National Scale Real-Time Surveillance of SARS-CoV-2 Variants Dynamics by Wastewater Monitoring in Israel.

In this report, we describe a national-scale monitoring of the SARS-CoV-2 (SC-2) variant dynamics in Israel, using multiple-time sampling of 13 wastewater treatment plants. We used a combination of inclusive and selective quantitative PCR assays that specifically identify variants A19/A20 or B.1.1.7 and tested each sample for the presence and relative viral RNA load of each variant. We show that between December 2020 and March 2021, a complete shift in the SC-2 variant circulation was observed, where the B.1.1.7 replaced the A19 in all examined test points. We further show that the normalized viral load (NVL) values and the average new cases per week reached a peak in January 2021 and then decreased gradually in almost all test points, in parallel with the progression of the national vaccination campaign, during February-March 2021. This study demonstrates the importance of monitoring SC-2 variant by using a combination of inclusive and selective PCR tests on a national scale through wastewater sampling, which is far more amendable for high-throughput monitoring compared with sequencing. This approach may be useful for real-time dynamics surveillance of current and future variants, such as the Omicron (BA.1, BA.2) and other variants.

Monitoring of Enterovirus D68 Outbreak in Israel by a Parallel Clinical and Wastewater Based Surveillance.

Enterovirus D68 (EVD68) was recently identified as an important cause of respiratory illness and acute flaccid myelitis (AFM), mostly in children. Here, we examined 472 pediatric patients diagnosed with severe respiratory illness and screened for EVD68 between April and October 2021. In parallel, samples collected from a wastewater treatment plant (WWTP) covering the residential area of the hospitalized patients were also tested for EVD68. Of the 472 clinical samples evaluated, 33 (7%) patients were positive for EVD68 RNA. All wastewater samples were positive for EVD68, with varying viral genome copy loads. Calculated EVD68 genome copies increased from the end of May until July 2021 and dramatically decreased at the beginning of August. A similar trend was observed in both clinical and wastewater samples during the period tested. Sequence analysis of EVD68-positive samples indicated that all samples originated from the same branch of subclade B3. This study is the first to use wastewater-based epidemiology (WBE) to monitor EVD68 dynamics by quantitative detection and shows a clear correlation with clinically diagnosed cases. These findings highlight the potential of WBE as an important tool for continuous surveillance of EVD68 and other enteroviruses.

Specific Detection of SARS-CoV-2 Variants B.1.1.7 (Alpha) and B.1.617.2 (Delta) Using a One-Step Quantitative PCR Assay.

In this report, we describe the development of a reverse transcription-quantitative PCR (RT-qPCR) assay, termed Alpha-Delta assay, which can detect all severe acute respiratory syndrome coronavirus 2 (SC-2) variants and distinguish between the Alpha (B.1.1.7) and Delta (B.1.617.2) variants. The Alpha- and Delta-specific reactions in the assay target mutations that are strongly linked to the target variant. The Alpha reaction targets the D3L substitution in the N gene, and the Delta reaction targets the spike gene 156 to 158 mutations. Additionally, we describe a second Delta-specific assay that we use as a confirmatory test for the Alpha-Delta assay that targets the 119 to 120 deletion in the Orf8 gene. Both reactions have similar sensitivities of 15 to 25 copies per reaction, similar to the sensitivity of commercial SC-2 detection tests. The Alpha-Delta assay and the Orf8119del assay were successfully used to classify clinical samples that were subsequently analyzed by whole-genome sequencing. Lastly, the capability of the Alpha-Delta assay and Orf8119del assay to identify correctly the presence of Delta RNA in wastewater samples was demonstrated. This study provides a rapid, sensitive, and cost-effective tool for detecting and classifying two worldwide dominant SC-2 variants. It also highlights the importance of a timely diagnostic response to the emergence of new SC-2 variants with significant consequences on global health. IMPORTANCE The new assays described herein enable rapid, straightforward, and cost-effective detection of severe acute respiratory syndrome coronavirus 2 (SC-2) with immediate classification of the examined sample as Alpha, Delta, non-Alpha, or non-Delta variant. This is highly important for two main reasons: (i) it provides the scientific and medical community with a novel diagnostic tool to rapidly detect and classify any SC-2 sample of interest as Alpha, Delta, or none and can be applied to both clinical and environmental samples, and (ii) it demonstrates how to respond to the emergence of new variants of concern by developing a variant-specific assay. Such assays should improve our preparedness and adjust the diagnostic capacity to serve clinical, epidemiological, and research needs.

