Since its first identification in the United Kingdom in late 2020, the highly
transmissible B.1.1.7 variant of SARS-CoV-2 has become dominant in several countries
raising great concern. We developed a duplex real-time RT-qPCR assay to detect,
discriminate, and quantitate SARS-CoV-2 variants containing one of its mutation
signatures, the ΔHV69/70 deletion, and used it to trace the community circulation
of the B.1.1.7 variant in Spain through the Spanish National SARS-CoV-2 Wastewater
Surveillance System (VATar COVID-19). The B.1.1.7 variant was detected earlier than
clinical epidemiological reporting by the local authorities, first in the southern city
of Málaga (Andalucía) in week 20_52 (year_week), and multiple
introductions during Christmas holidays were inferred in different parts of the country.
Wastewater-based B.1.1.7 tracking showed a good correlation with clinical data and
provided information at the local level. Data from wastewater treatment plants, which
reached B.1.1.7 prevalences higher than 90% for ≥2 consecutive weeks showed that
8.1 ± 2.0 weeks were required for B.1.1.7 to become dominant. The study highlights
the applicability of RT-qPCR-based strategies to track specific mutations of variants of
concern as soon as they are identified by clinical sequencing and their integration into
existing wastewater surveillance programs, as a cost-effective approach to complement
clinical testing during the COVID-19 pandemic.
Wastewater-based epidemiology (WBE) has proven to be an effective tool for epidemiological surveillance of SARS-CoV-2 during the current COVID-19 pandemic. Furthermore, combining WBE together with high-throughput sequencing techniques can be useful for the analysis of SARS-CoV-2 viral diversity present in a given sample. The present study focuses on the genomic analysis of SARS-CoV-2 in 76 sewage samples collected during the three epidemiological waves that occurred in Spain from 14 wastewater treatment plants distributed throughout the country. The results obtained demonstrate that the metagenomic analysis of SARS-CoV-2 in wastewater allows the detection of mutations that define the B.1.1.7 lineage and the ability of the technique to anticipate the detection of certain mutations before they are detected in clinical samples. The study proves the usefulness of sewage sequencing to track Variants of Concern that can complement clinical testing to help in decision-making and in the analysis of the evolution of the pandemic.
Isolation, contact tracing and restrictions on social movement are being globally implemented to prevent and control onward spread of SARS-CoV-2, even though the infection risk modelled on RNA detection by RT-qPCR remains biased as viral shedding and infectivity are not discerned. Thus, we aimed to develop a rapid viability RT-qPCR procedure to infer SARS-CoV-2 infectivity in clinical specimens and environmental samples. We screened monoazide dyes and platinum compounds as viability molecular markers on five SARS-CoV-2 RNA targets. A platinum chloride-based viability RT-qPCR was then optimized using genomic RNA, and inactivated SARS-CoV-2 particles inoculated in buffer, stool, and urine. Our results were finally validated in nasopharyngeal swabs from persons who tested positive for COVID-19 and in wastewater samples positive for SARS-CoV-2 RNA. We established a rapid viability RT-qPCR that selectively detects potentially infectious SARS-CoV-2 particles in complex matrices. In particular, the confirmed positivity of nasopharyngeal swabs following the viability procedure suggests their potential infectivity, while the complete prevention of amplification in wastewater indicated either non-infectious particles or free RNA. The viability RT-qPCR approach provides a more accurate ascertainment of the infectious viruses detection and it may complement analyses to foster risk-based investigations for the prevention and control of new or re-occurring outbreaks with a broad application spectrum.
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