Association of SARS-COV-2 viral RNAemia, IL- 6 gene polymorphism, serum IL-6 and peripheral blood lymphocytes and monocytes with disease severity in COVID-19 patients

Coronavirus disease 2019 (COVID-19) is a fatal pandemic viral disease caused by the severe acute respiratory syndrome corona virus type-2 (SARS-CoV-2). The aim of this study is to observe the associations of IL-6, SARS-COV-2 viral load (RNAemia), IL- 6 gene polymorphism and lymphocytes and monocytes in peripheral blood with disease severity in COVID-19 patients. This study was carried out from March 2021 to January 2022. RT-PCR positive 84 COVID-19 patients and 28 healthy subjects were enrolled. Blood was collected to detect SARS-COV-2 viral RNA (RNAemia) by rRT-PCR, serum IL-6 level by chemiluminescence method, SNPs of IL-6 by SSP-PCR, immunophenotyping of lymphocytes and monocyte by flow cytometry. Serum IL-6 level (pg/ml) was considerably high among critical patients (102.02 +/- 149.7) compared to severe (67.20 +/- 129.5) and moderate patients (47.04 +/- 106.5) and healthy controls (3.5 +/- 1.8). Serum SARS-CoV-2 nucleic acid positive cases detected mostly in critical patients (39.28%) and was correlated with extremely high IL-6 level and high mortality (R =.912, P < 0.001). Correlation between IL-6 and monocyte was statistically significant with disease severity (severe group, p < 0.001, and 0.867*** and critical group p < 0.001 and 0.887***). In healthy controls, moderate, severe and critically ill COVID-19 patients, IL-6 174G/C (rs 1800795) GG genotype was 82.14%, 89.20%, 67.85% and 53.57% respectively. CC and GC genotype had strong association with severity of COVID-19 when compared with GG genotype. Significant statistical difference found in genotypes between critical and moderate groups (p < 0.001, OR-10.316, CI-3.22-23.86), where CC genotype was associated with COVID-19 severity and mortality. The absolute count of T cell, B cell, NK cell, CD4+ T cells and CD8+ T cells were significantly decreased in critical group compared to healthy, moderate and severe group (P < 0.001). Exhaustion marker CD94/NKG2A was increased on NK cells and CD8+ cytotoxic T cell among critical and severe group. Absolute count of monocyte was significantly increased in critical group (P < 0.001). Serum IL-6, IL-6 174 G/C gene and SARS-CoV-2 RNAaemia can be used in clinical practice for risk assessment;T cell subsets and monocyte as biomarkers for monitoring COVID-19 severity. Monoclonal antibody targeting IL-6 receptor and NKG2A for therapeutics may prevent disease progression and decrease morbidity and mortality.Copyright © 2023 Elsevier Inc.

Identification of Torquetenovirus Species in Patients with Kawasaki Disease Using a Newly Developed Species-Specific PCR Method.

A next-generation sequencing (NGS) study identified a very high viral load of Torquetenovirus (TTV) in KD patients. We aimed to evaluate the feasibility of a newly developed quantitative species-specific TTV-PCR (ssTTV-PCR) method to identify the etiology of KD. We applied ssTTV-PCR to samples collected from 11 KD patients and 22 matched control subjects who participated in our previous prospective study. We used the NGS dataset from the previous study to validate ssTTV-PCR. The TTV loads in whole blood and nasopharyngeal aspirates correlated highly (Spearman's R = 0.8931, p < 0.0001, n = 33), supporting the validity of ssTTV-PCR. The ssTTV-PCR and NGS results were largely consistent. However, inconsistencies occurred when ssTTV-PCR was more sensitive than NGS, when the PCR primer sequences mismatched the viral sequences in the participants, and when the NGS quality score was low. Interpretation of NGS requires complex procedures. ssTTV-PCR is more sensitive than NGS but may fail to detect a fast-evolving TTV species. It would be prudent to update primer sets using NGS data. With this precaution, ssTTV-PCR can be used reliably in a future large-scale etiological study for KD.

