A comparative study on the detection of Mycobacterium leprae DNA in urine samples of leprosy patients using Rlep-PCR with other conventional samples.

BACKGROUND: Mycobacterium leprae causes leprosy that is highly stigmatized and chronic infectious skin disease. Only some diagnostic tools are being used for the identification M. leprae in clinical samples, such as bacillary detection, and histopathological tests. These methods are invasive and often have low sensitivity. Currently, the PCR technique has been used as an effective tool fordetecting M. leprae DNA across different clinical samples. The current study aims to detect M. leprae DNA in urine samples of untreated and treated leprosy patients using the Rlep gene (129 bp) and compared the detection among Ridley-Jopling Classification. METHODS: Clinical samples (Blood, Urine, and Slit Skin Smears (SSS)) were collected from leprosy and Non-leprosy patients. DNA extraction was performed using standard laboratory protocol and Conventional PCR was carried out for all samples using Rlep gene target and the amplicons of urine samples were sequenced by Sanger sequencing to confirm the Rlep gene target. RESULTS: The M. leprae DNA was successfully detected in all clinical samples across all types of leprosy among all the study groups using RLEP-PCR. Rlep gene target was able to detect the presence of M. leprae DNA in 79.17% of urine, 58.33% of blood, and 50% of SSS samples of untreated Smear-Negative leprosy patients. The statistical significant difference (p = 0.004) was observed between BI Negative (Slit Skin Smear test) and RLEP PCR positivity in urine samples of untreated leprosy group. CONCLUSION: The PCR positivity using Rlep gene target (129 bp) was highest in all clinical samples among the study groups, across all types of leprosy. Untreated tuberculoid and PNL leprosy patients showed the highest PCR positivity in urine samples, indicating its potential as a non-invasive diagnostic tool for leprosy and even for contact screening.

Partial ORF1ab Gene Target Failure with Omicron BA.2.12.1.

Mutations in the genome of SARS-CoV-2 can affect the performance of molecular diagnostic assays. In some cases, such as S-gene target failure, the impact can serve as a unique indicator of a particular SARS-CoV-2 variant and provide a method for rapid detection. Here, we describe partial ORF1ab gene target failure (pOGTF) on the cobas SARS-CoV-2 assays, defined by a ≥2-thermocycle delay in detection of the ORF1ab gene compared to that of the E-gene. We demonstrate that pOGTF is 98.6% sensitive and 99.9% specific for SARS-CoV-2 lineage BA.2.12.1, an emerging variant in the United States with spike L452Q and S704L mutations that may affect transmission, infectivity, and/or immune evasion. Increasing rates of pOGTF closely mirrored rates of BA.2.12.1 sequences uploaded to public databases, and, importantly, increasing local rates of pOGTF also mirrored increasing overall test positivity. Use of pOGTF as a proxy for BA.2.12.1 provides faster tracking of the variant than whole-genome sequencing and can benefit laboratories without sequencing capabilities.

Alteration of circulating platelet-related and diabetes-related microRNAs in individuals with type 2 diabetes mellitus: a stepwise hypoglycaemic clamp study.

