SARS-CoV-2 N Gene G29195T Point Mutation May Affect Diagnostic Reverse Transcription-PCR Detection

Accurate diagnostic detection of SARS-CoV-2 currently depends on the large-scale deployment of RT-PCR assays. SARS-CoV-2 RT-PCR assays target predetermined regions in the viral genomes by complementary binding of primers and probes to nucleic acid sequences in the clinical samples.

Replacement of the Gamma by the Delta variant in Brazil: impact of lineage displacement on the ongoing pandemic

The COVID-19 epidemic in Brazil was driven mainly by the spread of Gamma (P.1), a locally emerged Variant of Concern (VOC) that was first detected in early January 2021. This variant was estimated to be responsible for more than 96% of cases reported between January and June 2021, being associated with increased transmissibility and disease severity, a reduction in neutralization antibodies and effectiveness of treatments or vaccines, as well as diagnostic detection failure. Here we show that, following several importations predominantly from the USA, the Delta variant rapidly replaced Gamma after July 2021. However, in contrast to what was seen in other countries, the rapid spread of Delta did not lead to a large increase in the number of cases and deaths reported in Brazil. We suggest that this was likely due to the relatively successful early vaccination campaign coupled with natural immunity acquired following prior infection with Gamma. Our data reinforces reports of the increased transmissibility of the Delta variant and, considering the increasing concern due to the recently identified Omicron variant, argues for the necessity to strengthen genomic monitoring on a national level to quickly detect and curb the emergence and spread of other VOCs that might threaten global health.

A Strategy to Detect Emerging Non-Delta SARS-CoV-2 Variants with a Monoclonal Antibody Specific for the N501 Spike Residue

Efforts to control SARS-CoV-2 have been challenged by the emergence of variant strains that have important implications for clinical and epidemiological decision making. Four variants of concern (VOCs) have been designated by the Centers for Disease Control and Prevention (CDC), namely, B.1.617.2 (delta), B.1.1.7 (alpha), B.1.351 (beta), and P.1 (gamma), although the last three have been downgraded to variants being monitored (VBMs). VOCs and VBMs have shown increased transmissibility and/or disease severity, resistance to convalescent SARS-CoV-2 immunity and antibody therapeutics, and the potential to evade diagnostic detection. Methods are needed for point-of-care (POC) testing to rapidly identify these variants, protect vulnerable populations, and improve surveillance. Antigen-detection rapid diagnostic tests (Ag-RDTs) are ideal for POC use, but Ag-RDTs that recognize specific variants have not yet been implemented. Here, we describe a mAb (2E8) that is specific for the SARS-CoV-2 spike protein N501 residue. The 2E8 mAb can distinguish the delta VOC from variants with the N501Y meta-signature, which is characterized by convergent mutations that contribute to increased virulence and evasion of host immunity. Among the N501Y-containing mutants formerly designated as VOCs (alpha, beta, and gamma), a previously described mAb, CB6, can distinguish beta from alpha and gamma. When used in a sandwich ELISA, these mAbs sort these important SARS-CoV-2 variants into three diagnostic categories, namely, (1) delta, (2) alpha or gamma, and (3) beta. As delta is currently the predominant variant globally, they will be useful for POC testing to identify N501Y meta-signature variants, protect individuals in high-risk settings, and help detect epidemiological shifts among SARS-CoV-2 variants.

Distribution of cycle threshold values in RT-qPCR tests during the autumn 2020 peak of the COVID-19 pandemic in the Czech Republic

Reverse-transcription quantitative PCR (RT-qPCR) is currently the most sensitive method to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19). We analysed 1927 samples collected in a local public hospital during the autumn 2020 peak of the pandemic in the Czech Republic. The tests were performed using the Seegene Allplex 2019-nCov assay, which simultaneously detects three SARS-CoV-2 genes. In all samples analysed, 44.5 % were negative for all three genes, and 37.6 % were undoubtedly positive, with all three viral genes being amplified. A high degree of correlation between C t values among the genes confirmed the internal consistency of testing. Most of the positive samples were detected between the 15th and 35th cycles. We also registered a small number of samples with only one (13.2 %) or two (4.7 %) amplified genes, which may have originated from either freshly infected or already recovering patients. In addition, we did not detect any potentially false-positive samples from low-prevalence settings. Our results document that PCR testing represents a reliable and robust method for routine diagnostic detection of SARS-CoV-2.