Do People Use Games to Compensate for Psychological Needs During Crises? A Mixed-Methods Study of Gaming During COVID-19 Lockdowns

Do people use games to cope with adverse life events and crises? Research informed by self-determination theory proposes that people might compensate for thwarted basic psychological needs in daily life by seeking out games that satisfy those lacking needs. To test this, we conducted a preregistered mixed-method survey study (n = 285) on people's gaming behaviours and need states during early stages of the COVID-19 pandemic (May 2020). We found qualitative evidence that gaming was an often actively sought out and successful means of replenishing particular needs, but one that could 'backfre' for some through an appraisal process discounting gaming as 'unreal'. Meanwhile, contrary to our predictions, the quantitative data showed a "rich get richer, poor get poorer" pattern: need satisfaction in daily life positively correlated with need satisfaction in games. We derive methodological considerations and propose three potential explanations for this contradictory data pattern to pursue in future research.

CRISPR-Cas assisted diagnostics: A broad application biosensing approach

In addition to its remarkable genome editing capability, the CRISPR-Cas system has proven to be very effective in many fields of application, including the biosensing of pathogenic infections, mutagenic defects, or early cancer diagnosis. Thanks to their many advantages in terms of simplicity, efficiency, and reduced time, several CRISPR-Cas systems have been described for the design of sensitive and selective analytical tools, paving the way for the development and further commercialization of next-generation diagnostics. However, CRISPR-Cas-based biosensors still need further research efforts to improve some drawbacks, such as the need for target amplification, low reproducibility, and lack of knowledge of exploited element robustness. This review aims to describe the latest trends in the design of CRISPR-Cas biosensing technologies to better highlight the insights of their advantages and to point out the limitations that still need to be overcome for their future market entry as medical diagnostics.Copyright © 2023 Elsevier B.V.

Lifelong vaccination strategy as a tool against pandemics in Hungary / Élethosszan át tartó oltási stratégia mint eszköz a pandémiák elleni védekezésben Magyarországon.

Összefoglaló. Különbözo tényezok következtében az új és újra felbukkanó fertozo betegségek megjelenésére a 21. században egyre nagyobb az esély, ezzel párhuzamosan a pandémiák kialakulása is nagyobb valószínuségu. A 2019-ben felbukkant COVID-19-járvány azt is közvetíti számunkra, hogy egyes új és újra jelentkezo fertozo betegségek - az eredményes intézkedések elmaradása, késlekedése esetén - gyorsan terjedhetnek. A fertozo betegségek elleni harc egyik fo eszköze a védooltás segítségével történo immunizáció. A jelen tanulmány célja bemutatni a védooltások elonyeit, fókuszba helyezve az elöregedo társadalomban az élethosszan tartó immunizációs stratégiának a személyes egészségre ható, közegészségügyi, gazdasági, valamint társadalmi érdekeit. Az oltás elonyeinek minél nagyobb fokú kihasználásához egy élethosszan tartó immunizációs stratégia felállítása javasolható, amelynek aspektusait és gyakorlatba ültetésének lehetséges lépéseit foglaltuk össze közleményünkben. Orv Hetil. 2022; 163(14): 535-543. Summary. Due to various factors, the chances of infectious disease emergence or re-emergence have increased in the 21st century, thus, the likelihood of new emerging pandemics has also increased. The COVID-19 pandemic, which appeared in 2019, has highlighted that certain new and re-emerging infectious diseases - in the case of lack or delay in effective measures - can spread very rapidly. The main tool for the fight against infectious diseases is immunization through vaccination. While focusing on the personal health, public health, economic and societal benefits of a lifelong immunization strategy, especially in light of the aging society, the goal of this paper is to present the benefits of vaccines. In order to increase the added value of vaccinations it is recommended to create a lifelong immunization strategy. Our paper summarizes the relevant aspects of such a strategy, highlighting potential practical steps towards implementation. Orv Hetil. 2022; 163(14): 535-543.

COVID-19 in patients with primary immunodeficiency. / COVID-19 primer immunhiányos betegekben.

