Nirmatrelvir Resistance-de Novo E166V/L50V Mutations in an Immunocompromised Patient Treated With Prolonged Nirmatrelvir/Ritonavir Monotherapy Leading to Clinical and Virological Treatment Failure-a Case Report.

Resistance of SARS-CoV-2 to antivirals was shown to develop in immunocompromised individuals receiving remdesivir. We describe an immunocompromised patient who was treated with repeated and prolonged courses of nirmatrelvir and developed de-novo E166V/L50F mutations in the Mpro region. These mutations were associated with clinical and virological treatment failure.

Rapid molecular epidemiology investigations into two recent measles outbreaks in Israel detected from October 2023 to January 2024.

Between late 2023 and early 2024, two measles outbreaks occurred in Israel, each caused by importation of measles virus strains of respective B3 and D8 genotypes. In this study, we validate transmission pathways uncovered by epidemiological investigations using a rapid molecular approach, based on complete measles virus genomes. The presented findings support this rapid molecular approach in complementing conventional contact tracing and highlight its potential for informing public health interventions.

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.

Emergence of genetically linked vaccine-originated poliovirus type 2 in the absence of oral polio vaccine, Jerusalem, April to July 2022.

We report an emergence and increase in poliovirus type 2 detection via routine wastewater surveillance in three non-overlapping regions in the Jerusalem region, Israel, between April and July 2022. Sequencing showed genetic linkage among isolates and accumulation of mutations over time, with two isolates defined as vaccine-derived polioviruses (VDPV). This demonstrates the emergence and potential circulation of type 2 VDPV in a high-income country with high vaccine coverage and underscores the importance of routine wastewater surveillance during the polio eradication.

Nosocomial outbreak caused by the SARS-CoV-2 Delta variant in a highly vaccinated population, Israel, July 2021.

A nosocomial outbreak of SARS-CoV-2 Delta variant infected 42 patients, staff and family members; 39 were fully vaccinated. The attack rate was 10.6% (16/151) among exposed staff and reached 23.7% (23/97) among exposed patients in a highly vaccinated population, 16-26 weeks after vaccination (median: 25 weeks). All cases were linked and traced to one patient. Several transmissions occurred between individuals wearing face masks. Fourteen of 23 patients became severely sick or died, raising a question about possible waning immunity.

JC virus identified in a patient with persistent and severe West Nile virus disease.

West Nile virus is a notable cause of neuroinvasive disease, damage to the central nervous system, or even death. In this study, using metagenomics analysis and quantitative real-time PCR validation, we identified a JC virus infection in urine and cerebrospinal fluid samples of a West Nile virus patient with severe neurological symptoms and extended disease. JC virus is known to be involved in neurological complications, especially in immunocompromised individuals thus suggesting that the coinfection with JC virus is involved with the West Nile virus infection persistence and severe symptoms. JC virus was identified in urine samples from additional West Nile virus patients via quantitative real-time PCR, however, JC virus was not found in any cerebrospinal fluid samples of West Nile virus patients, suggesting that JC virus does not regularly infect the central nervous system of WNV patients. Overall, this study highlights the importance of identifying infection by opportunistic viruses in already-diagnosed patients and highlights the advantages of next-generation sequencing and metagenomics for viral diagnosis.

The V(H) repertoire and clonal diversification of B cells in inflammatory myopathies.

The contribution of antigen-driven B-cell adaptive immune responses within the inflamed muscle of inflammatory myopathies (IMs) is largely unknown. In this study, we investigated the immunoglobulin V(H) gene repertoire, somatic hypermutation, clonal diversification, and selection of infiltrating B cells in muscle biopsies from IM patients (dermatomyositis and polymyositis), to determine whether B cells and/or plasma cells contribute to the associated pathologies of these diseases. The data reveal that Ig V(H) gene repertoires of muscle-infiltrating B cells deviate from the normal V(H) gene repertoire in individual patients, and differ between different types of IMs. Analysis of somatic mutations revealed clonal diversification of muscle-infiltrating B cells and evidence for a chronic B-cell response within the inflamed muscle. We conclude that muscle-infiltrating B cells undergo selection, somatic hypermutation and clonal diversification in situ during antigen-driven immune responses in patients with IMs, providing insight into the contribution of B cells to the pathological mechanisms of these disorders.

