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. 2021 Sep 7;55(17):11756-11766.
doi: 10.1021/acs.est.1c03589. Epub 2021 Aug 16.

Monitoring Emergence of the SARS-CoV-2 B.1.1.7 Variant through the Spanish National SARS-CoV-2 Wastewater Surveillance System (VATar COVID-19)

Albert Carcereny  1   2 , Adán Martínez-Velázquez  1   2 , Albert Bosch  1   2 , Ana Allende  3 , Pilar Truchado  3 , Jenifer Cascales  3 , Jesús L Romalde  4 , Marta Lois  4 , David Polo  4 , Gloria Sánchez  5 , Alba Pérez-Cataluña  5 , Azahara Díaz-Reolid  5 , Andrés Antón  6 , Josep Gregori  7   8 , Damir Garcia-Cehic  7   8 , Josep Quer  7   8 , Margarita Palau  9 , Cristina González Ruano  10 , Rosa M Pintó  1   2 , Susana Guix  1   2
Affiliations

Monitoring Emergence of the SARS-CoV-2 B.1.1.7 Variant through the Spanish National SARS-CoV-2 Wastewater Surveillance System (VATar COVID-19)

Albert Carcereny et al. Environ Sci Technol. .

Abstract

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.

Keywords: B.1.1.7 variant; COVID-19; NGS; RT-qPCR; SARS-CoV-2; wastewater-based epidemiology (WBE).

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Estimated GC corresponding to wildtype SARS-CoV-2 sequences without ΔHV69/70 deletion (gray bars) and sequences containing ΔHV69/70 deletion in the S gene (yellow bars) from nine preparations at three different total concentration levels ((A): 1 × 104 GC/rxn, (B): 1 × 103 GC/rxn, and (C): 1 × 102 GC/rxn) and three different proportions of Wuhan-Hu-1 and B.1.1.7 GC (90:10, 50:50, and 10:90). Data correspond to mean values ± standard deviations from duplicate samples. Each sample corresponds to an independent preparation containing the indicated proportions of B.1.1.7 and Wuhan-Hu-1 synthetic control RNAs. Samples at different proportions of synthetic control RNAs were prepared in duplicate and were further diluted at the indicated concentration levels.
Figure 2
Figure 2
Overview of the nucleotide substitutions detected in SARS-CoV-2 S gene sequences from wastewater samples (n = 8) as compared to the SARS-CoV-2 isolate Wuhan-Hu-1 reference genome (MN908947.3). Percentages before each line indicate the proportion of the B.1.1.7 variant measured in each sample. B.1.1.7-specific markers are shown in light orange, yellow markers show mutations described in the B.1.177 variant, and blue markers indicate others. The RBD is indicated with a dotted square. Amplicon numbers are shown at the bottom. Shaded green colors indicate sequence coverage in a logarithmic scale for each amplicon.
Figure 3
Figure 3
Concentration of SARS-CoV-2 RNA in wastewater samples collected in Spain from December 2020 to March 2021, as measured by N1 RT-qPCR (dark blue), and duplex S gene allelic discrimination RT-qPCR [wildtype S (light blue) and B.1.1.7 S (red)]. WWTPs are alphabetically grouped by autonomous communities in Spain ((A): Andalucía, (B): Aragón, (C): Baleares, (D): Canarias, (E): Cantabria, (F): Castilla-La Mancha, (G): Castilla y León, (H): Cataluña, (I): Com. De Madrid, (J): Com. Valenciana, (K): Extremadura, (L): Galicia, M: La Rioja, (N): País Vasco, and (O): Pr. Asturias). Data represent average values, and error bars represent the standard deviation of the RT-qPCR replicates used for calculation. Dotted lines correspond to the LOQ of assays.
Figure 4
Figure 4
Evolution of B.1.1.7 SARS-CoV-2 prevalence over time, as measured by duplex RT-qPCR in wastewater samples from 32 WWTPs. As in Figure 3, data are alphabetically shown according to autonomous communities. * indicates samples with detection of a single variant but with titers
Figure 5
Figure 5
Comparison of B.1.1.7 estimates from wastewater testing and clinical epidemiological surveillance. (A) Correlation between B.1.1.7 proportions estimated by duplex RT-qPCR from wastewater and data reported by local authorities from clinical specimen sequencing. (B) Geographic and temporal evolution of B.1.1.7 SARS-CoV-2 emergence in Spain during the study period, estimated from wastewater samples (left panels) and reported in clinical data (right panels). For wastewater data, percentages are indicated for each WWTP. * indicates samples with detection of a single variant but with titers
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