Dynamics of SARS-CoV-2 Alpha (B.1.1.7) variant spread: The wastewater surveillance approach.

Wastewater based epidemiology (WBE) offers an overview of the SARS-CoV-2 variants circulating among the population thereby serving as a proper surveillance method. The variant of concern (VOC) Alpha was first identified in September 2020 in the United Kingdom, and rapidly became dominant across Europe. Our objective was to elucidate the Alpha VOC outcompetition rate and identify mutations in the spike glycoprotein (S) gene, indicative of the circulation of the Alpha VOC and/or other variants in the population through wastewater analysis. In the period covered by this study (November 2020-April 2021), forteen wastewater treatment plants (WWTPs) were weekly sampled. The total number of SARS-CoV-2 genome copies per L (GC/L) was determined with a Real-Time qPCR, targeting the N gene. Surveillance of the Alpha VOC circulation was ascertained using a duplex RT-qPCR, targeting and discriminating the S gene. Our results showed that in a period of 6 weeks the Alpha VOC was present in all the studied WWTPs, and became dominant in 11 weeks on average. The outcompetition rates of the Alpha VOC were estimated, and their relationship with different parameters statistically analyzed. The rapid spread of the Alpha VOC was influenced by its initial input and by the previous circulation of SARS-COV-2 in the population. This latter point could be explained by its higher transmissibility, particularly advantadgeous when a certain degree of herd immunity exists. Moreover, the presence of signature mutations of SARS-COV-2 variants were established by deep-sequencing of the complete S gene. The circulation of the Alpha VOC in the area under study was confirmed, and additionally two combinations of mutations in the S glycoprotein (T73A and D253N, and S477N and A522S) that could affect antibody binding were identified.

Time Evolution of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in Wastewater during the First Pandemic Wave of COVID-19 in the Metropolitan Area of Barcelona, Spain.

Two large wastewater treatment plants (WWTP), covering around 2.7 million inhabitants, which represents around 85% of the metropolitan area of Barcelona, were sampled before, during, and after the implementation of a complete lockdown. Five one-step reverse transcriptase quantitative PCR (RT-qPCR) assays, targeting the polymerase (IP2 and IP4), the envelope (E), and the nucleoprotein (N1 and N2) genome regions, were employed for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA detection in 24-h composite wastewater samples concentrated by polyethylene glycol (PEG) precipitation. SARS-CoV-2 was detected in a sewage sample collected 41 days ahead of the declaration of the first COVID-19 case. The evolution of SARS-CoV-2 genome copies in wastewater evidenced the validity of water-based epidemiology (WBE) to anticipate COVID-19 outbreaks, to evaluate the impact of control measures, and even to estimate the burden of shedders, including presymptomatic, asymptomatic, symptomatic, and undiagnosed cases. For the latter objective, a model was applied for the estimation of the total number of shedders, evidencing a high proportion of asymptomatic infected individuals. In this way, an infection prevalence of 2.0 to 6.5% was figured. On the other hand, proportions of around 0.12% and 0.09% of the total population were determined to be required for positive detection in the two WWTPs. At the end of the lockdown, SARS-CoV-2 RNA apparently disappeared in the WWTPs but could still be detected in grab samples from four urban sewers. Sewer monitoring allowed for location of specific hot spots of COVID-19, enabling the rapid adoption of appropriate mitigation measures.IMPORTANCE Water-based epidemiology (WBE) is a valuable early warning tool for tracking the circulation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) among the population, including not only symptomatic patients but also asymptomatic, presymptomatic, and misdiagnosed carriers, which represent a high proportion of the infected population. In the specific case of Barcelona, wastewater surveillance anticipated by several weeks not only the original COVID-19 pandemic wave but also the onset of the second wave. In addition, SARS-CoV-2 occurrence in wastewater evidenced the efficacy of the adopted lockdown measures on the circulation of the virus. Health authorities profited from WBE to complement other inputs and adopt rapid and adequate measures to mitigate the effects of the pandemic. For example, sentinel surveillance of specific sewers helped to locate COVID-19 hot spots and to conduct massive numbers of RT-PCR tests among the population.

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).

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.

Investigation on the Evolution of Shiga Toxin-Converting Phages Based on Whole Genome Sequencing.

Bacteriophages are pivotal elements in the dissemination of virulence genes. The main virulence determinants of Shiga Toxin producing E. coli, Shiga Toxins (Stx), are encoded by genes localized in the genome of lambdoid bacteriophages. Stx comprise two antigenically different types, Stx1 and Stx2, further divided into subtypes. Among these, certain Stx2 subtypes appear to be more commonly occurring in the most severe forms of the STEC disease, haemorrhagic colitis and haemolytic uremic syndrome (HUS). This study aimed at obtaining insights on the evolution of Stx2 bacteriophages, due to their relevance in public health, and we report here on the analysis of the genomic structure of Stx2 converting phages in relation with the known reservoir of the E. coli strains harboring them. Stx2-converting phages conveying the genes encoding different stx2 subtypes have been isolated from STEC strains and their whole genomes have been sequenced, analyzed and compared to those of other Stx2 phages available in the public domain. The phages' regions containing the stx2 genes have been analyzed in depth allowing to make inference on the possible mechanisms of selection and maintenance of certain Stx2 phages in the reservoir. The "stx regions" of different stx2 gene subtypes grouped into three different evolutionary lines in the comparative analysis, reflecting the frequency with which these subtypes are found in different animal niches, suggesting that the colonization of specific reservoir by STEC strains could be influenced by the Stx phage that they carry. Noteworthy, we could identify the presence of nanS-p gene exclusively in the "stx regions" of the phages identified in STEC strains commonly found in cattle. As a matter of fact, this gene encodes an esterase capable of metabolizing sialic acids produced by submaxillary glands of bovines and present in great quantities in their gastrointestinal tract.