Összefoglaló. Az új típusú koronavírus (SARS-CoV-2) okozta pandémia súlyos terhet és nagy kihívást jelent a fertozésekkel szemben általában is fogékony, szerteágazó immunológiai és genetikai hátteru, primer immundeficiens (PID-) betegek számára. Az eddigi megfigyelések arra utalnak, hogy a SARS-CoV-2-fertozés és a súlyos COVID-19 mortalitása nem elsosorban az immunológiai alapbetegséggel, hanem sokkal inkább egyéb, a PID talaján megelozoen kialakult (például bronchiectasia, asthma, autoimmun betegség stb.) vagy attól független krónikus társbetegséggel (például diabetes, krónikus szív- és érrendszeri vagy vesebetegség) és szervi károsodással függ össze. A betegek egy kis csoportjában az I. típusú interferon-immunitás zavarát okozhatják génmutációk vagy autoantitestek termelése. A közleményben az eddig közölt adatok alapján beszámolunk a SARS-CoV-2-fertozés és a COVID-19 lefolyásáról és mortalitásáról PID-betegekben. Orv Hetil. 2022; 163(5): 166-170. Summary. The pandemic caused by the novel coronavirus (SARS-CoV-2) has resulted in tremendous challenges to the management of patients with primary immunodeficiencies (PIDs) representing a wide range of immunological and genetic entities. Preliminary data suggest that patients with PID would be at increased risk of severe disease and mortality from this newly emerged coronavirus. However, morbidity and mortality by SARS-CoV-2 may depend only partly on specific defect of immunity. Most of disease morbidity and mortality has been published to be related to previous damage of organs and tissues that had developed on the bases of PID before contracting SARS-CoV-2 or other, PID-independent disorders. In a small fraction of patients, impaired type I interferon immunity was found to predispose PID patients to severe coronavirus disease. In this review, we provide an update on published data about SARS-CoV-2 infections and COVID-19 in various PIDs. Orv Hetil. 2022; 163(5): 166-170.

Advances in RT-LAMP for COVID-19 testing and diagnosis.

INTRODUCTION: The SARS-CoV-2 pandemic, and the subsequent limitations on standard diagnostics, has vastly expanded the user base of Reverse Transcription Loop-mediated isothermal Amplification (RT-LAMP) in fundamental research and development. RT-LAMP has also penetrated commercial markets, with emergency use authorizations for clinical diagnosis. AREAS COVERED: This review discusses the role of RT-LAMP within the context of other technologies like RT-qPCR and rapid antigen tests, progress in sample preparation strategies to enable simplified workflow for RT-LAMP directly from clinical specimens, new challenges with primer and assay design for the evolving pandemic, prominent detection modalities including colorimetric and CRISPR-mediated methods, and translational research and commercial development of RT-LAMP for clinical applications. EXPERT OPINION: RT-LAMP occupies a middle ground between RT-qPCR and rapid antigen tests. The simplicity approaches that of rapid antigen tests, making it suitable for point-of-care use, but the sensitivity nears that of RT-qPCR. RT-LAMP still lags RT-qPCR in fundamental understanding of the mechanism, and the interplay between sample preparation and assay performance. Industry is now beginning to address issues around scalability and usability, which could finally enable LAMP and RT-LAMP to find future widespread application as a diagnostic for other conditions, including other pathogens with pandemic potential.

Comparison five primer sets from different genome region of COVID-19 for detection of virus infection by conventional RT-PCR.