Improved SARS-CoV-2 PCR detection and genotyping with double-bubble primers

A new approach for improved RT-PCR is described. It is based on primers designed to form controlled stem–loop and homodimer configurations, hence the name ‘double-bubble’ primers. The primers contain three main regions for efficient RT-PCR: a 3′ short overhang to allow reverse transcription, a stem region for hot start and a template-specific region for PCR amplification. As proof of principle, GAPDH, SARS-CoV-2 synthetic RNA and SARS-CoV-2 virus-positive nasopharyngeal swabs were used as templates. Additionally, these primers were used to positively confirm the N501Y mutation from nasopharyngeal swabs. Evidence is presented that the double-bubble primers offer fast, specific, robust and cost-effective improvement in RT-PCR amplification for detection of gene expression in general and for diagnostic detection and genotyping of SARS-CoV-2 in particular.

Characterization of Campylobacter spp. Strains Isolated From Wild Birds in Turkey

Turkey is an important stopover site for many migrating birds between Europe, Asia and Africa. Campylobacter spp. are frequently found in wildlife, in particular waterfowl, and distinct strains are disseminated within this reservoir. In this study, 183 wild birds of hunting areas in Turkey were collected and thermophilic Campylobacter spp. from cloacal swabs were isolated at a prevalence of 5.2% from song thrushes (6/116) and 93% from Eurasian coots (41/44). After PCR species differentiation and flaA restriction profiles determination, C. jejuni and C. coli strains were further investigated by whole genome sequencing. PCR target amplification of the ceuE gene, commonly used for C. coli species-identification was inefficient and even hampered in one isolate. A close look on the ceuE sequence revealed that various mismatches in the ceuE oligo annealing sites caused less efficient diagnostic detection. All C. coli isolates belonged to the environmental clade II and clade III, for which thirty-six novel MLST types were identified. Further single nucleotide polymorphism (SNP) analysis showed a high genomic divergence between the C. coli isolates. High variability was also implicated for putative plasmid-located genes detected in 51% of the C. coli isolates. Distinct gene variants in clades II and III C. coli were identified by a k-mer analysis. After substracting k-mers in common with C. coli clade I database, 11 and 35 distinct genes were identified in clades II and III isolates, mainly involved in surface structures and modifications as well as signal transduction, suggesting niche adaptation of C. coli strains in wild birds. All strains were susceptible against (fluoro-)quinolones, erythromycin, tetracycline, gentamicin and only one isolate was resistant against streptomycin, suggesting that the sensitive phenotype was due to absence of selective pressure and niche separation in wild birds in Turkey. We conclude that Campylobacter spp. isolates from wildlife and environmental sources are still scarce in the databases and that there is a need for more studies on thermophilic Campylobacter spp. from different places all over the world in order to complement our understanding on dissemination and adaptation to distinct niches of this global food-borne pathogen.

DeepSARS: simultaneous diagnostic detection and genomic surveillance of SARS-CoV-2

The continued spread of SARS-CoV-2 and emergence of new variants with higher transmission rates and/or partial resistance to vaccines has further highlighted the need for large-scale testing and genomic surveillance. However, current diagnostic testing (e.g., PCR) and genomic surveillance methods (e.g., whole genome sequencing) are performed separately, thus limiting the detection and tracing of SARS-CoV-2 and emerging variants. Here, we developed DeepSARS, a high-throughput platform for simultaneous diagnostic detection and genomic surveillance of SARS-CoV-2 by the integration of molecular barcoding, targeted deep sequencing, and computational phylogenetics. DeepSARS enables highly sensitive viral detection, while also capturing genomic diversity and viral evolution. We show that DeepSARS can be rapidly adapted for identification of emerging variants, such as alpha, beta, gamma, and delta strains, and profile mutational changes at the population level. DeepSARS sets the foundation for quantitative diagnostics that capture viral evolution and diversity.Abstract FigureGraphical abstractDeepSARS uses molecular barcodes (BCs) and multiplexed targeted deep sequencing (NGS) to enable simultaneous diagnostic detection and genomic surveillance of SARS-CoV-2.

Extracellular Vesicles and Pancreatic Cancer: Insights on the Roles of miRNA, lncRNA, and Protein Cargos in Cancer Progression

Pancreatic cancer (PC) is among the most devastating digestive tract cancers worldwide. This cancer is characterized by poor diagnostic detection, lack of therapy, and difficulty in predicting tumorigenesis progression. Although mutations of key oncogenes and oncosuppressor involved in tumor growth and in immunosurveillance escape are known, the underlying mechanisms that orchestrate PC initiation and progression are poorly understood or still under debate. In recent years, the attention of many researchers has been concentrated on the role of extracellular vesicles and of a particular subset of extracellular vesicles, known as exosomes. Literature data report that these nanovesicles are able to deliver their cargos to recipient cells playing key roles in the pathogenesis and progression of many pancreatic precancerous conditions. In this review, we have summarized and discussed principal cargos of extracellular vesicles characterized in PC, such as miRNAs, lncRNAs, and several proteins, to offer a systematic overview of their function in PC progression. The study of extracellular vesicles is allowing to understand that investigation of their secretion and analysis of their content might represent a new and potential diagnostic and prognostic tools for PC.