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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 twelve wastewater treatment plants. We used a combination of inclusive and selective quantitative PCR assays that specifically identify variants A19 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 dynamics on a national scale through wastewater sampling. It also provides a proof-of-concept methodology for continuous surveillance by using a combination of inclusive and selective PCR tests, 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) variant. SynopsisThis study describes the continuous monitoring of the SARS CoV-2 variant B.1.1.7 circulation in wastewater in Israel using a positive/negative quantitative PCR assay.
RESUMEN
Emerging SARS-CoV-2 (SC-2) variants with increased infectivity and vaccine resistance are of major concern. Rapid identification of such variants is important for the public health activities and provide valuable data for epidemiological and policy decision making. We developed a multiplex quantitative RT-qPCR (qPCR) assay that can specifically identify and differentiate between the emerging B.1.1.7 and B.1.351 SC-2 variants. In a single assay, we combined four reactions: one that detects SC-2 RNA independently of the strain, one that detects the D3L mutation, which is specific to variant B.1.1.7, and one that detects the 242-244 deletion, which is specific to variant B.1.351. The fourth reaction identifies human RNAseP gene, serving as an endogenous control for RNA extraction integrity. We show that the strain-specific reactions target mutations that are strongly associated with the target variants, and not with other major known variants. The assays specificity was tested against a panel of respiratory pathogens (n=16), showing high specificity towards SC-2 RNA. The assays sensitivity was assessed using both In-vitro transcribed RNA and clinical samples, and was determined to be between 20 and 40 viral RNA copies per reaction. The assay performance was corroborated with Sanger and whole genome sequencing, showing complete agreement with the sequencing results. The new assay is currently implemented in the routine diagnostic work at the Central Virology Laboratory, and may be used in other laboratories to facilitate the diagnosis of these major worldwide circulating SC-2 variants.
RESUMEN
The COVID-19 pandemic created a global crisis impacting not only healthcare systems, but also world economies and society. 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, 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. This was expressed as 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 tested area. Our index showed the rise in viral load between the two sampling periods (one week apart) and indicated an increase in morbidity that was evident 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. HIGHLIGHTSO_LIDetecting the presence of SARS-CoV-2 virus RNA in urban wastewater C_LIO_LIThe city sewer system may provide an early indication for SARS-CoV-2 infection and may be used as early warning for SARS-CoV-2 outbreaks C_LIO_LINVL index defines various infected urban zones from red (high) to green (low) C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC="FIGDIR/small/20215244v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@360a84org.highwire.dtl.DTLVardef@1ec8004org.highwire.dtl.DTLVardef@1c8ae93org.highwire.dtl.DTLVardef@3d670c_HPS_FORMAT_FIGEXP M_FIG C_FIG
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Conducting numerous, rapid, and reliable PCR tests for SARS-CoV-2 is essential for our ability to monitor and control the current COVID-19 pandemic. Here, we tested the sensitivity and efficiency of SARS-CoV-2 detection in clinical samples collected directly into a mix of lysis buffer and RNA preservative, thus inactivating the virus immediately after sampling. We tested 79 COVID-19 patients and 20 healthy controls. We collected two samples (nasopharyngeal swabs) from each participant: one swab was inserted into a test tube with Viral Transport Medium (VTM), following the standard guideline used as the recommended method for sample collection; the other swab was inserted into a lysis buffer supplemented with nucleic acid stabilization mix (coined NSLB). We found that RT-qPCR tests of patients were significantly more sensitive with NSLB sampling, reaching detection threshold 2.1{+/-}0.6 (Mean{+/-}SE) PCR cycles earlier then VTM samples from the same patient. We show that this improvement is most likely since NSLB samples are not diluted in lysis buffer before RNA extraction. Re-extracting RNA from NSLB samples after 72 hours at room temperature did not affect the sensitivity of detection, demonstrating that NSLB allows for long periods of sample preservation without special cooling equipment. We also show that swirling the swab in NSLB and discarding it did not reduce sensitivity compared to retaining the swab in the tube, thus allowing improved automation of COVID-19 tests. Overall, we show that using NSLB instead of VTM can improve the sensitivity, safety, and rapidity of COVID-19 tests at a time most needed.
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SARS-CoV-2 is an RNA virus, a member of the coronavirus family of respiratory viruses that includes SARS-CoV-1 and MERS. COVID-19, the clinical syndrome caused by SARSCoV-2, has evolved into a global pandemic with more than 2,900,000 people infected. It has had an acute and dramatic impact on health care systems, economies, and societies of affected countries within these few months. Widespread testing and tracing efforts are employed in many countries in order to contain and mitigate this pandemic. Recent data has indicated that fecal shedding of SARS-CoV-2 is common, and that the virus can be detected in wastewater. This indicates that wastewater monitoring is a potentially efficient tool for epidemiological surveillance of SARS-CoV-2 infection in large populations at relevant scales. Collecting raw sewage data, representing specific districts, and crosslinking this data with the number of infected people from each location, will enable us to derive and provide quantitative surveillance tools. In particular, this will provide important means to (i) estimate the extent of outbreaks and their spatial distributions, based primarily on in-sewer measurements (ii) manage the early-warning system quantitatively and efficiently (and similarly, verify disease elimination). Here we report the development of a virus concentration method using PEG or alum, providing an important a tool for detection of SARS-CoV-2 RNA in sewage and relating it to the local populations and geographic information. This will provide a proof of concept for the use of sewage associated virus data as a reliable epidemiological tool.