Marlee Shaffer

Bio: Marlee Shaffer is an academic researcher from University of Notre Dame. The author has contributed to research in topics: Virology & Sodium hypochlorite. The author has an hindex of 3, co-authored 4 publications receiving 21 citations.

Within- and between-Day Variability of SARS-CoV-2 RNA in Municipal Wastewater during Periods of Varying COVID-19 Prevalence and Positivity

Abstract: Wastewater surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA is being used to monitor Coronavirus Disease 2019 (COVID-19) trends in communities;however, within- and between-day variation of SARS-CoV-2 RNA concentration in primary influent remains largely uncharacterized. In the current study, grab sampling of primary influent was performed every 2 h over two 24-h periods at two wastewater treatment plants (WWTPs) in northern Indiana, USA. The recovery efficiency of endogenous SARS-CoV-2 RNA in wastewater was confirmed to be similar to the recovery efficiency of the process control, bovine respiratory syncytial virus (BRSV). Recovery-corrected SARS-CoV-2 RNA concentrations in primary influent indicate diurnal loading patterns and confirm monitoring dependent on grab samples should target daytime periods with high fecal loading. Importantly, manual compositing performed at the WWTP resulted in concentrations that were consistently lower than grab sample averages indicating potential bias. Uncorrected, recovery-corrected, and pepper mild mottle virus (PMMoV)-normalized SARS-CoV-2 RNA concentrations demonstrated an ordinal agreement with increasing clinical COVID-19 positivity but not COVID-19 cases. In areas where geolocated COVID-19 case data are not available, the COVID-19 positivity rate could provide a useful county-level metric for comparison with wastewater. Nonetheless, large variation both within- and between-days may preclude robust quantitative analyses beyond correlation.

Building-Level Wastewater Monitoring for COVID-19 Using Tampon Swabs and RT-LAMP for Rapid SARS-Cov-2 RNA Detection

Abstract: Building-level wastewater monitoring for COVID-19 using tampon 1 swabs and RT-LAMP for rapid SARS-CoV-2 RNA detection 2 3 Aaron Bivins1,2, Megan Lott3, Marlee Shaffer1, Zhenyu Wu1, Devin North1, Erin K. Lipp3, Kyle 4 Bibby1,2* 5 6 1 Department of Civil & Environmental Engineering & Earth Sciences, University of Notre Dame, 7 156 Fitzpatrick Hall, Notre Dame, IN 46556 8 2 Environmental Change Initiative, University of Notre Dame, 721 Flanner Hall, Notre Dame, IN 9 46556 10 3 Department of Environmental Health Science, University of Georgia, 150 East Green Street, 11 Athens, GA 30602 12 13 *kbibby@nd.edu 14 15 Abstract 16 Community-level wastewater monitoring for severe acute respiratory syndrome coronavirus 2 17 (SARS-CoV-2) RNA has demonstrated useful correlation with both coronavirus disease 2019 18 (COVID-19) case numbers and clinical testing positivity. Wastewater monitoring on college 19 campuses has demonstrated promising predictive capacity for the presence and absence of 20 COVID-19 cases. However, to date, such monitoring has largely relied upon composite or grab 21 samples and reverse transcription quantitative PCR (RT-qPCR) techniques, which limits the 22 accessibility and scalability of wastewater monitoring. In this study, we piloted a workflow that 23 uses tampons as passive swabs for collection and reverse transcription loop-mediated isothermal 24 amplification (RT-LAMP) to detect SARS-CoV-2 RNA in wastewater. Results for the developed 25 workflow were available same day, with a time to result following tampon swab collection of 26 approximately three hours. The RT-LAMP 95% limit of detection (76 gene copies reaction-1) was 27 greater than RT-droplet digital PCR (ddPCR; 3.3 gene copies reaction-1). Nonetheless, during a 28 building-level wastewater monitoring campaign conducted in the midst of weekly clinical testing 29 of all students, the workflow demonstrated a same-day positive predictive value (PPV) of 33% 30 and negative predictive value (NPV) of 80% for incident COVID-19 cases. The NPV is comparable 31 to that reported by wastewater monitoring using RT-qPCR. These observations suggest that even 32 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 17 May 2021 doi:10.20944/preprints202105.0381.v1