A self-directed method for cell-type identification and separation of gene expression microarrays.

Gene expression analysis is generally performed on heterogeneous tissue samples consisting of multiple cell types. Current methods developed to separate heterogeneous gene expression rely on prior knowledge of the cell-type composition and/or signatures--these are not available in most public datasets. We present a novel method to identify the cell-type composition, signatures and proportions per sample without need for a-priori information. The method was successfully tested on controlled and semi-controlled datasets and performed as accurately as current methods that do require additional information. As such, this method enables the analysis of cell-type specific gene expression using existing large pools of publically available microarray datasets.

Parvovirus B19 Outbreak in Israel: Retrospective Molecular Analysis from 2010 to 2023.

This study presents an analysis of the epidemiological trends of parvovirus B19 (B19V) in Israel from 2010 to 2023, with particular emphasis on the outbreak in 2023. The analysis utilized molecular diagnostic data from individual patients obtained at the Central Virology Laboratory. Between 2010 and 2022, 8.5% of PCR-tested samples were positive for B19V, whereas in 2023, this percentage surged to 31% of PCR-tested samples. Throughout the study period, annual cycles consistently peaked in early spring/summer, with the most recent prominent outbreak occurring in 2016. Predominantly, diagnoses were made in children and women aged 20-39. Despite the notable surge in 2023, over 80% of positive cases continued to be observed in children and young women, with a decrease in cases during winter months. Furthermore, genotype 1a of the virus remained the predominant strain circulating during the outbreak. In light of these circumstances, consideration should be given to implementing screening measures, particularly among high-risk groups such as pregnant women.

Incursion of SARS-CoV-2 BA.2.86.1 variant into Israel: National-scale wastewater surveillance using a novel quantitative real-time PCR assay.

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.

Altered local and systemic immune profiles underlie lymph node metastasis in breast cancer patients.

Cancer-mediated immune dysfunction contributes to tumor progression and correlates with patient outcome. Metastasis to tumor draining lymph nodes (TDLNs) is an important step in breast cancer progression and is used to predict patient outcome and survival. Although lymph nodes are important immune organs, the role of immune cells in TDLNs has not been thoroughly investigated. We hypothesized that the host immune response in node negative (NN) patients is more intact and thereby can resist tumor invasion compared to node positive (NP) patients. As such, lymph node metastasis requires breakdown of the host immune response in addition to escape of cancer cells from the tumor. To investigate the immunological differences between NN and NP breast cancer patients, we purified and profiled immune cells from the three major compartments where cancer and immune cells interact: tumor, TDLNs and peripheral blood. Significant down-regulation of genes associated with immune-related pathways and up-regulation of genes associated with tumor-promoting pathways was consistently observed in NP patients' TDLNs compared to NN patients. Importantly, these signatures were seen even in NP patients' tumor-free TDLNs, suggesting that such immune changes are not driven solely by local tumor invasion. Furthermore, similar patterns were also observed in NP patients' tumor and blood immune cells, suggesting that immunological differences between NN and NP patients are systemic. Together, these findings suggest that alterations in overall immune function may underlie risk for LN metastasis in breast cancer patients.

Dengue Types 1 and 3 Identified in Travelers Returning from Kathmandu, Nepal, during the October 2022 Outbreak Are Related to Strains Recently Identified in India.

Phylogenetic analysis of dengue serotypes 1 and 3, which were diagnosed in travelers and Nepalese infected in Kathmandu during the October 2022 outbreak, revealed that both serotypes were clustered closest to the sequences sampled in India. This suggests both serotypes may have originated in India.

Environmental surveillance detected type 3 vaccine-derived polioviruses in increasing frequency at multiple sites prior to detection of a poliomyelitis case.

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.

Factors Associated with Virological Failure in First-Line Antiretroviral Therapy in Patients Diagnosed with HIV-1 between 2010 and 2018 in Israel.

