Highly sensitive extraction-free saliva-based molecular assay for rapid diagnosis of SARS-CoV-2.

The COVID-19 pandemic highlighted the necessity of fast, sensitive, and efficient methods to test large populations for respiratory viruses. The "gold standard" molecular assays for detecting respiratory viruses, such as quantitative polymerase chain reaction (qPCR) and reverse transcription qPCR (RT-qPCR), rely on invasive swab samples and require time-consuming and labor-intensive extraction processes. Moreover, the turnaround time for RT-qPCR-based assays is too lengthy for rapid screening. Extraction-free saliva-based methods provide a non-invasive sampling process with a fast turnaround time and are suitable for high-throughput applications. However, when used with a standard RT-qPCR system, the absence of extraction significantly reduces the assays' sensitivity. Here, using a novel optical modulation biosensing (OMB) platform, we developed a rapid and highly sensitive extraction-free saliva-based molecular assay. We blindly tested 364 paired nasopharyngeal swabs and saliva samples from suspected SARS-CoV-2 cases in Israel. Compared with the gold standard swab-based RT-qPCR assay, the sensitivity of the extraction-free saliva-based OMB assay is 90.7%, much higher than the sensitivity of extraction-free saliva-based RT-qPCR assay (77.8%) with similar specificity (95.3% and 97.6%, respectively). Moreover, out of 12 samples identified by the OMB-based assay as positive, 8 samples were collected from hospitalized patients in a COVID-19 ward and were verified to be SARS-CoV-2-positive upon admission, indicating that the actual clinical sensitivity and specificity of the OMB assay are higher. Considering its user-friendly saliva-based protocol, short and cost-effective extraction-free process, and high clinical accuracy, the OMB-based molecular assay is very suitable for high-throughput testing of large populations for respiratory viruses. IMPORTANCE: Three years after the SARS-CoV-2 outbreak, there are no molecular tests that combine low-cost and straightforward sample preparation, effective sample handling, minimal reagent and disposable requirements, high sensitivity, and high throughput required for mass screening. Existing rapid molecular techniques typically sacrifice certain requirements to meet others. Yet, localized outbreaks of novel viral diseases happen daily in different parts of the world. In this context, respiratory diseases are of specific importance, as they are frequently airborne and highly contagious, with the potential for a rapid global spread. The widely accepted opinion is that another pandemic is just a question of time. To ensure that the containment efforts for the upcoming "disease X" are successful, introducing rapid, high-throughput, and highly sensitive diagnostic methods for detecting and identifying pathogens is critical. A few months into the pandemic, saliva was suggested as a diagnostic matrix for SARS-CoV-2 detection. The collection of saliva does not require swabs and is minimally invasive. In particular, extraction-free saliva-based assays require fewer reagents and disposables, and therefore are faster and cheaper, offering an appealing alternative for low-income countries. Unfortunately, current extraction-free saliva-based detection methods, such as direct RT-qPCR or isothermal amplification, have either low sensitivity or low throughput. Therefore, we believe that the presented highly sensitive ht-OMBi platform and the extraction-free saliva-based molecular assay can become an essential tool in the infectious disease monitoring toolbox.

The Outbreak of Unexplained Acute Hepatitis in Children: The Role of Viral Infections in View of the COVID-19 Pandemic.

BACKGROUND AND AIMS: An increase in the number of cases of acute hepatitis of unknown origin (HUO) in children was observed in 2021. Adenovirus and adeno-associated virus 2 (AAV2) infections have been suggested as possible triggers. However, the potential etiology is still unclear. We aimed to characterize a cohort of children with HUO in Israel in view of the COVID-19 pandemic. METHOD: Demographics, clinical data, and laboratory results on the children compatible with the CDC criteria for HUO were collected by the established registry of the Ministry of Health. Available specimens were sent to the Central Virology Laboratory. RESULTS: A total of 39 children were included in the registry. A total of 20 were enrolled prospectively, in which human herpes virus 6 (HHV6) infection or reactivation was identified in 11/19, adenovirus was found in 4/19 of the cases, and AAV2 was detected in 2/16. Past COVID-19 exposure was recorded for 24/39 of the children. A total of 10 children underwent liver biopsy, and 8 were successfully treated with steroids and 2 underwent liver transplantation. CONCLUSIONS: The COVID-19 pandemic and the related containment measures combined with reactivation or active infection with other viruses could have been a trigger for the HUO outbreak. In our cohort, HHV6 was the most abundant finding.

