Factors Associated With Protection From SARS-CoV-2 Omicron Variant Infection and Disease Among Vaccinated Health Care Workers in Israel.

Importance: A correlation between antibody levels and risk of infection has been demonstrated for the wild-type, Alpha, and Delta SARS-COV-2 variants. High rates of breakthrough infections by the Omicron variant emphasized the need to investigate whether the humoral response elicited by mRNA vaccines is also associated with reduced risk of Omicron infection and disease. Objective: To investigate whether the high antibody levels in individuals who have received at least 3 doses of an mRNA vaccine are associated with reduced risk of Omicron infection and disease. Design, Setting, and Participants: This prospective cohort study used serial real time-polymerase chain reaction (RT-PCR) and serological test data from January and May 2022 to assess the association of preinfection immunoglobin G (IgG) and neutralizing antibody titers with incidence of Omicron variant infection, incidence of symptomatic disease, and infectivity. Participants included health care workers who had received 3 or 4 doses of an mRNA COVID-19 vaccine. Data were analyzed from May to August 2022. Exposures: Levels of SARS-CoV-2 anti-receptor binding domain IgG and neutralizing antibodies. Main Outcomes and Measures: The main outcomes were incidence of Omicron infection, incidence of symptomatic disease, and infectivity. Outcomes were measured using SARS-COV-2 PCR and antigen testing and daily online surveys regarding symptomatic disease. Results: This study included 3 cohorts for 3 different analyses: 2310 participants were included in the protection from infection analysis (4689 exposure events; median [IQR] age, 50 [40-60] years; 3590 [76.6%] among female health care workers), 667 participants (median [IQR] age, 46.28 (37.44,54.8); 516 [77.4%] female) in the symptomatic disease analysis, and 532 participants (median [IQR] age, 48 [39-56] years; 403 [75.8%] female) in the infectivity analysis. Lower odds of infection were observed for each 10-fold increase in preinfection IgG (odds ratio [OR], 0.71; 95% CI, 0.56-0.90) and for each 2-fold increase in neutralizing antibody titers (OR, 0.89; 95% CI, 0.83-0.95). The odds of substantial symptomatic disease were reduced for each 10-fold increase in IgG levels (OR, 0.48; 95% CI, 0.29-0.78) and for each 2-fold increase in neutralizing antibodies levels (OR, 0.86; 95% CI, 0.76-0.96). Infectivity, assessed by mean cycle threshold value, was not significantly decreased with increasing IgG or neutralizing antibodies titers. Conclusions and Relevance: In this cohort study of vaccinated health care workers, IgG and neutralizing antibody titer levels were associated with protection against infection with the Omicron variant and against symptomatic disease.

Disinfection of SARS-CoV-2 by UV-LED 267 nm: comparing different variants.

UV irradiation is an efficient tool for the disinfection of viruses in general and coronavirus specifically. This study explores the disinfection kinetics of SARS-CoV-2 variants wild type (similar to the Wuhan strain) and three variants (Alpha, Delta, and Omicron) by 267 nm UV-LED. All variants showed more than 5 logs average reduction in copy number at 5 mJ/cm2 but inconsistency was evident, especially for the Alpha variant. Increasing the dose to 7 mJ/cm2 did not increase average inactivation but did result in a dramatic decrease in the inactivation inconsistency making this dose the recommended minimum. Sequence analysis suggests that the difference between the variants is likely due to small differences in the frequency of specific UV extra-sensitive nucleotide sequence motifs although this hypothesis requires further experimental testing. In summary, the use of UV-LED with their simple electricity need (can be operated from a battery or photovoltaic panel) and geometrical flexibility could offer many advantages in the prevention of SARS-CoV-2 spread, but minimal UV dose should be carefully considered.

Global disparities in SARS-CoV-2 genomic surveillance.

Genomic sequencing is essential to track the evolution and spread of SARS-CoV-2, optimize molecular tests, treatments, vaccines, and guide public health responses. To investigate the global SARS-CoV-2 genomic surveillance, we used sequences shared via GISAID to estimate the impact of sequencing intensity and turnaround times on variant detection in 189 countries. In the first two years of the pandemic, 78% of high-income countries sequenced >0.5% of their COVID-19 cases, while 42% of low- and middle-income countries reached that mark. Around 25% of the genomes from high income countries were submitted within 21 days, a pattern observed in 5% of the genomes from low- and middle-income countries. We found that sequencing around 0.5% of the cases, with a turnaround time <21 days, could provide a benchmark for SARS-CoV-2 genomic surveillance. Socioeconomic inequalities undermine the global pandemic preparedness, and efforts must be made to support low- and middle-income countries improve their local sequencing capacity.