BACKGROUND AND OBJECTIVES: The new beta-coronavirus, which caused Severe Acute Respiratory Coronavirus-2 Syndrome (SARS-CoV-2), a major respiratory outbreak in Wuhan, China in December 2019, is now prevalent in many countries around the world. Identifying PCR-based viruses is a well-known and relatively stable protocol. Unfortunately, the high mutation rates may lead to widespread changes in viral nucleic acid sequences, and so using specific primers for PCR can be recommended. In this study, we evaluated the power of a conventional RT-PCR to detect SARS-CoV-2 RNA among the five set primer sets. MATERIALS AND METHODS: The five genomic regions of the Coronavirus SARS-2 virus including Nucleocapsids (N), Envelope (E), RNA depended RNA Polymerase (RdRp), ORF1ab and Spike (S) were selected for primer designing. A conventional RT-PCR was performed to compare sensitivity, specificity and other analytical characteristics of primers designed against two Real Time PCR commercial kits. RESULTS: The result of the comparative analysis showed that the ORF1ab, N and RdRp primers had a sensitivity, specificity and positive predictive value higher than other primers. A significant difference in the analytical sensitivity between the studied primer sets in RT-PCR kits was observed. CONCLUSION: In this study, the ORF1ab, Nucleocapsid and RdRp regions have the best primers for identifying the SARS-CoV-2 RNA between different genes that have been suggested.

Caracterizacao Sorologica E Molecular Em Amostra De Paciente Com Ausencia De Dois Antigenos De Alta Frequencia De Diferentes Grupos Sanguineos

Objetivo: Relatar um caso imunohematologico complexo, descrevendo as estrategias sorologicas e moleculares realizadas para a caracterizacao fenotipica de um paciente com ausencia de dois antigenos de alta frequencia U (MNS:-5) e Yta (YT:-1), assim como, a determinacao do(s) anticorpo(s) de alta frequencia presente no soro. Materiais e metodos: Trata-se de um trabalho descritivo em que foram utilizados dados clinicos de prontuario disponiveis no laboratorio de referencia de imunohematologia (LRI) onde foram testadas as amostras. Resultados: Paciente de 84 anos, com anemia sintomatica, COVID-19 e abdomen agudo obstrutivo, teve a amostra encaminhada para o LRI para confirmacao de anticorpo anti-U no soro e genotipagem eritrocitaria. O soro do paciente foi testado com painel de hemacias comerciais e raras sendo reativo com todas as hemacias do painel em Gel-Liss/Enzima, exceto com hemacias S-s-U-/Yt(a+) demonstrando apenas a especificidade de anti-U. O teste de adsorcao alogenica com hemacias de doador tratadas com ZZAP excluiu a presenca de anticorpo anti-Yta no soro aloadsorvido. Para avaliar a importancia clinica do anticorpo, foi realizada a tecnica de MMA (Monocyte Monolayer Assay), sendo que o teste apresentou menos que 5% de atividade fagocitica dos Monocitos, alem disso, o fato da classe da Imunoglobulina nao pertencer a IgG1 ou IgG3 (cartao ID-Card DAT IgG1/IgG3, BioRad), indicaram que esse anticorpo nao possui importancia clinica. O DNA do paciente foi extraido do sangue total utilizando o Kit MagNa Pure, Roche. A genotipagem realizada pelas tecnicas do BloodChip Kit ID CoreXT - Grifol, PCR-SSP e PCR-RFLP demonstrou o seguinte resultado: GYPB*delecao;YT*2/2. O fenotipo foi confirmado como U- e Yt(a-) com antissoros provenientes do SCARF. Discussao: Antigenos de alta frequencia quando ausentes, resultam na dificuldade da busca de sangue compativel para a transfusao, assim como na disponibilidade de insumos e tecnicas bem padronizadas para a investigacao imunohematologica. Determinados antigenos de alta frequencia quando ausentes na membrana eritrocitaria podem afetar a expressao de antigenos de outros sistemas. Atraves de uma tecnica semi-automatizada de genotipagem identificamos que a amostra de um paciente tinha a ausencia de dois antigenos de alta frequencia U (MNS:-5) e Yta(YT:-1). Por se tratar de um caso complexo, estrategias sorologicas e moleculares foram realizadas para a caracterizacao fenotipica do paciente, determinacao do anticorpo de alta frequencia presente no soro e sua importancia clinica. Conclusao: A tecnica do BloodChip permitiu uma analise molecular mais completa, sendo possivel verificar a ausencia de dois antigenos de alta frequencia: U e Yta, o que tornou mais dificil a busca por doador fenotipo compativel para o paciente. Paineis de hemacias e soros raros foram capazes de definir a especificidade do anti-U e excluir a presenca do anti-Yta e de outros anticorpos que poderiam estar mascarados no soro. Apesar do anti-U nao aparentar importancia clinica, recomendamos a transfusao de concentrado de hemacias U- (MNS:-5), uma vez que esse anticorpo esta implicado em reacoes transfusionais hemoliticas. Copyright © 2022

Direct Reverse Transcription Real-Time PCR of Viral RNA from Saliva Samples Using Hydrogel Microparticles.