Within-Day Variability of SARS-CoV-2 RNA in Municipal Wastewater Influent During Periods of Varying COVID-19 Prevalence and Positivity

Abstract: Wastewater surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA is being used to monitor Coronavirus Disease 2019 (COVID-19) trends in communities; however, within-day variation in primary influent concentrations of SARS-CoV-2 RNA remain largely uncharacterized. In the current study, grab sampling of primary influent was performed every 2 hours over two different 24-hour periods at two wastewater treatment plants (WWTPs) in northern Indiana, USA. In primary influent, uncorrected, recovery-corrected, and pepper mild mottle virus (PMMoV)-normalized SARS-CoV-2 RNA concentrations demonstrated ordinal agreement with increasing clinical COVID-19 positivity, but not COVID-19 cases. Primary influent SARS-CoV-2 RNA concentrations exhibited greater variation than PMMoV RNA concentrations as expected for lower shedding prevalence. The bovine respiratory syncytial virus (BRSV) process control recovery efficiency was low (mean: 0.91%) and highly variable (coefficient of variation: 51% - 206%) over the four sampling events with significant differences between the two WWTPs (p <0.0001). The process control recovery was similar to the independently assessed SARS-CoV-2 RNA recovery efficiency, which was also significantly different between the two WWTPs (p <0.0001). Recovery-corrected SARS-CoV-2 RNA concentrations better reflected within-day changes in primary influent flow rate and fecal content, as indicated by PMMoV concentrations. These observations highlight the importance of assessing the process recovery efficiency, which is highly variable, using an appropriate process control. Despite large variations, both recovery-corrected and PMMoV-normalized SARS-CoV-2 RNA concentrations in primary influent demonstrate potential for monitoring COVID-19 positivity trends in WWTPs serving peri-urban and rural areas.

Viable Norovirus Persistence in Water Microcosms.

Abstract: Human noroviruses are one of the leading causes of acute gastroenteritis worldwide. Based on quantitative microbial risk assessments, norovirus contributes the greatest infectious risk of any pathogen from exposure to sewage-contaminated water; however, these estimates have been based upon molecular (i.e., RNA-based) data as human norovirus has remained largely unculturable in the laboratory. Current approaches to assess the environmental fate of noroviruses rely on the use of culturable surrogate viruses and molecular methods. Human intestinal enteroids (HIEs) are an emerging cell culture system capable of amplifying viable norovirus. Here, we applied the HIE assay to assess both viable norovirus and norovirus RNA persistence in surface, tap, and deionized water microcosms. Viable norovirus decreased to below the detection limit in tap and deionized water microcosms and was measured in a single replicate in the surface water microcosm at study conclusion (28 days). Conversely, the norovirus RNA signal remained constant over the duration of the study, even when viable norovirus was below the limit of detection. Our findings demonstrate the disconnect between current environmental norovirus detection via molecular methods and viability as assessed through the HIE assay. These results imply that molecular norovirus monitoring is not inherently representative of infectious norovirus.

Comparison of RT-qPCR and RT-dPCR Platforms for the Trace Detection of SARS-CoV-2 RNA in Wastewater

Abstract: We compared reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and RT digital PCR (RT-dPCR) platforms for the trace detection of SARS-CoV-2 RNA in low-prevalence COVID-19 locations in Queensland, Australia, using CDC N1 and CDC N2 assays. The assay limit of detection (ALOD), PCR inhibition rates, and performance characteristics of each assay, along with the positivity rates with the RT-qPCR and RT-dPCR platforms, were evaluated by seeding known concentrations of exogenous SARS-CoV-2 in wastewater. The ALODs using RT-dPCR were approximately 2–5 times lower than those using RT-qPCR. During sample processing, the endogenous (n = 96) and exogenous (n = 24) SARS-CoV-2 wastewater samples were separated, and RNA was extracted from both wastewater eluates and pellets (solids). The RT-dPCR platform demonstrated a detection rate significantly greater than that of RT-qPCR for the CDC N1 and CDC N2 assays in the eluate (N1, p = 0.0029; N2, p = 0.0003) and pellet (N1, p = 0.0015; N2, p = 0.0067) samples. The positivity results also indicated that for the analysis of SARS-CoV-2 RNA in wastewater, including the eluate and pellet samples may further increase the detection sensitivity using RT-dPCR.