Omicron BA.2.75 variant is efficiently neutralised following BA.1 and BA.5 breakthrough infection in vaccinated individuals, Israel, June to September 2022.

We evaluated neutralising antibody titres against wild type (WT) SARS-CoV-2 and four Omicron variants (BA.1, BA.2, BA.5 and BA.2.75) in fully vaccinated (three doses of Comirnaty vaccine) healthcare workers (HCW) in Israel who had breakthrough BA.1/BA5 infections. Omicron breakthrough infections in vaccinated individuals resulted in increased neutralising antibodies against the WT and Omicron variants compared with vaccinated uninfected HCW. HCW who recovered from BA.1 or BA.5 infections showed similar neutralising antibodies levels against BA.2.75.

COVID-19 vaccination and BA.1 breakthrough infection induce neutralising antibodies which are less efficient against BA.4 and BA.5 Omicron variants, Israel, March to June 2022.

This work evaluated neutralising antibody titres against wild type (WT) SARS-CoV-2 and four Omicron variants (BA.1, BA.2, BA.4 and BA.5) in healthcare workers who had breakthrough BA.1 infection. Omicron breakthrough infection in individuals vaccinated three or four times before infection resulted in increased neutralising antibodies against the WT virus. The fourth vaccine dose did not further improve the neutralising efficiency over the third dose against all Omicron variants, especially BA.4 and BA.5. An Omicron-specific vaccine may be indicated.

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.

Human metapneumovirus prevalence during 2019-2021 in Israel is influenced by the COVID-19 pandemic.

OBJECTIVES: To compare infection rates and circulating subtypes of human metapneumovirus (hMPV) before (2019-2020) and after the emergence of coronavirus disease 2019 (COVID-19) (2021) in Israel. METHODS: In total, 12,718 respiratory samples were collected from hospitalized patients of all ages during the years 2019 to 2021 at the Sheba Medical Center in Israel and subjected to reverse transcription-polymerase chain reaction analysis. In addition, whole-genome sequencing was performed to characterize the subtypes of hMPV circulating in Israel between 2019 and 2021. RESULTS: A total of 481 samples were found positive for hMPV. Before the emergence of COVID-19, hMPV peaked in winter months and declined thereafter. In sharp contrast, during the COVID-19 pandemic, we observed a delayed peak in hMPV infection cases and higher infection of young children. Viral sequencing showed a shift in the most prevalent circulating hMPV strain from A2b to B1 during the years 2019, 2020, and 2021. CONCLUSION: Compared with the years before the COVID-19 pandemic, in 2021, hMPV mostly affected young children, and the most prevalent circulating subtype shifted from A2b in 2019 to B1.

The impact of SARS-CoV-2 on respiratory syndromic and sentinel surveillance in Israel, 2020: a new perspective on established systems.

BackgroundThe COVID-19 pandemic presented new challenges for the existing respiratory surveillance systems, and adaptations were implemented. Systematic assessment of the syndromic and sentinel surveillance platforms during the pandemic is essential for understanding the value of each platform in the context of an emerging pathogen with rapid global spread.AimWe aimed to evaluate systematically the performance of various respiratory syndromic surveillance platforms and the sentinel surveillance system in Israel from 1 January to 31 December 2020.MethodsWe compared the 2020 syndromic surveillance trends to those of the previous 3 years, using Poisson regression adjusted for overdispersion. To assess the performance of the sentinel clinic system as compared with the national SARS-CoV-2 repository, a cubic spline with 7 knots and 95% confidence intervals were applied to the sentinel network's weekly percentage of positive SARS-CoV-2 cases.ResultsSyndromic surveillance trends changed substantially during 2020, with a statistically significant reduction in the rates of visits to physicians and emergency departments to below previous years' levels. Morbidity patterns of the syndromic surveillance platforms were inconsistent with the progress of the pandemic, while the sentinel surveillance platform was found to reflect the national circulation of SARS-CoV-2 in the population.ConclusionOur findings reveal the robustness of the sentinel clinics platform for the surveillance of the main respiratory viruses during the pandemic and possibly beyond. The robustness of the sentinel clinics platform during 2020 supports its use in locations with insufficient resources for widespread testing of respiratory viruses.

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.

First reported nosocomial SARS-CoV-2 outbreak in a hospital-based laundry facility.

