army installations. Wastewater samples are heat inactivated first for safe handling of these samples in the subsequent steps. Heat inactivation is carried out at 65 °C for 30 minutes to inactivate the SARS-CoV-2 virus and render these samples non-infectious, followed by a filtration step (0.45-micron filtration to remove potentially interfering particulates). The filtrates are used in subsequent steps (Method-1 and Method-2). Method-1 is a medium throughput process in which the virus is concentrated from 40 milliliters (mL) of filtrate by polyethylene glycol/sodium chloride (PEG/NaCl) precipitation followed by centrifugation. The pellets were resuspended and nucleic acids were extracted using the MagMAX™ Viral/Pathogen Nucleic Acid Isolation Kit on a MagMAX-96 instrument. Appropriate extraction controls were included to monitor extraction efficiencies. Method-2 is a high throughput direct nucleic acid extraction method capable of processing 96–192 wastewater samples (4 mL each) on an automated nucleic acid extraction system MagNAPure<sup>TM</sup> 96. We used SARS-CoV-2 specific molecular assays developed by the U.S. Centers for Disease Control and Prevention (CDC) for real time reverse transcriptase PCR (RT-qPCR) based detection of the virus from the extracted nucleic acids (in both methods). Additionally, we quantified the levels of pepper mild mottle virus (PMMoV), a fecal indicator virus RNA, by RT-qPCR to facilitate normalization of the variabilities associated with dilution of the wastewater samples collected on different days.  Based on the results we report the SARS-CoV-2 levels as genomic equivalents (GE) per liter of wastewater collected. Our PEG/NaCl methodology and automated direct extraction methodology achieved a limit of detection (LOD) of 2,500 GE/liter and 12,500 GE/liter of wastewater, respectively. We applied these approaches to determine the temporal trends in viral loads in samples collected every week over a period of 1 year (and ongoing). Our results closely matched those reported by an external testing facility contracted throughout the testing period on the same samples. Such data can be used to model against positivity rates and can serve as an indicator of impending outbreaks within sampled populations at different Army installations. Results of these studies will be presented.

Adaptable wastewater based disease surveillance approaches for army installations: A SARS-CoV-2 pilot study

Fall 2022 abstracts, posters, and presentations will launch in mid-September.

ACS Fall 2022

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.