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.

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.

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.

Proof-of-concept for effective antiviral activity of an in silico designed decoy synthetic mRNA against SARS-CoV-2 in the Vero E6 cell-based infection model.

The positive-sense single-stranded (ss) RNA viruses of the Betacoronavirus (beta-CoV) genus can spillover from mammals to humans and are an ongoing threat to global health and commerce, as demonstrated by the current zoonotic pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Current anti-viral strategies focus on vaccination or targeting key viral proteins with antibodies and drugs. However, the ongoing evolution of new variants that evade vaccination or may become drug-resistant is a major challenge. Thus, antiviral compounds that circumvent these obstacles are needed. Here we describe an innovative antiviral modality based on in silico designed fully synthetic mRNA that is replication incompetent in uninfected cells (termed herein PSCT: parasitic anti-SARS-CoV-2 transcript). The PSCT sequence was engineered to include key untranslated cis-acting regulatory RNA elements of the SARS-CoV-2 genome, so as to effectively compete for replication and packaging with the standard viral genome. Using the Vero E6 cell-culture based SARS-CoV-2 infection model, we determined that the intracellular delivery of liposome-encapsulated PSCT at 1 hour post infection significantly reduced intercellular SARS-CoV-2 replication and release into the extracellular milieu as compared to mock treatment. In summary, our findings are a proof-of-concept for the therapeutic feasibility of in silico designed mRNA compounds formulated to hinder the replication and packaging of ssRNA viruses sharing a comparable genomic-structure with beta-CoVs.

A Multi-Laboratory Evaluation of Commercial Monkeypox Virus Molecular Tests.

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.

Increased detection of Echovirus 6-associated meningitis in patients hospitalized during the COVID-19 pandemic, Israel 2021-2022.

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.

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.

Overlooked monkeypox cases among men having sex with men during the 2022 outbreak - a retrospective study.

OBJECTIVES: The aim of this study was to characterize overlooked cases of patients with monkeypox infection in the 2022 outbreak. METHODS: Clinical characteristics of 26 patients who were misdiagnosed with other diseases were described. RESULTS: Of the 26 patients who were misdiagnosed, six (23%) were given a diagnosis of bacterial tonsillitis, six (23%) were diagnosed with primary syphilis, five (19.2%) with oral or genital herpes, and four (15.3%) with bacterial proctitis or anal abscess. The average time interval between missed and right diagnosis was 4.4 days. There was no difference in the missed cases between the early and the later month of the outbreak. CONCLUSION: Monkeypox might still be commonly overlooked, especially in patients presenting with fever and sore throat or solitary ulcer as sole manifestations.

Detection and Analysis of West Nile Virus Structural Protein Genes in Animal or Bird Samples.

West Nile virus (WNV) is an important zoonotic pathogen, which is detected mainly by identification of its RNA using PCR. Genetic differentiation between WNV lineages is usually performed by complete genome sequencing, which is not available in many research and diagnostic laboratories. In this chapter, we describe a protocol for detection and analysis of WNV samples by sequencing the entire region of their structural genes capsid (C), preM/membrane, and envelope. The primary step is the detection of WNV RNA by quantitative PCR of the NS2A gene or the C gene regions. Next, the entire region containing the structural protein genes is amplified by PCR. The primary PCR product is then amplified again in parallel reactions, and these secondary PCR products are sequenced. Finally, bioinformatic analysis enables detection of mutations and classification of the samples of interest. This protocol is designed to be used by any laboratory equipped for endpoint and quantitative PCR. The sequencing can be performed either in-house or outsourced to a third-party service provider. This protocol may therefore be useful for rapid and affordable classification of WNV samples, obviating the need for complete genome sequencing.

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.

The effect of ivermectin on the viral load and culture viability in early treatment of nonhospitalized patients with mild COVID-19 - a double-blind, randomized placebo-controlled trial.