Nosocomial severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreaks among health care workers have been scarcely reported so far. This report presents the results of an epidemiologic and molecular investigation of a SARS-CoV-2 outbreak among laundromat facility workers in a large tertiary centre in Israel. Following the first three reported cases of SARS-CoV-2 among laundromat workers, all 49 laundromat personnel were screened by qRT-PCR tests using naso- and oropharingeal swabs. Epidemiologic investigations included questionnaires, interviews and observations of the laundromat facility. Eleven viral RNA samples were then sequenced, and a phylogenetic analysis was performed using MEGAX.The integrated investigation defined three genetic clusters and helped identify the index cases and the assumed routes of transmission. It was then deduced that shared commute and public showers played a role in SARS-CoV-2 transmission in this outbreak, in addition to improper PPE use and social gatherings (such as social eating and drinking). In this study, we present an integrated epidemiologic and molecular investigation may help detect the routes of SARS-CoV-2 transmission, emphasising such routes that are less frequently discussed. Our work reinforces the notion that person-to-person transmission is more likely to cause infections than environmental contamination (e.g. from handling dirty laundry).

HiSpike Method for High-Throughput Cost Effective Sequencing of the SARS-CoV-2 Spike Gene

The changing nature of the SARS-CoV-2 pandemic poses unprecedented challenges to the world's health systems. Emerging spike gene variants jeopardize global efforts to produce immunity and reduce morbidity and mortality. These challenges require effective real-time genomic surveillance solutions that the medical community can quickly adopt. The SARS-CoV-2 spike protein mediates host receptor recognition and entry into the cell and is susceptible to generation of variants with increased transmissibility and pathogenicity. The spike protein is the primary target of neutralizing antibodies in COVID-19 patients and the most common antigen for induction of effective vaccine immunity. Tight monitoring of spike protein gene variants is key to mitigating COVID-19 spread and generation of vaccine escape mutants. Currently, SARS-CoV-2 sequencing methods are labor intensive and expensive. When sequence demands are high sequencing resources are quickly exhausted. Consequently, most SARS-CoV-2 strains are sequenced in only a few developed countries and rarely in developing regions. This poses the risk that undetected, dangerous variants will emerge. In this work, we present HiSpike, a method for high-throughput cost effective targeted next generation sequencing of the spike gene. This simple three-step method can be completed in < 30 h, can sequence 10-fold more samples compared to conventional methods and at a fraction of their cost. HiSpike has been validated in Israel, and has identified multiple spike variants from real-time field samples including Alpha, Beta, Delta and the emerging Omicron variants. HiSpike provides affordable sequencing options to help laboratories conserve resources for widespread high-throughput, near real-time monitoring of spike gene variants.

Covid-19 Breakthrough Infections in Vaccinated Health Care Workers.

BACKGROUND: Despite the high efficacy of the BNT162b2 messenger RNA vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), rare breakthrough infections have been reported, including infections among health care workers. Data are needed to characterize these infections and define correlates of breakthrough and infectivity. METHODS: At the largest medical center in Israel, we identified breakthrough infections by performing extensive evaluations of health care workers who were symptomatic (including mild symptoms) or had known infection exposure. These evaluations included epidemiologic investigations, repeat reverse-transcriptase-polymerase-chain-reaction (RT-PCR) assays, antigen-detecting rapid diagnostic testing (Ag-RDT), serologic assays, and genomic sequencing. Correlates of breakthrough infection were assessed in a case-control analysis. We matched patients with breakthrough infection who had antibody titers obtained within a week before SARS-CoV-2 detection (peri-infection period) with four to five uninfected controls and used generalized estimating equations to predict the geometric mean titers among cases and controls and the ratio between the titers in the two groups. We also assessed the correlation between neutralizing antibody titers and N gene cycle threshold (Ct) values with respect to infectivity. RESULTS: Among 1497 fully vaccinated health care workers for whom RT-PCR data were available, 39 SARS-CoV-2 breakthrough infections were documented. Neutralizing antibody titers in case patients during the peri-infection period were lower than those in matched uninfected controls (case-to-control ratio, 0.361; 95% confidence interval, 0.165 to 0.787). Higher peri-infection neutralizing antibody titers were associated with lower infectivity (higher Ct values). Most breakthrough cases were mild or asymptomatic, although 19% had persistent symptoms (>6 weeks). The B.1.1.7 (alpha) variant was found in 85% of samples tested. A total of 74% of case patients had a high viral load (Ct value, <30) at some point during their infection; however, of these patients, only 17 (59%) had a positive result on concurrent Ag-RDT. No secondary infections were documented. CONCLUSIONS: Among fully vaccinated health care workers, the occurrence of breakthrough infections with SARS-CoV-2 was correlated with neutralizing antibody titers during the peri-infection period. Most breakthrough infections were mild or asymptomatic, although persistent symptoms did occur.