BACKGROUND: In patients with type 2 diabetes mellitus (T2DM) an association between severe hypoglycaemic episodes and the risk of cardiovascular (CV) morbidity and mortality has been previously established. METHODS: We aimed to investigate the influence of hypoglycaemia on several diabetes-related and platelet-related miRNAs selected based on bioinformatic analysis and literature search, including hsa-miR-16, hsa-miR-34a, hsa-miR-129-2, hsa-miR-15a, hsa-miR-15b, hsa-miR-106a, miR-223, miR-126. Selected miRNAs were validated by qRT-PCR in 14 patients with T2DM on metformin monotherapy, without established CV disease and antiplatelet therapy during a stepwise hypoglycaemic clamp experiment and a follow-up 7 days after the clamp event. In order to identify which pathways and phenotypes are associated with validated miRNAs we performed target prediction on genes expressed with high confidence in platelets. RESULTS: Circulating levels of miR-106a-5p, miR-15b, miR-15a, miR-16-5p, miR-223 and miR-126 were increased after euglycaemic clamp followed by hypoglycaemic clamp, each with its distinctive time trend. On the contrary, miR-129-2-3p, miR-92a-3p and miR-34a-3p remained unchanged. MiR-16-5p was negatively correlated with interleukin (IL)-6, intercellular adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM) (p = 0.002, p < 0.001, p = 0.016, respectively), whereas miR-126 was positively correlated with VCAM (p < 0.001). There were negative correlations between miR-16-5p, miR-126 and coagulation factors, including factor VIII and von Willebrand factor (vWF). Among all studied miRNAs, miR-126, miR-129-2-3p and miR-15b showed correlation with platelet function. Bioinformatic analysis of platelet-related targets of analyzed miRNAs showed strong enrichment of IL-2 signaling. We also observed significant enrichment of pathways and diseases related to cancer, CV diseases, hyperglycemia, and neurological diseases. CONCLUSIONS: Hypoglycaemia can significantly influence the expression of platelet-enriched miRNAs, with a time trend paralleling the time course of platelet activation. This suggests miRNAs could be exploited as biomarkers for platelet activation in response to hypoglycaemia, as they are probably released by platelets upon activation by hypoglycaemic episodes. Should they hold their promise in clinical endpoint studies, platelet-derived miRNAs might become helpful markers of CV risk in subjects with diabetes. Trial registration The study was registered at clinical trials.gov; Impact of Hypoglycaemia in Patients With DIAbetes Mellitus Type 2 on PLATElet Activation (Diaplate), trial number: NCT03460899.

The role of miRNAs in regulation of platelet activity and related diseases - a bioinformatic analysis.

MicroRNAs (miRNAs) are small, non-coding RNAs, able to regulate cellular functions by induction of mRNA degradation and post-transcriptional repression of gene expression. Platelets are the major source of circulating miRNAs, with significant regulatory potential on cardiovascular pathophysiology and other diseases. MiRNAs have been shown to modify the expression of platelet proteins, which influence the platelets reactivity. Circulating miRNAs can be determined from plasma, serum, or whole blood, and they can be used as diagnostic and prognostic biomarkers as well as therapeutic targets including cardiovascular diseases (CVDs). Herein, we present original results from bioinformatic analyses, which identified top 22 platelet-related miRNAs including hsa-miR-320a, hsa-miR-16-5p, hsa-miR-106a-5p, hsa-miR-320b, hsa-miR-15a-5p, hsa-miR-15b-5p, hsa-miR-195-5p, hsa-miR-92a-3p as widely involved in platelet reactivity and associated diseases, including CVDs, Alzheimer's and cerebrovascular diseases, cancer and hypertension. Analysis focused on the identification of the highly regulatory targets shared between those miRNAs identified 43 of them. Best ranked genes associated with overall platelet activity and most susceptible for noncoding regulation were PTEN, PIK3R1, CREB1, APP, and MAPK1. Top targets also strongly associated with CVDs were VEGFA, IGF1, ESR1, BDNF, and PPARG. Top targets associated with other platelet-related diseases including cancer identified in our study were TP53, KRAS, and CCND1. The most affected pathways by top miRNAs and top targets included diseases of signal transduction by Growth Factor Receptors (GDFRs) and second messengers, platelet activation, signaling, and aggregation, signaling by VEGF, MAPK family signaling cascades, and signaling by Interleukins. Terms specific only for platelet-related miRNAs included coronary artery disease, platelet degranulation, and neutrophil degranulation, while for the top platelet-related genes it was Estrogen Signaling Receptor (ESR) mediated signaling, extra-nuclear estrogen signaling, and endometriosis. Our results show the novel features of platelet physiology and may provide a basis for further clinical studies focused on platelet reactivity. They also show in which aspects miRNAs can be promising biomarkers of platelet-related pathological processes.

Enhancing epidemiological surveillance of the emergence of the SARS-CoV-2 Omicron variant using spike gene target failure data, England, 15 November to 31 December 2021.

