Early Detection of SARS-CoV-2 Omicron BA.4 and BA.5 in German Wastewater

Alexander Wilhelm; Shelesh Agrawal; Jens Schoth; Christina Meinert-Berning; Daniel Bastian; Laura Orschler; Sandra Ciesek; Burkhard Teichgräber; Thomas Wintgens; Susanne Lackner; Frank-Andreas Weber; Marek Widera

doi:10.3390/v14091876

Early Detection of SARS-CoV-2 Omicron BA.4 and BA.5 in German Wastewater

Viruses. 2022 Aug 25;14(9):1876. doi: 10.3390/v14091876.

Authors

Affiliations

¹ Institute for Medical Virology, University Hospital, Goethe University Frankfurt, Paul-Ehrlich-Str. 40, D-60596 Frankfurt, Germany.
² Institute IWAR, Water and Environmental Biotechnology, Technical University of Darmstadt, Franziska-Braun-Str. 7, D-64287 Darmstadt, Germany.
³ Emschergenossenschaft/Lippeverband, Kronprinzenstraße 24, D-45128 Essen, Germany.
⁴ Ruhrverband, Kronprinzenstraße 37, D-45128 Essen, Germany.
⁵ FiW e.V., Research Institute for Water Management and Climate Future at RWTH Aachen University, Kackertstraße 15-17, D-52056 Aachen, Germany.
⁶ German Center for Infection Research (DZIF), D-38124 Braunschweig, Germany.
⁷ Fraunhofer Institute for Translational Medicine and Pharmacology ITMP, Theodor Stern Kai 7, D-60595 Frankfurt am Main, Germany.
⁸ Institute of Environmental Engineering, RWTH Aachen University, Mies-van-der-Rohe-Strasse 1, D-52074 Aachen, Germany.

Abstract

Wastewater-based SARS-CoV-2 epidemiology (WBE) has been established as an important tool to support individual testing strategies. The Omicron sub-variants BA.4/BA.5 have spread globally, displacing the preceding variants. Due to the severe transmissibility and immune escape potential of BA.4/BA.5, early monitoring was required to assess and implement countermeasures in time. In this study, we monitored the prevalence of SARS-CoV-2 BA.4/BA.5 at six municipal wastewater treatment plants (WWTPs) in the Federal State of North Rhine-Westphalia (NRW, Germany) in May and June 2022. Initially, L452R-specific primers/probes originally designed for SARS-CoV-2 Delta detection were validated using inactivated authentic viruses and evaluated for their suitability for detecting BA.4/BA.5. Subsequently, the assay was used for RT-qPCR analysis of RNA purified from wastewater obtained twice a week at six WWTPs. The occurrence of L452R carrying RNA was detected in early May 2022, and the presence of BA.4/BA.5 was confirmed by variant-specific single nucleotide polymorphism PCR (SNP-PCR) targeting E484A/F486V and NGS sequencing. Finally, the mutant fractions were quantitatively monitored by digital PCR, confirming BA.4/BA.5 as the majority variant by 5 June 2022. In conclusion, the successive workflow using RT-qPCR, variant-specific SNP-PCR, and RT-dPCR demonstrates the strength of WBE as a versatile tool to rapidly monitor variants spreading independently of individual test capacities.

Keywords: BA.4; BA.5; COVID-19 surveillance; Omicron; SARS-CoV-2 monitoring; variant of concern; wastewater-based epidemiology (WBE).

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

COVID-19* / diagnosis
COVID-19* / epidemiology
Humans
RNA, Viral / analysis
RNA, Viral / genetics
Real-Time Polymerase Chain Reaction
SARS-CoV-2* / genetics
Wastewater

Substances

RNA, Viral
Waste Water

Grants and funding

This study has been performed with the support of the German Federal Ministry of Education and Research (BMBF) funding to the project COVIDready (grant number 02WRS1621A-D). Funding for the sequencing analysis was provided by BMBF under the project SARS-GenASeq (grant number 02WRS1602A-B). The responsibility for the content of this publication lies with the authors.