Enteroviruses (EVs) cause a spectrum of illnesses ranging from mild to severe, including neurological complications. In this study, the molecular epidemiology, clinical impact, and seasonality of EVs were evaluated among hospitalized patients in a large tertiary medical center in Israel from 2016 to 2024. A total of 11,246 cerebrospinal fluid (CSF) samples and 5,744 stool samples were tested for EV RNA using RT-PCR, with 7.1% (n = 798) and 19.3% (n = 1,110) testing positive, respectively. Positive samples were sequenced for genotype identification based on partial VP1 sequences with 70% genotype identification. The most commonly detected subtypes in CSF samples were: E-18 (16.4%), E-5 (80/567, 14.1%), E-6 (67/567, 11.8%), E-30 (64/567, 11.3%), CVB5 (37/567, 6.5%), E-4 (29/567, 5.1%), and CVB2 (28/567, 4.9%). These subtypes were also detected in stool samples during similar time periods. Seasonal analyses showed the expected summer peaks with unexpected off-season outbreaks in 2016, 2017, and 2022-2024. Additionally, EV positivity rates declined significantly during the SARS-CoV-2 pandemic (p = 0.006) followed by a resurgence in 2022-2023. Phylogenic analysis revealed genomic shifts in CVB2, E-5, E-6, E-18, and E-30, whereas CVB5 and E-4 showed no significant variations. CVB2 infections were particularly associated with severe illness in infants, with 10.6% of all sequenced cases requiring ICU admission. These findings demonstrate dynamic changes in EV epidemiology in Israel, including shifts in seasonality, genotype variability, and the impact of SARS-CoV-2 on EV circulation, highlighting the importance of continued molecular surveillance for identifying emerging strains and better understand trends relevant to public health.

An outbreak of enteroviral meningitis occurred in Israel between August 2025 and May 2026. Echovirus type 17 was identified in 72 hospitalised patients, most of them infants (n = 44). Many patients (n = 45) were from densely populated Haredi (strictly Orthodox) communities. Clinical course was mild. Phylogenetic analysis suggests extensive local transmission. Echovirus 17 infections declined abruptly following the implementation of community restrictions imposed during a military conflict. This outbreak highlights the value of routine molecular enterovirus surveillance.

An ongoing measles outbreak in Israel, involving ≈3,200 cases and 16 deaths, threatens the country’s measles elimination status and reflects declining vaccination rates observed globally and within Israel. Epidemiologic investigations supported by sequencing suggest that a single importation triggered the outbreak, underscoring the critical role of rapid genomic surveillance in outbreak control.

Background. An increase in the number of circulating vaccine-derived poliovirus (cVDPV) outbreaks has been reported worldwide in recent years, necessitating improved surveillance and diagnostic tools. The current standard protocol for polio detection is lengthy, labor-intensive, and inefficient for direct wastewater testing. Methods. Here we describe a multiplex poliovirus 2 (PV2)-specific assay that directly detects PV2 RNA derived from Sabin-2 vaccine in wastewater and stool samples. The new PV2-specific assay was used for direct detection of PV2 in environmental and clinical samples. Results. In silico analysis suggested that the assay is specific for PV2 originating from Sabin-2 vaccine and identifies 98.3% of available sequences, including some with <95% similarity to the vaccine reference sequence. We show that the assay is specific for PV2 and does not identify 19 other enterovirus strains, including Sabin-1 and Sabin-3 RNA. The new assay enabled fast detection of PV2 in suspected clinical samples, which was subsequently confirmed by isolation and sequencing. By analyzing 183 concentrated wastewater samples, we further show that the new assay specifically and directly identifies PV2 RNA, in the presence of PV1, PV3, and other enterovirus RNA. Conclusions. The assay provides quantitative results, allowing continuous monitoring of viral load dynamics. Comparison of the standard isolation protocol and the new assay showed excellent agreement in terms of specificity and sensitivity. This specific and rapid assay may therefore be a valuable tool for monitoring cVDPV2 outbreaks in real time, and promote global efforts for eradication and containment of poliovirus circulation.

Vaccine-derived polio virus (VDPV) strains can lead to acute flaccid paralysis (AFP), posing a significant challenge to public health and the eradication of poliovirus. In early 2025, two distinct cVDPV2 strains were identified in wastewater samples from separate regions in Israel as part of ongoing poliovirus environmental surveillance. Each strain was genetically linked to different African-origin emergences actively circulating in Africa. The first strain, detected in January in central Israel, was linked to the NIE-ZAS-1 emergence, while the second strain, identified one month later in Jerusalem, was genetically related to the SUD-RED-1 emergence. Whole-genome sequencing (WGS) and phylogenetic analyses revealed unique mutation patterns and recombination events, contributing valuable data for tracking further international detections of these strains. These events underscore the global risk of poliovirus importation and highlight the critical role of environmental surveillance in early detection.