When SARS-CoV-2 Omicron emerged in 2021, S gene target failure enabled differentiation between Omicron and the dominant Delta variant. In England, where S gene target surveillance (SGTS) was already established, this led to rapid identification (within ca 3 days of sample collection) of possible Omicron cases, alongside real-time surveillance and modelling of Omicron growth. SGTS was key to public health action (including case identification and incident management), and we share applied insights on how and when to use SGTS.

Current Scenario of Exogenously Induced RNAi for Lepidopteran Agricultural Pest Control: From dsRNA Design to Topical Application.

Invasive insects cost the global economy around USD 70 billion per year. Moreover, increasing agricultural insect pests raise concerns about global food security constraining and infestation rising after climate changes. Current agricultural pest management largely relies on plant breeding-with or without transgenes-and chemical pesticides. Both approaches face serious technological obsolescence in the field due to plant resistance breakdown or development of insecticide resistance. The need for new modes of action (MoA) for managing crop health is growing each year, driven by market demands to reduce economic losses and by consumer demand for phytosanitary measures. The disabling of pest genes through sequence-specific expression silencing is a promising tool in the development of environmentally-friendly and safe biopesticides. The specificity conferred by long dsRNA-base solutions helps minimize effects on off-target genes in the insect pest genome and the target gene in non-target organisms (NTOs). In this review, we summarize the status of gene silencing by RNA interference (RNAi) for agricultural control. More specifically, we focus on the engineering, development and application of gene silencing to control Lepidoptera through non-transforming dsRNA technologies. Despite some delivery and stability drawbacks of topical applications, we reviewed works showing convincing proof-of-concept results that point to innovative solutions. Considerations about the regulation of the ongoing research on dsRNA-based pesticides to produce commercialized products for exogenous application are discussed. Academic and industry initiatives have revealed a worthy effort to control Lepidoptera pests with this new mode of action, which provides more sustainable and reliable technologies for field management. New data on the genomics of this taxon may contribute to a future customized target gene portfolio. As a case study, we illustrate how dsRNA and associated methodologies could be applied to control an important lepidopteran coffee pest.

Tracking SARS-CoV-2 lineage B.1.1.7 dissemination: insights from nationwide spike gene target failure (SGTF) and spike gene late detection (SGTL) data, Portugal, week 49 2020 to week 3 2021.

We show that the SARS-CoV-2 B.1.1.7 lineage is highly disseminated in Portugal, with the odds of B.1.1.7 proportion increasing at an estimated 89% (95% confidence interval: 83-95%) per week until week 3 2021. RT-PCR spike gene target late detection (SGTL) can constitute a useful surrogate to track B.1.1.7 spread, besides the spike gene target failure (SGTF) proxy. SGTL/SGTF samples were associated with statistically significant higher viral loads, but not with substantial shift in age distribution compared to non-SGTF/SGTL cases.

Secreted breast tumor interstitial fluid microRNAs and their target genes are associated with triple-negative breast cancer, tumor grade, and immune infiltration.

BACKGROUND: Studies on tumor-secreted microRNAs point to a functional role of these in cellular communication and reprogramming of the tumor microenvironment. Uptake of tumor-secreted microRNAs by neighboring cells may result in the silencing of mRNA targets and, in turn, modulation of the transcriptome. Studying miRNAs externalized from tumors could improve cancer patient diagnosis and disease monitoring and help to pinpoint which miRNA-gene interactions are central for tumor properties such as invasiveness and metastasis. METHODS: Using a bioinformatics approach, we analyzed the profiles of secreted tumor and normal interstitial fluid (IF) microRNAs, from women with breast cancer (BC). We carried out differential abundance analysis (DAA), to obtain miRNAs, which were enriched or depleted in IFs, from patients with different clinical traits. Subsequently, miRNA family enrichment analysis was performed to assess whether any families were over-represented in the specific sets. We identified dysregulated genes in tumor tissues from the same cohort of patients and constructed weighted gene co-expression networks, to extract sets of co-expressed genes and co-abundant miRNAs. Lastly, we integrated miRNAs and mRNAs to obtain interaction networks and supported our findings using prediction tools and cancer gene databases. RESULTS: Network analysis showed co-expressed genes and miRNA regulators, associated with tumor lymphocyte infiltration. All of the genes were involved in immune system processes, and many had previously been associated with cancer immunity. A subset of these, BTLA, CXCL13, IL7R, LAMP3, and LTB, was linked to the presence of tertiary lymphoid structures and high endothelial venules within tumors. Co-abundant tumor interstitial fluid miRNAs within this network, including miR-146a and miR-494, were annotated as negative regulators of immune-stimulatory responses. One co-expression network encompassed differences between BC subtypes. Genes differentially co-expressed between luminal B and triple-negative breast cancer (TNBC) were connected with sphingolipid metabolism and predicted to be co-regulated by miR-23a. Co-expressed genes and TIF miRNAs associated with tumor grade were BTRC, CHST1, miR-10a/b, miR-107, miR-301a, and miR-454. CONCLUSION: Integration of IF miRNAs and mRNAs unveiled networks associated with patient clinicopathological traits, and underlined molecular mechanisms, specific to BC sub-groups. Our results highlight the benefits of an integrative approach to biomarker discovery, placing secreted miRNAs within a biological context.