The SARS-CoV-2 Lambda variant and its neutralisation efficiency following vaccination with Comirnaty, Israel, April to June 2021.

The SARS-CoV-2 Lambda (Pango lineage designation C.37) variant of interest, initially identified in Peru, has spread to additional countries. First detected in Israel in April 2021 following importations from Argentina and several European countries, the Lambda variant infected 18 individuals belonging to two main transmission chains without further spread. Micro-neutralisation assays following Comirnaty (BNT162b2 mRNA, BioNTech-Pfizer) vaccination demonstrated a significant 1.6-fold reduction in neutralising titres compared with the wild type virus, suggesting increased susceptibility of vaccinated individuals to infection.

Genomic variation and epidemiology of SARS-CoV-2 importation and early circulation in Israel.

Severe acute respiratory disease coronavirus 2 (SARS-CoV-2) which causes corona virus disease (COVID-19) was first identified in Wuhan, China in December 2019 and has since led to a global pandemic. Importations of SARS-CoV-2 to Israel in late February from multiple countries initiated a rapid outbreak across the country. In this study, SARS-CoV-2 whole genomes were sequenced from 59 imported samples with a recorded country of importation and 101 early circulating samples in February to mid-March 2020 and analyzed to infer clades and mutational patterns with additional sequences identified Israel available in public databases. Recorded importations in February to mid-March, mostly from Europe, led to multiple transmissions in all districts in Israel. Although all SARS-CoV-2 defined clades were imported, clade 20C became the dominating clade in the circulating samples. Identification of novel, frequently altered mutated positions correlating with clade-defining positions provide data for surveillance of this evolving pandemic and spread of specific clades of this virus. SARS-CoV-2 continues to spread and mutate in Israel and across the globe. With economy and travel resuming, surveillance of clades and accumulating mutations is crucial for understanding its evolution and spread patterns and may aid in decision making concerning public health issues.

Early effectiveness of BNT162b2 Covid-19 vaccine in preventing SARS-CoV-2 infection in healthcare personnel in six Israeli hospitals (CoVEHPI).

BACKGROUND: Methodologically rigorous studies on Covid-19 vaccine effectiveness (VE) in preventing SARS-CoV-2 infection are critically needed to inform national and global policy on Covid-19 vaccine use. In Israel, healthcare personnel (HCP) were initially prioritized for Covid-19 vaccination, creating an ideal setting to evaluate early real-world VE in a closely monitored population. METHODS: We conducted a prospective study among HCP in 6 hospitals to estimate the effectiveness of the BNT162b2 mRNA Covid-19 vaccine in preventing SARS-CoV-2 infection. Participants filled out weekly symptom questionnaires, provided weekly nasal specimens, and three serology samples - at enrollment, 30 days and 90 days. We estimated VE against PCR-confirmed SARS-CoV-2 infection using the Cox Proportional Hazards model and against a combined PCR/serology endpoint using Fisher's exact test. RESULTS: Of the 1567 HCP enrolled between December 27, 2020 and February 15, 2021, 1250 previously uninfected participants were included in the primary analysis; 998 (79.8%) were vaccinated with their first dose prior to or at enrollment, all with Pfizer BNT162b2 mRNA vaccine. There were four PCR-positive events among vaccinated participants, and nine among unvaccinated participants. Adjusted two-dose VE against any PCR-confirmed infection was 94.5% (95% CI: 82.6%-98.2%); adjusted two-dose VE against a combined endpoint of PCR and seroconversion for a 60-day follow-up period was 94.5% (95% CI: 63.0%-99.0%). Five PCR-positive samples from study participants were sequenced; all were alpha variant. CONCLUSIONS: Our prospective VE study of HCP in Israel with rigorous weekly surveillance found very high VE for two doses of Pfizer BNT162b2 mRNA vaccine against SARS-CoV-2 infection in recently vaccinated HCP during a period of predominant alpha variant circulation. FUNDING: Clalit Health Services.

Effectiveness of BNT162b2 mRNA COVID-19 vaccine against SARS-CoV-2 variant Beta (B.1.351) among persons identified through contact tracing in Israel: A prospective cohort study.