BACKGROUND:In 2024, Israel experienced its largest West Nile Virus (WNV) outbreak, reporting 934 cases. Diagnosis primarily relies on serological testing for IgM antibodies; however, cross-reactivity with other flaviviruses and prolonged IgM persistence complicate interpretation. Molecular testing is less utilized due to concerns about the short duration of viremia and potential false negatives. OBJECTIVES:To evaluate the diagnostic reliability and persistence of WNV molecular testing across different sample types compared to serological testing, leveraging the extensive sample collection during the 2024 outbreak in Israel. STUDY DESIGN:Samples from 919 WNV cases, including whole blood (WB), serum, urine, and cerebrospinal fluid (CSF) were evaluated. WNV RNA, IgM antibodies and IgG avidity were tested on all sample types, serum and CSF and serum, respectively. RESULTS:WNV RNA was detected in 91 % of WB samples, and observed up to 52 days post-symptom onset. Detection rates were lower in serum (82 %), urine (71 %), and CSF (53 %), with shorter detection windows. Viral concentrations were highest in urine, followed by WB with serum, and CSF, the lowest. IgM antibodies were present in 83 % of serum and 86 % of CSF samples. RNA detection rates were comparable between hospitals (91 %) and health maintenance organizations (HMOs) (92 %), but IgM positivity was significantly higher in hospitals (91 %) than in HMOs (69 %). CONCLUSIONS:Molecular testing on WB offers the highest sensitivity and longest detection window for WNV RNA. Molecular diagnosis enhances accuracy, facilitates earlier detection, and improves clinical and public health response strategies compared to serological diagnosis.

We report the emergence and evolution of a circulating vaccine-derived poliovirus type 1 (cVDPV1) outbreak in Israel, linked to a vaccine-associated paralytic poliomyelitis case. Whole genome sequencing revealed a strong genetic link between the Sabin-like poliovirus type 1 variant from the case and pre-VDPV1 and VDPV1 isolated from environmental samples collected in October 2024–April 2025, mostly in Jerusalem. Early detection was made possible by Israel’s robust environmental surveillance and advanced sequencing technologies, enabling a rapid public health response.

Background An increase in the number of circulating vaccine derived poliovirus (VDPV) outbreaks is reported worldwide in recent years, necessitating improved surveillance and diagnostic tools. The current standard protocol for polio detection is lengthy, labor-intensive, and inefficient for direct wastewater testing. Methods Here we describe a multiplex poliovirus-2 (PV2) specific assay that directly detects PV2 RNA derived from Sabin-2 vaccine in wastewater and stool samples. The new PV2-specific assay was used for direct detection of PV2 in environmental and clinical samples. Results In-silico analysis suggested that the assay is specific for PV2 originated from Sabin-2 vaccine and identifies 98.3% of available sequences, including some with <95% similarity to the vaccine reference sequence. We show that the assay is specific for PV2 and does not identify 19 other enterovirus strains, including sabin-1 and Sabin-3 RNA. The new assay enabled fast detection of PV2 in suspected clinical samples, which was subsequently confirmed by isolation and sequencing. By analyzing 183 concentrated wastewater samples, we further show that the new assay specifically and directly identifies PV2 RNA, in the presence of PV1, PV3, and other enteroviruses RNA. Conclusions The assay provides quantitative results, allowing continuous monitoring of viral load dynamics. Comparison of the standard isolation protocol and the new assay showed excellent agreement in terms of specificity and sensitivity. This specific and rapid assay may therefore be a valuable tool for monitoring cVDPV2 outbreaks in real time, and promote the global efforts for eradication and containment poliovirus circulation