MicroRNA-target gene responses to root knot nematode (Meloidogyne incognita) infection in cotton (Gossypium hirsutum L.).

MicroRNAs (miRNAs) are a large class of small regulatory RNA molecules, however no study has been performed to elucidate the role of miRNAs in cotton (Gossypium hirsutum) response to the root knot nematode (RKN, Meloidogyne incognita) infection. We selected 28 miRNAs and 8 miRNA target genes to investigate the miRNA-target gene response to M. incognita infection. Our results show that RKN infection significantly affected the expression of several miRNAs and their targeted genes. After 10 days of RKN infection, expression fold changes on miRNA expressions ranged from down-regulated by 33% to upregulated by 406%; meanwhile the expression levels of miRNA target genes were 45.8% to 231%. Three miRNA-target pairs, miR159-MYB, miR319-TCP4 and miR167-ARF8, showed inverse expression patterns between gene targets and their corresponded miRNAs, suggesting miRNA-mediated gene regulation in cotton roots in response to RKN infection.

Regional differences in gallbladder cancer pathogenesis: Insights from a multi-institutional comparison of tumor mutations.

BACKGROUND: Although rare in the United States, gallbladder cancer (GBCA) is a common cause of cancer death in some parts of the world. To investigate regional differences in pathogenesis and outcomes for GBCA, tumor mutations were analyzed from a sampling of specimens. METHODS: Primary tumors from patients with GBCA who were treated in Chile, Japan, and the United States between 1999 and 2016 underwent targeted sequencing of known cancer-associated genes. Fisher exact and Kruskal-Wallis tests assessed differences in clinicopathologic and genetic factors. Kaplan-Meier methods evaluated differences in overall survival from the time of surgery between mutations. RESULTS: A total of 81 patients were included. Japanese patients (11 patients) were older (median age, 72 years [range, 54-81 years]) compared with patients from Chile (21 patients; median age, 59 years [range, 32-73 years]) and the United States (49 patients; median age, 66 years [range, 46-87 years]) (P = .002) and had more well-differentiated tumors (46% vs 0% for Chile/United States; P < .001) and fewer gallstone-associated cancers (36% vs 67% for Chile and 69% for the United States; P = .13). Japanese patients had a median mutation burden of 6 (range, 1-23) compared with Chile (median mutation burden, 7 [range, 3-20]) and the United States (median mutation burden, 4 [range, 0-27]) (P = .006). Tumors from Japanese patients lacked AT-rich interaction domain 1A (ARID1A) and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) mutations, whereas Chilean tumors lacked Erb-B2 receptor tyrosine kinase 3 (ERBB3) and AT-rich interaction domain 2 (ARID2) mutations. SMAD family member 4 (SMAD4) was found to be mutated similarly across centers (38% in Chile, 36% in Japan, and 27% in the United States; P = .68) and was univariately associated with worse overall survival (median, 10 months vs 25 months; P = .039). At least one potentially actionable gene was found to be altered in 80% of tumors. CONCLUSIONS: Differences in clinicopathologic variables suggest the possibility of distinct GBCA pathogenesis in Japanese patients, which may be supported by differences in mutation pattern. Among all centers, SMAD4 mutations were detected in approximately one-third of patients and may represent a converging factor associated with worse survival. The majority of patients carried mutations in actionable gene targets, which may inform the design of future trials.

