Suchergebnisse

AbstractWastewater monitoring has proven to be an important approach to detecting and controlling the development of the SARS-CoV-2 pandemic. Various tests based on reverse transcription real-time PCR (qRT-PCR) have been developed and used for the detection of SARS-CoV-2 in wastewater samples. In this study, we attempted to increase the sensitivity of qRT-PCR by developing a one-step single-tube nested qRT-PCR assay (OSN-qRT-PCR). Two variants were developed, oriented to nucleocapsid phosphoprotein gene (N) and to spike protein gene (S), respectively. The performance of conventional qRT-PCR assays oriented to these genes with two novel OSN-qRT-PCR assays were firstly optimized using wastewater artificially contaminated with two encapsidated RNA mimic systems harboring a portion either N or S gene (ENRM and ESRM, respectively). The assays were coupled to a polyethylene glycol–based RNA precipitation/extraction method and applied to detect SARS-CoV-2 in wastewater samples from four cities in Slovakia. Both novel OSN-qRT-PCR assays demonstrated higher detection rates than the ordinary qRT-PCR counterparts. The virus levels in the analyzed wastewater samples had a high or very high relation with the numbers of clinical cases in the monitored regions. In fact, correlation with a 3-, 4-, or 5-day temporal offset was revealed. The OSN-qRT-PCR assays demonstrated robustness, mainly in samples with low viral loads.

The presence of SARS-CoV-2 RNA in wastewater was first reported in March 2020. Over the subsequent months, the potential for wastewater surveillance to contribute to COVID-19 mitigation programmes has been the focus of intense national and international research activities, gaining the attention of policy makers and the public. As a new application of an established methodology, focused collaboration between public health practitioners and wastewater researchers is essential to developing a common understanding on how, when and where the outputs of this non-invasive community-level approach can deliver actionable outcomes for public health authorities. Within this context, the NORMAN SCORE "SARS-CoV-2 in sewage" database provides a platform for rapid, open access data sharing, validated by the uploading of 276 data sets from nine countries to-date. Through offering direct access to underpinning meta-data sets (and describing its use in data interpretation), the NORMAN SCORE database is a resource for the development of recommendations on minimum data requirements for wastewater pathogen surveillance. It is also a tool to engage public health practitioners in discussions on use of the approach, providing an opportunity to build mutual understanding of the demand and supply for data and facilitate the translation of this promising research application into public health practice. ; All authors wish to thank the WWTP operators for providing samples. LL, AH and MV would like to acknowledge the VINNOVA (Swedish Governmental Agency for Innovation Systems) DRIZZLE – Centre for Stormwater Management (Grant no. 2016-05176) and the technical expertise provided by the Stormwater&Sewers network, Nireas-International Water Research Center of the University of Cyprus would like to thank the Sewerage Board of Limassol-Amathus (SBLA), the Sewerage Board of Nicosia (SBN) and the Paralimni Sewerage Board (PSB) for the provision of influent samples, for the purpose of performing this work. MPD wishes to thank COVIDBENS Inv04020 financed by EDAR Bens S.A, A Coruña, FYL wishes to thank Z Cetecioglu Gurol (KTH) and P Haglund (Umeå University) and TM would like to acknowledge financial support from APVV-19-0250, PP-COVID-20-0019, ASS8 and VIR-SCAN. Authors from the Univ Jaume LB, FH, MB and RdL acknowledge the financial support from Dirección General del Agua, Generalitat Valenciana, to develop the project "Covid_Wastewater", as well as the help E. Santateresa and N. Zamorano from FACSA, for the invaluable support in performing this work. RdL. was funded through a Beatriz Galindo Fellowship of the Ministerio de Educación y Formación Profesional, Spanish Government (BEAGAL18/00042). TM wishes to thank the generous support of the Operational Program Integrated Infrastructure for the project "Strategic research in the field of SMART monitoring, treatment and preventive protection against coronavirus (SARS-CoV-2) ", Project no. 313011ASS8 (co-financed by the European Regional Development Fund) and the project VIR-SCAN - Wastewater monitoring data as an early warning tool to alert COVID-19 in the population (EOSCsecretariat.eu has received funding from the European Union's Horizon Program call H2020-INFRAEOSC- 05-2018-2019, grant Agreement number 831644). SK (IBISS) acknowledges the financial support from Ministry of Education, Science and Technological Development of Republic of Serbia grant No 451-03-9/2021-14/ 200007.