BACKGROUND: SARS-CoV-2 variant Beta (B.1.351) was designated as a Variant of Concern (VoC) after becoming the dominant strain in South Africa and spreading internationally. BNT162b2 showed lower levels of neutralizing antibodies against Beta than against other strains raising concerns about effectiveness of vaccines against infections caused by Beta. We estimated BNT162b2 vaccine effectiveness (VE) against Beta infections in Israel, a country with high vaccine uptake. METHODS: The Ministry of Health (MoH) identified Beta cases through mandatory reporting of SARS-CoV-2 cases and whole genome sequencing (WGS) of specimens from vaccination-breakthrough infections, reinfections, arriving international travelers, and a selection of other infected persons. A cohort analysis was conducted of exposure events of contacts of primary Beta cases. WGS was conducted on available PCR-positive specimens collected from contacts. VE estimates with 95% confidence intervals (CIs) against confirmed and probable Beta infections were determined by comparing infection risk between unvaccinated and fully-vaccinated (≥7 days after the second dose) contacts, and between unvaccinated and partially-vaccinated (<7 days after the second dose) contacts. FINDINGS: MoH identified 310 Beta cases through Jun 27, 2021. During the study period (Dec 11, 2020 - Mar 25, 2021), 164 non-institutionalized primary Beta cases, with 552 contacts aged ≥16 years, were identified. 343/552 (62%) contacts were interviewed and tested. 71/343 (21%) contacts were PCR-positive. WGS was performed on 7/71 (10%) PCR-positive specimens; all were Beta. Among SARS-CoV-2-infected contacts, 48/71 (68%) were symptomatic, 10/71 (14%) hospitalized, and 2/71 (3%) died. Fully-vaccinated VE against confirmed or probable Beta infections was 72% (95% CI -5 - 97%; p=0·04) and against symptomatic confirmed or probable Beta infections was 100% (95% CI 19 - 100%; p=0·01). There was no evidence of protection in partially-vaccinated contacts. INTERPRETATION: In a prospective observational study, two doses of BNT162b2 were effective against confirmed and probable Beta infections. Through the end of June 2021, introductions of Beta did not interrupt control of the pandemic in Israel. FUNDING: Israel Ministry of Health and Pfizer.

BNT162b2 vaccine effectiveness was marginally affected by the SARS-CoV-2 beta variant in fully vaccinated individuals.

OBJECTIVE: To evaluate the effectiveness of the Pfizer BNT162b2 vaccine against the SARS-Cov-2 Beta variant. STUDY DESIGN AND SETTING: Israel's mass vaccination program, using two doses of the Pfizer BNT162b2 vaccine, successfully curtailed the Alpha variant outbreak during winter 2020-2021, However, the virus may mutate and partially evade the immune system. To monitor this, sequencing of selected positive swab samples of interest was initiated. Comparing vaccinated with unvaccinated PCR positive persons, we estimated the odds ratio for a vaccinated case to have the Beta vs. the Alpha variant, using logistic regression, controlling for important confounders. RESULTS: There were 19 cases of Beta variant (3.2%) among those vaccinated more than 14 days before the positive sample and 79 (3.4%) among the unvaccinated. The estimated odds ratio was 1.26 (95% CI: 0.65-2.46). Assuming the effectiveness against the Alpha variant to be 95%, the estimated effectiveness against the Beta variant was 94% (95% CI: 88%-98%). CONCLUSION: Despite concerns over the Beta variant, the BNT162b2 vaccine seemed to provide substantial immunity against both the Beta and the Alpha variants. From 14 days following the second vaccine dose, the effectiveness of BNT162b2 vaccine was at most marginally affected by the Beta variant.

Rapid and High-Throughput Reverse Transcriptase Quantitative PCR (RT-qPCR) Assay for Identification and Differentiation between SARS-CoV-2 Variants B.1.1.7 and B.1.351.

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 decision making and to provide valuable data for epidemiological and policy decision making. We developed a multiplex reverse transcriptase quantitative PCR (RT-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, one that detects the 242 to 244 deletion, which is specific to variant B.1.351, and the fourth reaction, which identifies the 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 assay's specificity was tested against a panel of respiratory pathogens (n = 16), showing high specificity toward SC-2 RNA. The assay's 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. IMPORTANCE This study describes the design and utilization of a multiplex reverse transcriptase quantitative PCR (RT-qPCR) to identify SARS-COV-2 (SC2) RNA in general and, specifically, to detect whether it is of lineage B.1.1.7 or B.1.351. Implementation of this method in diagnostic and research laboratories worldwide may help the efforts to contain the COVID-19 pandemic. The method can be easily scaled up and be used in high-throughput laboratories, as well as small ones. In addition to immediate help in diagnostic efforts, this method may also help in epidemiological studies focused on the spread of emerging SC-2 lineages.