In silico target network analysis of de novo-discovered, tick saliva-specific microRNAs reveals important combinatorial effects in their interference with vertebrate host physiology.

The hard tick Ixodes ricinus is an important disease vector whose salivary secretions mediate blood-feeding success on vertebrate hosts, including humans. Here we describe the expression profiles and downstream analysis of de novo-discovered microRNAs (miRNAs) expressed in I. ricinus salivary glands and saliva. Eleven tick-derived libraries were sequenced to produce 67,375,557 Illumina reads. De novo prediction yielded 67 bona fide miRNAs out of which 35 are currently not present in miRBase. We report for the first time the presence of microRNAs in tick saliva, obtaining furthermore molecular indicators that those might be of exosomal origin. Ten out of these microRNAs are at least 100 times more represented in saliva. For the four most expressed microRNAs from this subset, we analyzed their combinatorial effects upon their host transcriptome using a novel in silico target network approach. We show that only the inclusion of combinatorial effects reveals the functions in important pathways related to inflammation and pain sensing. A control set of highly abundant microRNAs in both saliva and salivary glands indicates no significant pathways and a far lower number of shared target genes. Therefore, the analysis of miRNAs from pure tick saliva strongly supports the hypothesis that tick saliva miRNAs can modulate vertebrate host homeostasis and represents the first direct evidence of tick miRNA-mediated regulation of vertebrate host gene expression at the tick-host interface. As such, the herein described miRNAs may support future drug discovery and development projects that will also experimentally question their predicted molecular targets in the vertebrate host.

Significance of circulating microRNAs in diabetes mellitus type 2 and platelet reactivity: bioinformatic analysis and review.

In the light of growing global epidemic of type 2 diabetes mellitus (T2DM), significant efforts are made to discover next-generation biomarkers for early detection of the disease. Multiple mechanisms including inflammatory response, abnormal insulin secretion and glucose metabolism contribute to the development of T2DM. Platelet activation, on the other hand, is known to be one of the underlying mechanisms of atherosclerosis, which is a common T2DM complication that frequently results in ischemic events at later stages of the disease. Available data suggest that platelets contain large amounts of microRNAs (miRNAs) that are found in circulating body fluids, including the blood. Since miRNAs have been illustrated to play an important role in metabolic homeostasis through regulation of multiple genes, they attracted substantial scientific interest as diagnostic and prognostic biomarkers in T2DM. Various miRNAs, as well as their target genes are implicated in the complex pathophysiology of T2DM. This article will first review the different miRNAs studied in the context of T2DM and platelet reactivity, and subsequently present original results from bioinformatic analyses of published reports, identifying a common gene (PRKAR1A) linked to glucose metabolism, blood coagulation and insulin signalling and targeted by miRNAs in T2DM. Moreover, miRNA-target gene interaction networks built upon Gene Ontology information from electronic databases were developed. According to our results, miR-30a-5p, miR-30d-5p and miR-30c-5p are the most widely regulated miRNAs across all specified ontologies, hence they are the most promising biomarkers of T2DM to be investigated in future clinical studies.

Short communication: Bioinformatics-based mining of novel gene targets for identification of Cronobacter turicensis using PCR.

Cronobacter turicensis is a food-borne pathogen found in dairy products. It has been reported to cause bacteremia and enteritis in immunocompromised people, especially infants. Cronobacter turicensis has been isolated from various food sources, and contaminated powdered infant formula was found to be the most common source of infection among infants. Although some gene targets are used for the identification of C. turicensis, they are not specific at the species level. In this study, we analyzed the genome sequence of C. turicensis by bioinformatics and identified 13 specific gene targets. Primer sets targeting these sequences were designed and selected based on their specificity. Finally, primer set CT11, targeting gene CTU_19580, which codes for a hypothetical protein, was selected for development of the PCR assay because it alone produced positive PCR results for C. turicensis. To our knowledge, this is the first time that this gene target has been used to develop PCR detection assays for C. turicensis. The specific PCR assay had detection limits as low as 760 fg/µL for genomic DNA (approximately 158 copies/µL of DNA) and could detect C. turicensis in powdered infant formula with initial cell concentrations as low as 8.5 cfu per 10 g of powdered infant formula after 10 h of enrichment. Thus, this PCR assay is highly sensitive and can be used for rapid detection of C. turicensis.

