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1.
medrxiv; 2023.
Preprint in English | medRxiv | ID: ppzbmed-10.1101.2023.04.14.23288559

ABSTRACT

Wastewater based epidemiology (WBE) is a useful method to detect pathogen prevalence and may serve to effectively monitor diseases at a broad scale. WBE has been used throughout the COVID-19 pandemic to track localized and population-level disease burden through the quantification of SARS-CoV-2 RNA present in wastewater. Aside from case load estimation, WBE is being used to assay viral genomic diversity and the emergence of potential SARS-CoV-2 variants. Here, we present a study in which we sequenced RNA extracted from sewage influent samples obtained from eight wastewater treatment plants representing 16 million people in Southern California over April 2020 - August 2021. We sequenced SARS-CoV-2 with two methods: Illumina Respiratory Virus Enrichment and metatranscriptomic sequencing (N = 269), and QIAseq SARS-CoV-2 tiled amplicon sequencing (N = 95). We were able to classify SARS-CoV-2 reads into lineages and sublineages that approximated several named variants across a full year, and we identified a diversity of single nucleotide variants (SNVs) of which many are putatively novel SNVs, and SNVs of unknown potential function and prevalence. Through our retrospective study, we also show that several sublineages of SARS-CoV-2 were detected in wastewater up to several months before clinical detection, which may assist in the prediction of future Variants of Concern. Lastly, we show that sublineage diversity was similar between wastewater treatment plants across Southern California, and that diversity changed by sampling month indicating that WBE is effective across megaregions. As the COVID-19 pandemic moves to new phases, and additional SARS-CoV-2 variants emerge, the ongoing monitoring of wastewater is important to understand local and population-level dynamics of the virus. Our study shows the potential of WBE to detect SARS-CoV-2 variants throughout Southern Californias wastewater and track the diversity of viral SNVs and strains in urban and suburban locations. These results will aid in our ability to monitor the evolutionary potential of SARS-CoV-2 and help understand circulating SNVs to further combat COVID-19.


Subject(s)
COVID-19 , Encephalitis, California
2.
medrxiv; 2021.
Preprint in English | medRxiv | ID: ppzbmed-10.1101.2021.06.16.21259063

ABSTRACT

The rapid emergence of wastewater based surveillance has led to a wide array of SARS-CoV-2 RNA quantification methodologies being employed. Here we compare methods to store samples, inactivate viruses, capture/concentrate viruses, and extract/measure viral RNA from primary influent into wastewater facilities. We found that heat inactivation of the viruses led to a 1-3 log10 decrease compared to chemical inactivation. Freezing influent prior to concentration caused a 1-4 log10 decrease compared to processing fresh samples, but viral capture by membrane adsorption prior to freezing was robust to freeze-thaw variability. Concentration vs. direct extraction, and PCR platform also affected outcome, but by a smaller amount. The choice of nucleocapsid gene target had nearly no effect. Pepper mild-mottle virus was much less sensitive to these methodological differences than was SARS-CoV-2, which challenges its use as a population-level control among studies using different methods. Better characterizing the variability associated with different methodologies, in particular the impact of methods on sensitivity, will aid decision makers in following the effects of vaccination campaigns, early detection of future outbreaks, and potentially monitoring the appearance of SARS-CoV-2 variants in the population.

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