In recent decades "saliva" has emerged as an important non-invasive biofluid for diagnostic purposes in both human and animal health sectors. However, with the rapid evolution of molecular detection technologies, the limitation has been the lack of an efficient method for the facile amplification of target RNA from such a complex matrix. Herein, we demonstrate the novel application of hydrogel microparticles of primer-immobilized networks (PIN) for direct quantitative reverse transcription PCR (dirRT-qPCR) of viral RNA from saliva samples without prior RNA purification. Each of these highly porous PIN particles operates as an independent reactor. They filter in micro-volumes of the analyte solution. Viral RNA is captured and converted to complementary DNA (cDNA) through the RT step using covalently incorporated RT primers. The PIN with cDNA of the viral target will be ready for subsequent highly specific qPCR. Preceded by heat-treatment for viral lysis, we were able to conduct PIN dirRT-qPCR with 95% efficiency of the matrix (M) gene for influenza A virus (IAV) and 5' untranslated region (5' UTR) for chicken coronavirus spiked into saliva samples. The addition of reverse transcriptase enzyme (RTase) and 10% dilution of the matrix improved the assay sensitivity considerably. PIN particles' compatibility with microfluidic PCR chip technology has significantly reduced total sample processing time to 50 min, instead of an average of 120 min that are normally used by other assays. We anticipate this technology will be useful for other viral RNA targets by changing the incorporated RT primer sequences and can be adapted for onsite diagnostics. Supplementary Information: The online version contains supplementary material available at 10.1007/s13206-022-00065-0.

Primer Design for Reverse Transcription-Quantitative PCR Simulation: an Online Lab Exercise Using a SARS-CoV-2 Model.

Since the start of the 2019 coronavirus disease (COVID-19) pandemic, many microbiology lab activities have been conducted online. We produced a simple PCR primer design and virtual PCR activity for introductory students to use as part of an online laboratory course or as an independent activity in a traditional laboratory setting. Most students are aware of basic PCR concepts but struggle with important details, such as how PCR is specific and how false positives and negatives can be generated in a diagnostic test that is not well designed. This exercise helps students review molecular biology concepts within the context of a test that was commonplace during the COVID-19 pandemic. We found that nursing students and Biology and non-Biology majors were able to complete the worksheet as a group with minimal instructor input. This could be used as a stand-alone activity, as a warm-up for other bioinformatics exercises, or as a prelab activity for actual in-lab quantitative PCR experiments, such as the one offered by miniPCR bio. With minor modifications, it could also be used with more advanced students.

Translocation pause of remdesivir-containing primer/template RNA duplex within SARS-CoV-2's RNA polymerase complexes.

The mechanism of remdesivir incorporation into the RNA primer by the RNA-dependent RNA polymerase (RdRp) of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) remains to be fully established at the molecular level. Here, we compare molecular dynamics (MD) simulations after incorporation of either remdesivir monophosphate (RMP) or adenosine monophosphate (AMP). We find that the Mg2+-pyrophosphate (PPi) binds more tightly to the polymerase when the added RMP is at the third primer position than in the AMP added complex. The increased affinity of Mg2+-PPi to the RMP-added primer/template (P/T) RNA duplex complex introduces a new hydrogen bond of a substituted cyano group in RMP with the K593 sidechain. The new interactions disrupt a switching mechanism of a hydrogen bond network that is essential for translocation of the P/T duplex product and for opening of a vacant NTP-binding site necessary for next primer extension. Furthermore, steric interactions between the sidechain of S861 and the 1'-cyano group of RMP at position i+3 hinders translocation of RMP to the i + 4 position, where i labels the insertion site. These findings are particularly valuable to guide the design of more effective inhibitors of SARS-CoV-2 RNA polymerase.