A novel PCR-based approach to discover miRNA target genes.

MiRNAs are potent regulators of gene expression, and most miRNAs have from several to several thousands of gene targets. Validating the numerous gene targets of a given miRNA remains challenging despite the existence of various tools and databases that predict candidate gene-miRNA pairs. In the present study, we present a high-throughput but flexible method that applies a PCR-based application to simulate the binding of miRNAs to their gene targets. Using hsa-miR-377 as an illustrative example, our method was able to identify 13 potential targets of hsa-miR-377. Moreover, our results include 2 genes (SOD2 and PPM1A) that have already been verified as targets of hsa-miR-377. Our method may provide an alternative way of identifying the gene targets of miRNAs for future research.

Identification of neural-relevant toxcast high-throughput assay intended gene targets: Applicability to neurotoxicity and neurotoxicant putative molecular initiating events.

The US EPA's Toxicity Forecaster (ToxCast) is a suite of high-throughput in vitro assays to screen environmental toxicants and predict potential toxicity of uncharacterized chemicals. This work examines the relevance of ToxCast assay intended gene targets to putative molecular initiating events (MIEs) of neurotoxicants. This effort is needed as there is growing interest in the regulatory and scientific communities about developing new approach methodologies (NAMs) to screen large numbers of chemicals for neurotoxicity and developmental neurotoxicity. Assay gene function (GeneCards, NCBI-PUBMED) was used to categorize gene target neural relevance (1 = neural, 2 = neural development, 3 = general cellular process, 3 A = cellular process critical during neural development, 4 = unlikely significance). Of 481 unique gene targets, 80 = category 1 (16.6 %); 16 = category 2 (3.3 %); 303 = category 3 (63.0 %); 97 = category 3 A (20.2 %); 82 = category 4 (17.0 %). A representative list of neurotoxicants (548) was researched (ex. PUBMED, PubChem) for neurotoxicity associated MIEs/Key Events (KEs). MIEs were identified for 375 compounds, whereas only KEs for 173. ToxCast gene targets associated with MIEs were primarily neurotransmitter (ex. dopaminergic, GABA)receptors and ion channels (calcium, sodium, potassium). Conversely, numerous MIEs associated with neurotoxicity were absent. Oxidative stress (OS) mechanisms were 79.1 % of KEs. In summary, 40 % of ToxCast assay gene targets are relevant to neurotoxicity mechanisms. Additional receptor and ion channel subtypes and increased OS pathway coverage are identified for potential future assay inclusion to provide more complete coverage of neural and developmental neural targets in assessing neurotoxicity.

S-Gene Target Failure as an Effective Tool for Tracking the Emergence of Dominant SARS-CoV-2 Variants in Switzerland and Liechtenstein, Including Alpha, Delta, and Omicron BA.1, BA.2, and BA.4/BA.5.