BACKGROUND:Similarly to wild poliovirus, vaccine-derived poliovirus (VDPV) strains can cause acute flaccid paralysis, posing a considerable challenge to public health and the eradication of poliovirus. VDPV outbreaks, particularly VDPV type 2 (VDPV2), are increasing worldwide, including in high-income countries with high vaccine coverage. We aimed to conduct a comprehensive analysis of the molecular epidemiology of a widespread VDPV2 outbreak in Israel in 2022-23 using conventional polio identification techniques and whole-genome sequencing. METHODS:In this genomic epidemiology study, we monitored and identified poliovirus type 2 (PV2) through the surveillance of stool samples from individuals with acute flaccid paralysis and related contacts, as well as environmental surveillance of sewage samples. Environmental surveillance involved 15 routine surveillance sites and an additional 30 sites dedicated to monitoring this outbreak, covering approximately 70% of Israel's population between April 1, 2022, and June 30, 2023. Additionally, we performed phylogenetic and mutation analyses using whole-genome, next-generation sequencing of PV2 isolates to identify recombination events, characterise VDPV2 lineages according to the capsid region, and establish the geographical distribution and linkage of PV2 isolates. FINDINGS:We detected 256 genetically linked samples from environmental surveillance, as well as one case of acute flaccid paralysis and four positive contacts associated with the Sabin type 2 oral vaccine strain. Most affected locations showed a high-density population of Jewish Ultra-Orthodox communities. Through high-resolution genomic characterisation and phylogenetic analysis of 202 representative sequences with complete capsid coverage, including isolates from both environmental surveillance and the case of acute flaccid paralysis, a conclusive linkage was established among all detections, confirming them to be part of a single VDPV2 outbreak. This strategy enabled the characterisation of three distinct lineages and established connections between different locations in Israel, including linking the case of acute flaccid paralysis and nearby environmental surveillance detections from the northern region with detections in the geographically distant central region. INTERPRETATION:This study highlights the role of environmental surveillance in the early detection and monitoring of poliovirus circulation, enabling a prompt public health response involving enhanced surveillance and a catch-up campaign with inactivated polio vaccine. Whole-genome sequencing offered valuable insights into the origins of the outbreak, linkage across detections, and the geographical distribution of the virus, with higher resolution than would have been possible with the standard analysis of the VP1 gene alone. FUNDING:None.

Coxsackievirus B2 (CVB2) is a member of the enterovirus group known to induce a spectrum of illnesses, from mild to severe. In the summer of 2022, an unusual outbreak of enteroviral central nervous system (CNS) infections occurred that was attributed to CVB2. Cerebrospinal fluid (CSF) samples collected from patients in 2015-2022 were tested for enterovirus via RT-PCR, followed by Sanger sequencing for positive cases. CVB2 samples were further sequenced in the P1 region using NGS. A total of 30 CSF samples were identified as CVB2, with 60% of these cases occurring between June and August 2022. The 2022 CVB2 variants were associated with severe clinical symptoms, including encephalitis (50%) and ataxia (27.8%). Samples from 2015 to 2020 were also included due to the absence of these symptoms. Phylogenetic analysis revealed that CVB2 strains from 2019 to 2020 were also distinct from those obtained in 2022. Amino acid analysis of the capsid proteins VP1, VP2, and VP3 identified three unique substitutions with potential antigenic significance in the 2022 variant: S67A in VP2, and T93A and E274D in VP1. These substitutions were not present in earlier strains or reported in the literature, suggesting they may influence the virus's virulence. The clinical observations from this study highlight new patterns of CVB2 infection in the CNS that had not been previously observed. Additionally, it identifies unique genetic changes in the 2022 CVB2 variant that may account for the increased virulence seen in the 2022 outbreak. However, further analysis is required to validate this assumption.

BACKGROUND:Varicella zoster virus (VZV) is among the leading pathogens causing meningitis and encephalitis. While VZV-PCR-positive CSF is considered a gold-standard for diagnosis, it is not-uncommon to detect VZV-DNA in CSF of patients with other acute or chronic illness. Our goal was to determine the clinical relevance of VZV-PCR-positive CSF when investigating patients with neurological symptoms.METHODS:In this retrospective cohort from the largest hospital in Israel, we collected demographic, clinical and laboratory data of patients with VZV-PCR-positive CSF, analyzing the significance of various parameters.RESULTS:During a 5-years study, 125 patient-unique VZV-PCR-positive CSFs were recorded, in which only 9 alternative diagnoses were noted. The commonest symptoms were headache (N = 104, 83 %) and rash (N = 96, 76 %). PCR-cycle-threshold (Ct), a surrogate of viral burden, did not significantly vary across the clinical manifestations; however, patients with rash and Ct<35 were prone to develop stroke in the following year (N = 6, 7 %). Empiric nucleoside-analogue treatment was not associated with a better outcome compared to treatment administered upon a positive-PCR result.DISCUSSION:Our findings suggest that in patients with neurological symptoms, detection of VZV-DNA in CSF renders VZV the probable culprit. Nevertheless, a systematic evaluation of treatment and follow-up algorithms of patients with suspected or proved VZV meningitis and encephalitis is needed. The benefits of a prompt treatment should be weighed against the potential complications of nucleoside-analogue. Conversely, the propensity for stroke in patients with higher viral-burden, necessitates further studies assessing VZV causal role, directing additional workup, treatment and monitoring policy.