Unique Molecular Identifiers and Multiplexing Amplicons Maximize the Utility of Deep Sequencing To Critically Assess Population Diversity in RNA Viruses.

Next generation sequencing (NGS)/deep sequencing has become an important tool in the study of viruses. The use of unique molecular identifiers (UMI) can overcome the limitations of PCR errors and PCR-mediated recombination and reveal the true sampling depth of a viral population being sequenced in an NGS experiment. This approach of enhanced sequence data represents an ideal tool to study both high and low abundance drug resistance mutations and more generally to explore the genetic structure of viral populations. Central to the use of the UMI/Primer ID approach is the creation of a template consensus sequence (TCS) for each genome sequenced. Here we describe a series of experiments to validate several aspects of the Multiplexed Primer ID (MPID) sequencing approach using the MiSeq platform. We have evaluated how multiplexing of cDNA synthesis and amplicons affects the sampling depth of the viral population for each individual cDNA and amplicon to understand the relationship between broader genome coverage versus maximal sequencing depth. We have validated reproducibility of the MPID assay in the detection of minority mutations in viral genomes. We have also examined the determinants that allow sequencing reads of PCR recombinants to contaminate the final TCS data set and show how such contamination can be limited. Finally, we provide several examples where we have applied MPID to analyze features of minority variants and describe limits on their detection in viral populations of HIV-1 and SARS-CoV-2 to demonstrate the generalizable utility of this approach with any RNA virus.

Mining and analysis of microsatellites in human coronavirus genomes using the in-house built Java pipeline.

Microsatellites or simple sequence repeats are motifs of 1 to 6 nucleotides in length present in both coding and non-coding regions of DNA. These are found widely distributed in the whole genome of prokaryotes, eukaryotes, bacteria, and viruses and are used as molecular markers in studying DNA variations, gene regulation, genetic diversity and evolutionary studies, etc. However, in vitro microsatellite identification proves to be time-consuming and expensive. Therefore, the present research has been focused on using an in-house built java pipeline to identify, analyse, design primers and find related statistics of perfect and compound microsatellites in the seven complete genome sequences of coronavirus, including the genome of coronavirus disease 2019, where the host is Homo sapiens. Based on search criteria among seven genomic sequences, it was revealed that the total number of perfect simple sequence repeats (SSRs) found to be in the range of 76 to 118 and compound SSRs from 01 to10, thus reflecting the low conversion of perfect simple sequence to compound repeats. Furthermore, the incidence of SSRs was insignificant but positively correlated with genome size (R2 = 0.45, p > 0.05), with simple sequence repeats relative abundance (R2 = 0.18, p > 0.05) and relative density (R2 = 0.23, p > 0.05). Dinucleotide repeats were the most abundant in the coding region of the genome, followed by tri, mono, and tetra. This comparative study would help us understand the evolutionary relationship, genetic diversity, and hypervariability in minimal time and cost.

SARS-CoV-2 Mutations and Variants May Muddle the Sensitivity of COVID-19 Diagnostic Assays.

The performance of diagnostic polymerase chain reaction (PCR) assays can be impacted by SARS-CoV-2 variability as this is dependent on the full complementarity between PCR primers/probes and viral target templates. Here, we investigate the genetic variability of SARS-CoV-2 regions recognized by primers/probes utilized by PCR diagnostic assays based on nucleotide mismatching analysis. We evaluated the genetic variation in the binding regions of 73 primers/probes targeting the Nucleocapsid (N, N = 36), Spike (S, N = 22), and RNA-dependent RNA-polymerase/Helicase (RdRp/Hel, N = 15) of the publicly available PCR-based assays. Over 4.9 million high-quality SARS-CoV-2 genome sequences were retrieved from GISAID and were divided into group-A (all except Omicron, >4.2 million) and group-B (only Omicron, >558 thousand). In group-A sequences, a large range of variability in primers/probes binding regions in most PCR assays was observed. Particularly, 87.7% (64/73) of primers/probes displayed ≥1 mismatch with their viral targets, while 8.2% (6/73) contained ≥2 mismatches and 2.7% (2/73) contained ≥3 mismatches. In group-B sequences, 32.9% (24/73) of primers/probes were characterized by ≥1 mismatch, 13.7% (10/73) by ≥2 mismatches, and 5.5% (4/73) by ≥3 mismatches. The high rate of single and multiple mismatches- found in the target regions of molecular assays used worldwide for SARS-CoV-2 diagnosis reinforces the need to optimize and constantly update these assays according to SARS-CoV-2 genetic evolution and the future emergence of novel variants.