OBJECTIVES: Ivermectin, an antiparasitic agent, also has antiviral properties. In this study, we aimed to assess whether ivermectin has anti-SARS-CoV-2 activity. METHODS: In this double-blinded trial, we compared patients receiving ivermectin for 3 days versus placebo in nonhospitalized adult patients with COVID-19. A reverse transcriptase-polymerase chain reaction from a nasopharyngeal swab was obtained at recruitment and every 2 days for at least 6 days. The primary endpoint was a reduction of viral load on the sixth day as reflected by cycle threshold level >30 (noninfectious level). The primary outcome was supported by the determination of viral-culture viability. RESULTS: Of 867 patients screened, 89 were ultimately evaluated per-protocol (47 ivermectin and 42 placeboes). On day 6, the odds ratio (OR) was 2.62 (95% confidence interval [CI]: 1.09-6.31) in the ivermectin arm, reaching the endpoint. In a multivariable logistic regression model, the odds of a negative test on day 6 were 2.28 times higher in the ivermectin group but reached significance only on day 8 (OR 3.70; 95% CI: 1.19-11.49, P = 0.02). Culture viability on days 2 to 6 was positive in 13.0% (3/23) of ivermectin samples versus 48.2% (14/29) in the placebo group (P = 0.008). CONCLUSION: There were lower viral loads and less viable cultures in the ivermectin group, which shows its anti-SARS-CoV-2 activity. It could reduce transmission in these patients and encourage further studies with this drug.

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

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

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

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

Virus Infection in Equine.

The relationship between men and horses has significantly evolved over the last century [...].

HBV-RNA, Quantitative HBsAg, Levels of HBV in Peripheral Lymphocytes and HBV Mutation Profiles in Chronic Hepatitis B.

A comprehensive characterization of chronic HBV (CHB) patients is required to guide therapeutic decisions. The cumulative impact of classical and novel biomarkers on the clinical categorization of these patients has not been rigorously assessed. We determined plasma HBV-RNA and HBsAg levels, HBV in peripheral lymphocytes (PBMCs) and HBV mutation profiles in CHB patients. Patient demographics (n = 139) and classical HBV biomarkers were determined during a clinical routine. HBV-RNA in plasma and HBV-DNA in PBMCs were determined by RT-PCR. HBsAg levels were determined using Architect. In samples with HBV-DNA viral load >1000 IU/mL, genotype mutations in precore (PC), basal core promoter (BCP), HBsAg and Pol regions were determined by sequencing. Most patients (n = 126) were HBeAg-negative (HBeAgNeg) with significantly lower levels of HBV-RNA, HBV-DNA and HBsAg compared to HBeAg-positive (HBeAgPos) patients (p < 0.05). HBV genotype D prevailed (61/68), and >95% had BCP/PC mutations. Escape mutations were identified in 22.6% (13/63). HBeAgNeg patients with low levels of HBsAg (log IU ≤ 3) were older and were characterized by undetectable plasma HBV-DNA and undetectable HBV-RNA but not undetectable HBV-DNA in PBMCs compared to those with high HBsAg levels. In >50% of the studied HBeAgNeg patients (66/126), the quantitation of HBsAg and HBV-RNA may impact clinical decisions. In conclusion, the combined assessment of classical and novel serum biomarkers, especially in HBeAgNeg patients, which is the largest group of CHB patients in many regions, may assist in clinical decisions. Prospective studies are required to determine the real-time additive clinical advantage of these biomarkers.

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.

Effective bubble-based testing for SARS-CoV-2 using swab-pooling.

OBJECTIVES: Despite the success in developing COVID-19 vaccines, containment of the disease is obstructed worldwide by vaccine production bottlenecks, logistics hurdles, vaccine refusal, transmission through unvaccinated children, and the appearance of new viral variants. This underscores the need for effective strategies for identifying carriers/patients, which was the main aim of this study. METHODS: We present a bubble-based PCR testing approach using swab-pooling into lysis buffer. A bubble is a cluster of people who can be periodically tested for SARS-CoV-2 by swab-pooling. A positive test of a pool mandates quarantining each of its members, who are then individually tested while in isolation to identify the carrier(s) for further epidemiological contact tracing. RESULTS: We tested an overall sample of 25 831 individuals, divided into 1273 bubbles, with an average size of 20.3 ± 7.7 swabs/test tube, obtaining for all pools (≤37 swabs/pool) a specificity of 97.5% (lower bound 96.6%) and a sensitivity of 86.3% (lower bound 78.2%) and a post hoc analyzed sensitivity of 94.6% (lower bound 86.7%) and a specificity of 97.2% (lower bound 96.2%) in pools with ≤25 swabs, relative to individual testing. DISCUSSION: This approach offers a significant scale-up in sampling and testing throughput and savings in testing cost, without reducing sensitivity or affecting the standard PCR testing laboratory routine. It can be used in school classes, airplanes, hospitals, military units, and workplaces, and may be applicable to future pandemics.