ABSTRACT
Wastewater-based surveillance (WBS) has become an effective tool around the globe for indirect monitoring of COVID-19 in communities. Quantities of viral fragments of SARS-CoV-2 in wastewater are related to numbers of clinical cases of COVID-19 reported within the corresponding sewershed. Variants of Concern (VOCs) have been detected in wastewater by use of reverse transcription quantitative polymerase chain reaction (RT-qPCR) or sequencing. A multiplex RT-qPCR assay to detect and estimate the prevalence of multiple VOCs, including Omicron/Alpha, Beta, Gamma, and Delta, in wastewater RNA extracts was developed and validated. The probe-based multiplex assay, named "N200" focuses on amino acids 199-202, a region of the N gene that contains several mutations that are associated with variants of SARS- CoV-2 within a single amplicon. Each of the probes in the N200 assay are specific to the targeted mutations and worked equally well in single- and multi-plex modes. To estimate prevalence of each VOC, the abundance of the targeted mutation was compared with a non- mutated region within the same amplified region. The N200 assay was applied to monitor frequencies of VOCs in wastewater extracts from six sewersheds in Ontario, Canada collected between December 1, 2021, and January 4, 2022. Using the N200 assay, the replacement of the Delta variant along with the introduction and rapid dominance of the Omicron variant were monitored in near real-time, as they occurred nearly simultaneously at all six locations. The N200 assay is robust and efficient for wastewater surveillance can be adopted into VOC monitoring programs or replace more laborious assays currently being used to monitor SARS- CoV-2 and its VOCs.
ABSTRACT
Curtailing the Spring 2020 COVID-19 surge required sweeping and stringent interventions by governments across the world. Wastewater-based COVID-19 epidemiology programs have been initiated in many countries to provide public health agencies with a complementary disease tracking metric and facile surveillance tool. However, their efficacy in prospectively capturing resurgence following a period of low prevalence is unclear. In this study, the SARS-CoV-2 viral signal was measured in primary clarified sludge harvested every two days at the City of Ottawas water resource recovery facility during the summer of 2020, when clinical testing recorded daily percent positivity below 1%. In late July, increases of >400% in normalized SARS-CoV-2 RNA signal in wastewater were identified 48 hours prior to reported >300% increases in positive cases that were retrospectively attributed to community-acquired infections. During this resurgence period, SARS-CoV-2 RNA signal in wastewater preceded the reported >160% increase in community hospitalizations by approximately 96 hours. This study supports wastewater-based COVID-19 surveillance of populations in augmenting the efficacy of diagnostic testing, which can suffer from sampling biases or timely reporting as in the case of hospitalization census.