Identification DNA Methylation Change of ABCC8 Gene in Type 2 Diabetes Mellitus as Predictive Biomarkers

Type 2 diabetes mellitus is the most common chronic endocrine disorder that affecting 5%-10% of adults globally. Recently, the disease has rapidly spread throughout the Kurdistan Region. This study investigates DNA methylation status in the ABCCS gene among the study population, and it possibly used as a biomarker. One hundred and thirteen individuals were included in this study, and they were divided into three categories (47 diabetes, 36 prediabetic, and 30 controls). Blood samples were collected to investigate DNA methylation status in patients who attended private clinical sectors in Koya city, Kurdistan Region of Iraq, between August and December 2021. Methylation-specific PCR (MSP) uses paired primers for each methylated and unmethylated region. In addition, the X2 Kruskal-Wallis statistical and Wilcoxon signed-rank tests were run with a significance level of p 0.05. In comparison to the healthy group, hypermethylation of DNA is detected in the promoter region of diabetes and prediabetes. In addition, age, gender, BMI, alcohol use, family history, and physical activity all influence the degree of DNA methylation in people who have had coronavirus illness. The above-mentioned findings suggest that DNA methylation alterations in the ABCC8 promoter region might be exploited as a possible predictive biomarker for type 2 diabetes mellitus diagnosis.

Sequencing during Times of Change: Evaluating SARS-CoV-2 Clinical Samples during the Transition from the Delta to Omicron Wave.

The pandemic of SARS-CoV-2 is characterized by the emergence of new variants of concern (VOCs) that supplant previous waves of infection. Here, we describe our investigation of the lineages and host-specific mutations identified in a particularly vulnerable population of predominantly older and immunosuppressed SARS-CoV-2-infected patients seen at our medical center in Chicago during the transition from the Delta to Omicron wave. We compare two primer schemes, ArticV4.1 and VarSkip2, used for short read amplicon sequencing, and describe our strategy for bioinformatics analysis that facilitates identifying lineage-associated mutations and host-specific mutations that arise during infection. This study illustrates the ongoing evolution of SARS-CoV-2 VOCs in our community and documents novel constellations of mutations that arise in individual patients. The ongoing evaluation of the evolution of SARS-CoV-2 during this pandemic is important for informing our public health strategies.

rprimer: an R/bioconductor package for design of degenerate oligos for sequence variable viruses.

BACKGROUND: This paper presents a new R/Bioconductor package, rprimer, for design of degenerate oligos and PCR assays for sequence variable viruses. A multiple DNA sequence alignment is used as input data, while the outputs consist of comprehensive tables (data frames) and dashboard-like plots. The workflow can be run directly from the R console or through a graphical user interface (Shiny application). Here, rprimer is demonstrated and evaluated by using it to design two norovirus genogroup I (GI) assays: one RT-qPCR assay for quantitative detection and one RT­PCR assay for Sanger sequencing and polymerase-capsid based genotyping. RESULTS: The assays generated were evaluated using stool samples testing positive for norovirus GI. The RT-qPCR assay accurately amplified and quantified all samples and showed comparable performance to a widely-used standardised assay, while the RT-PCR assay resulted in successful sequencing and genotyping of all samples. Merits and limitations of the package were identified through comparison with three similar freely available software packages. Several features were comparable across the different tools, but important advantages of rprimer were its speed, flexibility in oligo design and capacity for visualisation. CONCLUSIONS: An R/Bioconductor package, rprimer, was developed and shown to be successful in designing primers and probes for quantitative detection and genotyping of a sequence-variable virus. The package provides an efficient, flexible and visual approach to degenerate oligo design, and can therefore assist in virus research and method development.