During the SARS-CoV-2 pandemic, the Dr. Risch medical group employed the multiplex TaqPathTM COVID-19 CE-IVD RT-PCR Kit for large-scale routine diagnostic testing in Switzerland and the principality of Liechtenstein. The TaqPath Kit is a widely used multiplex assay targeting three genes (i.e., ORF1AB, N, S). With emergence of the B.1.1.7 (Alpha) variant, a diagnostic flaw became apparent as the amplification of the S-gene target was absent in these samples due to a deletion (ΔH69/V70) in the Alpha variant genome. This S-gene target failure (SGTF) was the earliest indication of a new variant emerging and was also observed in subsequent variants such as Omicron BA.1 and BA4/BA.5. The Delta variant and Omicron BA.2 did not present with SGTF. From September 2020 to November 2022, we investigated the applicability of the SGTF as a surrogate marker for emerging variants such as B.1.1.7, B.1.617.2 (Delta), and Omicron BA.1, BA.2, and BA.4/BA.5 in samples with cycle threshold (Ct) values < 30. Next to true SGTF-positive and SGTF-negative samples, there were also samples presenting with delayed-type S-gene amplification (higher Ct value for S-gene than ORF1ab gene). Among these, a difference of 3.8 Ct values between the S- and ORF1ab genes was found to best distinguish between "true" SGTF and the cycle threshold variability of the assay. Samples above the cutoff were subsequently termed partial SGTF (pSGTF). Variant confirmation was performed by whole-genome sequencing (Oxford Nanopore Technology, Oxford, UK) or mutation-specific PCR (TIB MOLBIOL). In total, 17,724 (7.4%) samples among 240,896 positives were variant-confirmed, resulting in an overall sensitivity and specificity of 93.2% [92.7%, 93.7%] and 99.3% [99.2%, 99.5%], respectively. Sensitivity was increased to 98.2% [97.9% to 98.4%] and specificity lowered to 98.9% [98.6% to 99.1%] when samples with pSGTF were included. Furthermore, weekly logistic growth rates (α) and sigmoid's midpoint (t0) were calculated based on SGTF data and did not significantly differ from calculations based on comprehensive data from GISAID. The SGTF therefore allowed for a valid real-time estimate for the introduction of all dominant variants in Switzerland and Liechtenstein.

Molecular biology of SARS-CoV-2 and techniques of diagnosis and surveillance.

The World Health Organization (WHO) declared coronavirus disease 2019 (COVID-19), a disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a global pandemic in March 2020. Reverse transcription-polymerase chain reaction (RT-PCR) is the reference technique for molecular diagnosis of SARS-CoV-2 infection. The SARS-CoV-2 virus is constantly mutating, and more transmissible variants have emerged, making genomic surveillance a crucial tool for investigating virus transmission dynamics, detecting novel genetic variants, and assessing mutation impact. The S gene, which encodes the spike protein, is frequently mutated, and it plays an important role in transmissibility. Spike protein mutations affect infectivity and vaccine effectiveness. SARS-CoV-2 variants are tracked using whole genome sequencing (WGS) and S-gene analysis. WGS, Sanger sequencing, and many S-gene-targeted RT-PCR methods have been developed. WGS and Sanger sequencing are standard methods for detecting mutations and can be used to identify known and unknown mutations. Melting curve analysis, endpoint genotyping assay, and S-gene target failure are used in the RT-PCR-based method for the rapid detection of specific mutations in SARS-CoV-2 variants. Therefore, these assays are suitable for high-throughput screening. The combinatorial use of RT-PCR-based assays, Sanger sequencing, and WGS enables rapid and accurate tracking of SARS-CoV-2 variants. In this review, we described RT-PCR-based detection and surveillance techniques for SARS-CoV-2.

A microRNA profile of the saliva in the argasid ticks Ornithodoros erraticus and Ornithodoros moubata and prediction of specific target genes.

Ornithodoros erraticus and Ornithodoros moubata ticks are the main vectors of the agents of human relapsing fever (TBRF) and African swine fever (ASF) in the Mediterranean Basin and Africa, respectively. Tick ​​saliva is crucial for complete tick feeding and pathogen transmission, as it contains numerous molecules such as proteins, lipids, and non-coding RNAs (ncRNA) including microRNAs (miRNA). MiRNAs are small ncRNAs capable of regulating the expression of their target messenger RNA (mRNA) leading to degradation or inhibition of its translation into protein. Research on miRNAs from ixodid ticks has revealed that miRNAs are involved in the regulation of different physiological processes of ticks, as well as in the modulation of host gene expression, immune response to tick bite and pathogen transmission. Regarding argasid ticks, there is not information about their miRNAs or their potential involvement in tick physiology and/or in the regulation of the tick-host-pathogen interactions. The aim of this work was to profile the miRNAs expressed in the saliva of O. erraticus and O. moubata, and the in silico prediction and functional analysis of their target genes in the swine host. As a whole, up to 72 conserved miRNAs families were identified in both species: 35 of them were shared and 23 and 14 families were unique to O. erraticus and O. moubata, respectively. The most abundant miRNAs families were mir-1, mir-10 and let-7 in O. erraticus and let-7, mir-252, mir-10 in O. moubata. Four miRNAs sequences of each species were validated by RT-qPCR confirming their presence in the saliva. Target gene prediction in the host (Sus scrofa) and functional analysis showed that the selected miRNAs are mainly involved in processes related to signal transduction, regulation of mRNA transcription and gene expression, synapse regulation, immune response, angiogenesis and vascular development. These results suggest that miRNAs could play an important role at the tick-host interface, providing new insights into this complex relationship that may contribute to a more precise selection of tick molecules for the development of therapeutic and immune strategies to control tick infestations and tick-borne pathogens.