Despite the progress in contemporary antiretroviral therapy (ART) and the continuous changes in treatment guidelines, virological failure (VF) is still an ongoing concern. The goal of this study was to assess factors related to VF after first-line ART. A longitudinal cohort retrospective study of individuals on first-line ART diagnosed with HIV-1 in 2010-2018 and followed-up for a median of two years was conducted. Demographics, baseline and longitudinal CD4 counts, treatment regimens, adherence and VF were recorded. The Cox proportional hazards regression and mixed models were used. A cohort of 1130 patients were included. Overall, 80% were males and 62% were Israeli-born individuals. Compared to individuals diagnosed in 2010-2014, when treatment was initiated according to CD4 levels, those diagnosed in 2015-2018 were older and had lower baseline CD4 counts. VF was recorded in 66 (5.8%) patients. Diagnosis with CD4 <200 cells/mmᶟ with AIDS-defining conditions (HR = 2.75, 95%CI:1.52-4.97, p < 0.001) and non-integrase strand transfer inhibitor regimens (non-INSTI, HR = 1.80, 95%CI:1.01-3.24, p = 0.047) increased VF risk. No impact of baseline resistance was observed. We concluded that the early detection of HIV-1 infection and usage of INSTI-based regimens are recommended to reduce VF.

Recurrent congenital cytomegalovirus infection in a sequential pregnancy with severe sequelae, and a possible association with prophylactic valacyclovir treatment: a case report.

Recurrent congenital cytomegalovirus infections in consecutive pregnancies are rarely reported. Due to the risk of fetal infection from preconception maternal infection, a 6-month interval after primary maternal infection is generally advised before a new conception. Recently, high-dose valacyclovir treatment was shown to prevent fetal infection in first trimester primary infections. We present a case of first trimester primary infection treated with high-dose valacyclovir but resulting in polymerase chain reaction-confirmed fetal infection. Cytomegalovirus-specific immunoglobulin G titers remained very low during treatment and rose only after cessation of antiviral treatment. Six months after primary seroconversion, in a sequential pregnancy, recurrent fetal infection was diagnosed and resulted in severe fetal sequella. Whole genome sequencing of both amniotic fluid isolates proved them to be identical. Both pregnancies were terminated. We hypothesize that valacyclovir treatment, although unsuccessful in preventing fetal infection, had delayed the adaptive maternal immune response and might have contributed to fetal infection during the sequential pregnancy. We suggest that a longer delay might be warranted after valacyclovir treatment and before a new conception.

HIV-1 Circulating Recombinant Forms (CRFs) and Unique Recombinant Forms (URFs) in Israel, 2010-2018.

Monitoring HIV-1 circulating recombinant forms (CRFs) and unique recombinant forms (URFs) is important for disease surveillance. Recombination may affect prevention efforts and interfere with the diagnosis and treatment of HIV-1 infection. Here, we characterized the epidemiology of HIV-1 CRFs and URFs in Israel. Partial pol sequences from treatment naïve patients diagnosed in 2010−2018 were assessed using the recombinant identification program (RIP), the recombinant detection program (RDP5), and using the maximum-likelihood phylogenetic method, using 410 reference sequences obtained from the Los Alamos database. CRFs and URFs were identified in 11% (213/1940) of all sequenced cases. The median age at diagnosis was 38 (30−47) years, 61% originated from Israel, and 82% were male. The most common were CRF02_AG (30.5%), CRF01_AE (16.9%), and the more complex forms CRF01_AE/CRF02_AG/A3 (10.8%) and B/F1 (7%). A significant increase in their overall proportion was observed in recent years (8.1% in 2010−2012, 20.3% in 2016−2018, p < 0.001). This increase was most prominent in individuals carrying CRF02_AG (2.5% in 2010−2015, 9.8% in 2016−2018, p < 0.001). Men who have sex with men (MSM) was the most common risk group; however, those infected with the secondary recombinant CRF02_AG/A6 were mainly injecting drug users (IDUs). The most common resistance mutations were K103N (5/213, 2.3%) and E138A (18/213, 8.5%) in the reverse transcriptase. Only E138A was more frequent in the recombinants compared with the classic subtypes and was significantly associated with a specific secondary CRF, CRF02_AG/A4. We concluded that CRFs and URFs were mainly detected in Israeli-born MSM and that an increase in the overall proportion of such HIV-1 sequences could be observed in more recent years.

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.

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.

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.

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.