Regressing SARS-CoV-2 Sewage Measurements Onto COVID-19 Burden in the Population: A Proof-of-Concept for Quantitative Environmental Surveillance.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an RNA virus, a member of the coronavirus family of respiratory viruses that includes severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) and the Middle East respiratory syndrome (MERS). It has had an acute and dramatic impact on health care systems, economies, and societies of affected countries during the past 8 months. Widespread testing and tracing efforts are being employed in many countries in attempts to contain and mitigate this pandemic. Recent data has indicated that fecal shedding of SARS-CoV-2 is common and that the virus RNA can be detected in wastewater. This indicates that wastewater monitoring may provide a potentially efficient tool for the epidemiological surveillance of SARS-CoV-2 infection in large populations at relevant scales. In particular, this provides important means of (i) estimating the extent of outbreaks and their spatial distributions, based primarily on in-sewer measurements, (ii) managing the early-warning system quantitatively and efficiently, and (iii) verifying disease elimination. Here we report different virus concentration methods using polyethylene glycol (PEG), alum, or filtration techniques as well as different RNA extraction methodologies, providing important insights regarding the detection of SARS-CoV-2 RNA in sewage. Virus RNA particles were detected in wastewater in several geographic locations in Israel. In addition, a correlation of virus RNA concentration to morbidity was detected in Bnei-Barak city during April 2020. This study presents a proof of concept for the use of direct raw sewage-associated virus data, during the pandemic in the country as a potential epidemiological tool.

City-level SARS-CoV-2 sewage surveillance.

The COVID-19 pandemic created a global crisis impacting not only healthcare systems, but also economics and society. Therefore, it is important to find novel methods for monitoring disease activity. Recent data have indicated that fecal shedding of SARS-CoV-2 is common, and that viral RNA can be detected in wastewater. This suggests that wastewater monitoring is a potentially efficient tool for both epidemiological surveillance, and early warning for SARS-CoV-2 circulation at the population level. In this study we sampled an urban wastewater infrastructure in the city of Ashkelon (Ì´ 150,000 population), Israel, during the end of the first COVID-19 wave in May 2020 when the number of infections seemed to be waning. We were able to show varying presence of SARS-CoV-2 RNA in wastewater from several locations in the city during two sampling periods, before the resurgence was clinically apparent. This was expressed with a new index, Normalized Viral Load (NVL) which can be used in different area scales to define levels of virus activity such as red (high) or green (no), and to follow morbidity in the population at the tested area. The rise in viral load between the two sampling periods (one week apart) indicated an increase in morbidity that was evident two weeks to a month later in the population. Thus, this methodology may provide an early indication for SARS-CoV-2 infection outbreak in a population before an outbreak is clinically apparent.

A Unique SARS-CoV-2 Spike Protein P681H Variant Detected in Israel.

The routine detection, surveillance, and reporting of novel SARS-CoV-2 variants is crucial, as these threaten to hinder global vaccination efforts. Herein we report a novel local variant with a non-synonymous mutation in the spike (S) protein P681H. This local Israeli variant was not associated with a higher infection rate or higher prevalence. Furthermore, the local variant was successfully neutralized by sera from fully vaccinated individuals at a comparable level to the B.1.1.7 variant and an Israel wild-type strain. While it is not a variant of concern, routine monitoring by sequencing is still required.

Detection of SARS-CoV-2 variants by genomic analysis of wastewater samples in Israel.

Investigation of SARS-CoV-2 spread and identification of variants in sewers has been demonstrated to accurately detect prevalence of viral strains and is advantageous to clinical sampling in population catchment size. Herein, we utilized an established nationwide system of wastewater sampling and viral concentration approaches to perform large-scale surveillance of SARS-CoV-2 variants in nine different locations across Israel that were sampled from August 2020 to February 2021 and sequenced (n = 58). Viral sequences obtained from the wastewater samples had high coverages of the genome, and mutation analyses successfully identified the penetration of the B.1.1.7 variant into Israel in December 2020 in the central and north regions, and its spread into additional regions in January and February 2021, corresponding with clinical sampling results. Moreover, the wastewater analysis identified the B.1.1.7 variant in December 2020 in regions in which non-sufficient clinical sampling was available. Other variants of concern examined, including P.1 (Brazil/Manaus), B.1.429 (USA/California), B.1.526 (USA/New York), A.23.1 (Uganda) and B.1.525 (Unknown origin), did not show consistently elevated frequencies. This study exemplifies that surveillance by sewage is a robust approach which allows to monitor the diversity of SARS-CoV-2 strains circulating in the community. Most importantly, this approach can pre-identify the emergence of epidemiologically or clinically relevant mutations/variants, aiding in public health decision making.

Rethinking wastewater risks and monitoring in light of the COVID-19 pandemic

The COVID-19 pandemic has severely impacted public health and the worldwide economy. Converging evidence from the current pandemic, previous outbreaks and controlled experiments indicates that SARS-CoVs are present in wastewater for several days, leading to potential health risks via waterborne and aerosolized wastewater pathways. Conventional wastewater treatment provides only partial removal of SARS-CoVs, thus safe disposal or reuse will depend on the efficacy of final disinfection. This underscores the need for a risk assessment and management framework tailored to SARS-CoV-2 transmission via wastewater, including new tools for environmental surveillance, ensuring adequate disinfection as a component of overall COVID-19 pandemic containment. Converging evidence indicates that SARS-CoVs are present in wastewater for several days with potential health risks. This Review analyses knowledge about such risks as well as the potential spread of SARS-CoVs in waterborne, waterborne-aerosolized and waterborne-foodborne pathways during a pandemic.