The emergence of the SARS-CoV-2 variant BA.2.86.1 raised a considerable concern, due to the large number of potentially virulent mutations. In this study, we developed a novel assay that specifically detects variant BA.2.86.1, and used it to screen environmental samples from wastewater treatment sites across Israel. By using a multiplex assay that included a general SARS-CoV-2 reaction, together with the BA.2.86.1-specific reaction and a control reaction, we quantified the absolute number of viral copies in each sample and its relative abundance, compared with the total copy number of circulating SARS-CoV-2. Evaluation of the new reactions showed that they are both sensitive and specific, detecting down to four copies per reaction, and maintain specificity in the presence of Omicron variants BA.1, 2 and 4 RNA. Examination of 279 samples from 30 wastewater collection sites during August -September 2023 showed that 35 samples (12.5%) were positive, from 18 sites. Quantitative analysis of the samples showed that the relative abundance of variant BA.2.86.1 with respect to the total viral load of SARS-CoV-2 was very low and consisted between 0.01 % and 0.6 % of the total SARS-CoV-2 circulation. This study demonstrates the importance of combining wastewater surveillance with the development of specialized diagnostic assays, when clinical testing is insufficient. This approach may be useful for timely response by public health authorities in future outbreaks.

Israel conducts routine environmental (15 sites) and acute flaccid paralysis (AFP) surveillance for poliovirus. During September 2021, increasing numbers of wastewater samples collected from more than one site in the Jerusalem region proved positive for ambiguous type 3 vaccine-derived poliovirus (aVDPV3), while environmental samples from remaining sampling sites were negative. In late February 2022, a VDPV3, genetically related to the Jerusalem environmental surveillance samples, was isolated from a stool sample collected from a non-immunodeficient, non-immunized child from Jerusalem who developed AFP, indicating that the aVDPV3s were circulating (cVDPV3s) rather than immunodeficiency-related VDPV3s (iVDPVs). In response to these isolations, the Israel Ministry of Health launched a catch-up immunization program.

Background: Enteroviruses (EV) comprise the single most common cause of aseptic meningitis with variable geographical and temporal epidemiology. While EV-PCR in CSF is considered a gold standard for diagnosis, it is not-uncommon to use stool EV as a surrogate. Our aim was to assess the clinical significance of EV-PCR-positive CSF and stool in the investigation of patients with neurological symptoms. Methods: In this retrospective study from Sheba Medical centre, the largest tertiary hospital in Israel, we collected demographic, clinical and laboratory data of patients with EV-PCR-positive between 2016 and 2020. A comparison between various combinations of EV-PCR-positive CSF and stool was conducted. Data regarding EV strain-type and cycle threshold (Ct) were crossed with clinical symptoms and temporal kinetics. Results: Between 2016-2020, 448 CSF samples with positive EV-PCR were recorded from unique patients, the vast majority of which were diagnosed with meningitis (98%, 443/448). Unlike the diverse strain types of EV background activity, meningitis-related EV showed a clear epidemic pattern. In comparison with the EV CSF+/ Stool+ group, the EV CSF-/Stool+ group had frequently more alternative pathogens detected and a higher stool Ct-value. Clinically, EV CSF-/Stool+ patients were less febrile and more lethargic and convulsive. Discussion: The comparison of the EV CSF+/Stool+ and CSF-/Stool+ groups suggests that putative diagnosis of EV meningitis is prudent in the febrile, non-lethargic non-convulsive patients with an EV-PCR-positive stool. Otherwise, the detection of stool EV only, in a non-epidemic setup, especially with a high Ct-value, may be incidental and mandate a continuous diagnostic effort for an alternative culprit.

