SARS-CoV-2 wastewater surveillance in Germany: Long-term RT-digital droplet PCR monitoring, suitability of primer/probe combinations and biomarker stability

Abstract

In recent months, wastewater-based epidemiology (WBE) has been shown to be an important tool for early detection of SARS-CoV-2 circulation in the population. In this study, a detection methodology for SARS-CoV-2 RNA (wildtype and variants of concern) in wastewater was developed based on the detection of different target genes (E and ORF1ab) by polyethylene glycol (PEG) precipitation and digital droplet PCR. This methodology was used to determine the SARS-CoV-2 concentration and the proportion of N501Y mutation in raw sewage of the wastewater treatment plant of the city of Karlsruhe in south-western Germany over a period of 1 year (June 2020 to July 2021). Comparison of SARS-CoV-2 concentrations with reported COVID-19 cases in the catchment area showed a significant correlation. As the clinical SARS-CoV-2 official case report chain takes time, viral RNA titre trends appeared more than 12 days earlier than clinical data, demonstrating the potential of wastewater-based epidemiology as an early warning system. Parallel PCR analysis using seven primer and probe systems revealed similar gene copy numbers with E, ORF, RdRP2 and NSP9 assays. RdPP1 and NSP3 generally resulted in lower copy numbers, and in particular for N1 there was low correlation with the other assays. The occurrence of the N501Y mutation in the wastewater of Karlsruhe was consistent with the occurrence of the alpha-variant (B.1.1.7) in the corresponding individual clinical tests. In batch experiments SARS-CoV-2 RNA was stable for several days under anaerobic conditions, but the copy numbers decreased rapidly in the presence of dissolved oxygen. Overall, this study shows that wastewater-based epidemiology is a sensitive and robust approach to detect trends in the spread of SARS-CoV-2 at an early stage, contributing to successful pandemic management.

Wastewater-Based Epidemiology for SARS-CoV-2 Biomarkers: Evaluation of Normalization Methods in Small and Large Communities in Southern Germany

“…In previous investigations, lead times of the SARS-CoV-2 biomarker data ahead of the clinical data were stated 7,13,16 . When accounting for this lead time for the sampling site Berchtesgaden, we observed a substantial improvement of the coefficients of determination for all normalizations for a time shift of 2 to 8 days with an optimum at 5 days (Fig.…”

“…As the goal of SARS-CoV-2 WBE is to derive valuable information on the infection dynamics in the human population from wastewater, the SARS-CoV-2 biomarker concentrations were frequently compared to epidemiological data. They were observed to strongly correlate with clinical prevalence data and to have lead times of 2 to 24 days ahead of the clinical data, depending on the location and the stage of the pandemic 7,8,[13][14][15][16] , although there are also cases, for which the correlations were low [17][18][19] . Furthermore, regression models to predict the viral incidence based on the SARS-CoV-2 biomarker concentrations in wastewater were suggested 14,16,17 .…”

“…Subsequently, numerous case studies investigated the applicability of SARS-CoV-2 WBE as monitoring and early warning system: from small pilot studies sampling only for several days in the early stage of the pandemic 10,11 , to long-term 12,13 and large-scale 14 monitoring programs gathering comprehensive datasets. In the course of the pandemic, knowledge on sampling strategies, quantification methods, data processing, and comparison to epidemiological data increased substantially; yet, there is still intensive ongoing research on various aspects of SARS-CoV-2 WBE.…”

Evaluation of RT-qPCR Primer-Probe Sets to Inform Public Health Interventions Based on COVID-19 Sewage Tests

“…Besides, there is another dilemma associated with the sewage sample volume and the test sensitivity. Recent studies have focused only on comparing detection rates 18 and stabilities 11 of some primer-probe sets in sewage samples, but to what extent the sewage matrix would impact the RT-qPCR detection across different primer-probe sets remains to be studied.…”

Early Detection of SARS-CoV-2 Omicron BA.4/5 in German wastewater

“…Using molecular reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) analysis the detection of SARS-CoV-2 gene fragments in wastewater was shown to correlate with the COVID-19 prevalence in the catchment area of the sewage treatment plant [14,15]. A wide variety of advanced detection methods including RT-qPCR, digital (droplet) PCR (d(d)PCR), and genomic sequencing (NGS) were described to detect and quantify variant specific RNA fragments [16][17][18][19][20][21][22]. For all approaches, substitutions absent in the predecessing variant were shown as suitable target for molecular detection.…”