Clinico-Epidemiological Profile of COVID-19 Patients with Omicron Variant Admitted in a Tertiary Care Center, South India.

Background and Objectives: Omicron, a variant of SARS COV2, is looming large as a cause of global concern. Its high transmissibility can pose challenges in healthcare allocation in a highly populous country like India. Studying the behaviour of the virus among the Indian population will definitely help in planning for the impending omicron surge, so we conducted a preliminary analysis of the clinical and epidemiological characteristics of the suspected omicron cases in the early part of the surge. Methodology: The study was conducted in the Rajiv Gandhi Government General Hospital, from 17th December 2021 to 11th January 2022. A total number of 159 consecutive patients ≥18 years of age with the S gene target failure were enrolled and clinically followed up during hospitalisation. Results: Nearly half (n = 79, 49.7%) were aged between 18 and 30 years and the mean (SD) age of the patients was 35.1 (14.9); 52.8% (n = 84) were males and 54.7% (n = 87) were healthcare workers. The NLR ratio and CRP were raised in unvaccinated individuals. Out of 159 patients, only 4 patients required oxygen and all the others showed a mild course of illness and there was no mortality. Conclusion: The clinical course of suspected omicron patients was mild in those who were vaccinated. Unvaccinated individuals with comorbid illness need to be closely monitored for prompt referral for acute care. Further studies are needed in the high-risk group with omicron.

Observational cohort study of the clinical outcomes associated with B.1.1.7/SGTF among hospitalized COVID-19 patients in Lebanon.

Background: The B.1.1.7 SARS-CoV-2 variant results in spike gene target failure (SGTF) in reverse transcription-quantitative polymerase chain reaction (RT-PCR) assays. Few studies have been published on the clinical impact of B.1.1.7/SGTF. Aims: To assess the incidence of B.1.1.7/SGTF and its associated clinical characteristics among hospitalized COVID-19 patients. Methods: This observational, single-centre, cohort study was conducted between December 2020 and February 2021 and included 387 hospitalized COVID-19 patients. The Kaplan-Meier method was used for survival analysis, and logistic regression to identify risk factors associated with B.1.1.7/SGTF. Results: By February 2021, B.1.1.7/SGTF (88%) dominated the SARS-CoV-2 PCR results in a Lebanese hospital. Of the 387 eligible COVID-19 patients confirmed by SARS-CoV-2 RT-PCR, 154 (40%) were non-SGTF and 233 (60%) were B.1.1.1.7/SGTF; this was associated with a higher mortality rate among female patients [22/51 (43%) vs 7/37 (19%); P = 0.0170]. Among patients in the B.1.1.7/SGTF group, most were aged ≥ 65 years [162/233 (70%) vs 74/154 (48%); P < 0.0001]. Independent predictors of B.1.1.7/SGTF infection were hypertension (OR = 0.415; CI: 0.242-0.711; P = 0.0010), age ≥ 65 years (OR = 0.379; CI: 0.231-0.622; P < 0.0001), smoking (OR = 1.698; CI: 1.023-2.819; P = 0.0410), and cardiovascular disease (OR = 3.812; CI: 2.215-6.389; P < 0.0001). Only non-SGTF patients experienced multi-organ failure [5/154 (4%) vs 0/233 (0%); P = 0.0096]. Conclusion: There was a clear difference between the clinical features associated with B.1.1.7/SGTF and non-SGTF lineages. Tracking viral evolution and its clinical impact is crucial for proper understanding and management of the COVID-19 pandemic.