Evaluation of RT-qPCR Primer-Probe Sets to Inform Public Health Interventions Based on COVID-19 Sewage Tests.

Sewage surveillance is increasingly employed as a supplementary tool for COVID-19 control. Experiences learnt from large-scale trials could guide better interpretation of the sewage data for public health interventions. Here, we compared the performance of seven commonly used primer-probe sets in RT-qPCR and evaluated the usefulness in the sewage surveillance program in Hong Kong. All selected primer-probe sets reliably detected SARS-CoV-2 in pure water at 7 copies per µL. Sewage matrix did not influence RT-qPCR determination of SARS-CoV-2 concentrated from a small-volume sewage (30 mL) but introduced inhibitory impacts on a large-volume sewage (920 mL) with a ΔCt of 0.2-10.8. Diagnostic performance evaluation in finding COVID-19 cases showed that N1 was the best single primer-probe set, while the ORF1ab set is not recommended. Sewage surveillance using the N1 set for over 3200 samples effectively caught the outbreak trend and, importantly, had a 56% sensitivity and a 96% specificity in uncovering the signal sources from new cases and/or convalescent patients in the community. Our study paves the way for selecting detection primer-probe sets in wider applications in responding to the COVID-19 pandemic.

Transición de clases presenciales a aprendizaje remoto asistido

Resumen La pandemia de COVID-19 obligó a las instituciones de educación superior a cambiar la modalidad de enseñanza tradicional presencial a la remota asistida con herramientas tecnológicas. Estudios recientes han descrito las experiencias existentes de la enseñanza remota asistida como una alternativa metodológica de transición emergente para cumplir con los lineamientos de distanciamiento social y seguridad estipulados a nivel mundial. Este estudio fusiona datos de dos fuentes por medio de una metodología paralela convergente y mixta. Se presenta una sistematización del plan de acción por parte de las autoridades docentes de la Universidad Nacional de Costa Rica y un análisis de seguimiento de las experiencias de los estudiantes de primer año de la carrera de Bachillerato en la Enseñanza del Inglés como Lengua Extranjera durante el primer ciclo 2020. Los resultados evidencian la experiencia de los estudiantes en cuanto a sus posibilidades tecnológicas, dispositivos electrónicos que cuenta para conectarse y los salientes problemas en los ámbitos de la salud, la economía, lo social o del espacio físico de aprendizaje. Por último, los investigadores proponen un estudio de investigación empático adicional basado en el modelo de design thinking para la integración de herramientas y recursos tecnológicos en la carrera. Alternate : Resumen Resumen La pandemia de COVID-19 obligó a las instituciones de educación superior a cambiar la modalidad de enseñanza tradicional presencial a la remota asistida con herramientas tecnológicas. Estudios recientes han descrito las experiencias existentes de la enseñanza remota asistida como una alternativa metodológica de transición emergente para cumplir con los lineamientos de distanciamiento social y seguridad estipulados a nivel mundial. Este estudio fusiona datos de dos fuentes por medio de una metodología paralela convergente y mixta. Se presenta una sistematización del plan de acción por parte de las autoridades docentes de la Universidad Nacional de Costa Rica y un análisis de seguimiento de las experiencias de los estudiantes de primer año de la carrera de Bachillerato en la Enseñanza del Inglés como Lengua Extranjera durante el primer ciclo 2020. Los resultados evidencian la experiencia de los estudiantes en cuanto a sus posibilidades tecnológicas, dispositivos electrónicos que cuenta para conectarse y los salientes problemas en los ámbitos de la salud, la economía, lo social o del espacio físico de aprendizaje. Por último, los investigadores proponen un estudio de investigación empático adicional basado en el modelo de design thinking para la integración de herramientas y recursos tecnológicos en la carrera.