Introduction: Inactivated polio virus (IPV) vaccinations are a mainstay of immunization schedules in developed countries, while oral polio vaccine (OPV) is administered in developing countries and is the main vaccine in outbreaks. Due to circulating wild poliovirus (WPV1) detection in Israel (2013), oral biva-lent polio vaccination (bOPV) was administered to IPV primed children and incorporated into the vacci-nation regimen.Objectives: We aimed to determine the extent and timeframe of fecal and salivary polio vaccine virus (Sabin strains) shedding following bOPV vaccination among IPV primed children.Methods: Fecal samples were collected from a convenience sample of infants and toddlers attending 11 Israeli daycare centers. Salivary samples were collected from infants and toddlers following bOPV vaccination.Results: 398 fecal samples were collected from 251 children (ages: 6-32 months), 168 received bOPV vaccination 4-55 days prior to sample collection. Fecal excretion continued among 80 %, 50 %, and 20 %, 2, 3, and 7 weeks following vaccination. There were no significant differences in the rate and duration of positive samples among children immunized with 3 or 4 IPV doses. Boys were 2.3-fold more likely to excrete the virus (p = 0.006). Salivary shedding of Sabin strains occurred in 1/47 (2 %) and 1/49 (2 %) sam-ples 4, and 6 days following vaccination respectively.Conclusions: Fecal detection of Sabin strains among IPV-primed children continues for 7 weeks; addi-tional doses of IPV do not augment intestinal immunity; limited salivary shedding occurs for up to a week. This data can enhance understanding of intestinal immunity achieved by different vaccination schedules and guide recommendations for contact precautions of children following bOPV vaccination.& COPY; 2023 Published by Elsevier Ltd.

BACKGROUND:Outbreaks of enteroviral meningitis occur periodically and may lead to hospitalization and severe disease.OBJECTIVE:To analyze and describe the meningitis outbreak in patients hospitalized in Israel in 2021-2022, during the COVID-19 pandemic.RESULTS:In December 2021, before the emergence of the SARS-CoV-2 omicron variant, an off-season increase in enterovirus (EV) infections was observed among patients hospitalized with meningitis. In January 2022, enterovirus cases decreased by 66% in parallel with the peak of the Omicron wave, and then increased rapidly by 78% in March (compared with February) after a decline in Omicron cases. Sequencing of the enterovirus-positive samples showed a dominance of echovirus 6 (E-6) (29%) before and after the Omicron wave. Phylogenetic analysis found that all 29 samples were very similar and all clustered in the E-6 C1 subtype. The main E-6 symptoms observed were fever and headache, along with vomiting and neck stiffness. The median patient age was 25 years, with a broad range (0-60 years).CONCLUSION:An upsurge in enterovirus cases was observed after the decline of the SARS-CoV-2 omicron wave. The dominant subtype was E-6, which was present prior to the emergence of the omicron variant, but increased rapidly only after the omicron wave decline. We hypothesize that the omicron wave delayed the rise in E-6-associated meningitis.

In this report, we describe the first national scale multi-laboratory evaluation of monkeypox virus (MPXV) DNA commercial PCR kits. The objective of this study was to evaluate 2 kits by different diagnostic laboratories across Israel. Ten standardized samples were tested simultaneously using the Novaplex (15 laboratories) and Bio-Speedy (seven laboratories) kits. An in-house assay based on previously published reactions was used as reference. Comparison of the results showed high intra-assay agreement between laboratories, with small variations for most samples. The in-house assay had an analytical detection limit of less than 10 copies per reaction. While the 2 commercial kits were able to detect specimens with low viral loads similarly to the in-house assay, significant differences were observed, in the Cq values and relative fluorescence (RF), between the assays. The RF signal of the in-house and Bio-Speedy assays ranged between 5,000 and 10,000 RFU, while the signal in the Novaplex assay was less than 600 RFU. Due to the kit measurement protocol, the Cq values of the Bio-Speedy kit were 5 to 7.5 cycles lower than those of the in-house assay. On the contrary, the Cq values of the Novaplex kit were significantly higher than those of the in-house assay, with differences of 3 to 5 cycles per sample. Our results suggest that while all assays were similar in their overall sensitivity, direct comparison of Cq values between them may be misleading. To our knowledge, this is the first methodical evaluation of commercial MPX test kits. We therefore anticipate that this study would help diagnostic laboratories in choosing a specific MPX detection assay. IMPORTANCE To the best of our knowledge, this study is the first methodical evaluation of commercial kits designed for Monkeypox virus detection. This was done by performing the same tests using the same sample set in multiple laboratories, simultaneously, on a national scale. It therefore provides important and unique information on the performance of such kits and provides a guideline for choosing the assay of choice for monkeypox virus diagnosis in a standard diagnostic laboratory. It also demonstrates potential complications when trying to compare the results of different assays, even when testing exactly the same samples, under identical conditions.

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.

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.

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.