An ultra-sensitive one-pot RNA-templated DNA ligation rolling circle amplification-assisted CRISPR/Cas12a detector assay for rapid detection of SARS-CoV-2.

Rapid, sensitive, and one-pot diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays an extremely important role in point-of-care testing (POCT). Herein, we report an ultra-sensitive and rapid one-pot enzyme-catalyzed rolling circle amplification-assisted CRISPR/FnCas12a assay, termed OPERATOR. OPERATOR employs a single well-designed single-strand padlock DNA, containing a protospacer adjacent motif (PAM) site and a sequence complementary to the target RNA which procedure converts and amplifies genomic RNA to DNA by RNA-templated DNA ligation and multiply-primed rolling circle amplification (MRCA). The MRCA amplicon of single-stranded DNA is cleaved by the FnCas12a/crRNA complex and detected via a fluorescence reader or lateral flow strip. OPERATOR presents outstanding advantages including ultra-sensitivity (1.625 copies per reaction), high specificity (100%), rapid reaction speed (∼30 min), easy operation, low cost, and on-spot visualization. Furthermore, we established a POCT platform by combining OPERATOR with rapid RNA release and a lateral flow strip without professional equipment. The high performance of OPERATOR in SARS-CoV-2 tests was confirmed using both reference materials and clinical samples, and the results suggest that is readily adaptable for point-of-care testing of other RNA viruses.

Rapid whole genome sequencing methods for RNA viruses.

RNA viruses are the etiological agents of many infectious diseases. Since RNA viruses are error-prone during genome replication, rapid, accurate and economical whole RNA viral genome sequence determination is highly demanded. Next-generation sequencing (NGS) techniques perform whole viral genome sequencing due to their high-throughput sequencing capacity. However, the NGS techniques involve a significant burden for sample preparation. Since to generate complete viral genome coverage, genomic nucleic acid enrichment is required by reverse transcription PCR using virus-specific primers or by viral particle concentration. Furthermore, conventional NGS techniques cannot determine the 5' and 3' terminal sequences of the RNA viral genome. Therefore, the terminal sequences are determined one by one using rapid amplification of cDNA ends (RACE). However, since some RNA viruses have segmented genomes, the burden of the determination using RACE is proportional to the number of segments. To date, there is only one study attempting whole genome sequencing of multiple RNA viruses without using above mentioned methods, but the generated sequences' accuracy compared to the reference sequences was up to 97% and did not reach 100% due to the low read depth. Hence, we established novel methods, named PCR-NGS and RCA-NGS, that were optimized for an NGS machine, MinION. These methods do not require nucleic acid amplification with virus-specific PCR primers, physical viral particle enrichment, and RACE. These methods enable whole RNA viral genome sequencing by combining the following techniques: (1) removal of unwanted DNA and RNA other than the RNA viral genome by nuclease treatment; (2) the terminal of viral genome sequence determination by barcoded linkers ligation; (3) amplification of the viral genomic cDNA using ligated linker sequences-specific PCR or an isothermal DNA amplification technique, such as rolling circle amplification (RCA). The established method was evaluated using isolated RNA viruses with single-stranded, double-stranded, positive-stranded, negative-stranded, non-segmented or multi-segmented genomes. As a result, all the viral genome sequences could be determined with 100% accuracy, and these mean read depths were greater than 2,500×, at least using either of the methods. This method should allow for easy and economical determination of accurate RNA viral genomes.

A Novel Rolling Circle Amplification-Based Detection of SARS-CoV-2 with Multi-Region Padlock Hybridization.

SARS-CoV-2 has remained a global health burden, primarily due to the continuous evolution of different mutant strains. These mutations present challenges to the detection of the virus, as the target genes of qPCR, the standard diagnostic method, may possess sequence alterations. In this study, we develop an isothermal one-step detection method using rolling circle amplification (RCA) for SARS-CoV-2. This novel strategy utilizes a multi-padlock (MP-RCA) approach to detect viral-RNA via a simplified procedure with the reliable detection of mutated strains over other procedures. We designed 40 padlock-based probes to target different sequences across the SARS-CoV-2 genome. We established an optimal one-step isothermal reaction protocol utilizing a fluorescent output detected via a plate reader to test a variety of padlock combinations. This method was tested on RNA samples collected from nasal swabs and validated via PCR. S-gene target failure (SGTF)-mutated strains of SARS-CoV-2 were included. We demonstrated that the sensitivity of our assay was linearly proportional to the number of padlock probes used. With the 40-padlock combination the MP-RCA assay was able to correctly detect 45 out 55 positive samples (81.8% efficiency). This included 10 samples with SGTF mutations which we were able to detect as positive with 100% efficiency. We found that the MP-RCA approach improves the sensitivity of the MP-RCA assay, and critically, allows for the detection of SARS-CoV-2 variants with SGTF. Our method offers the simplicity of the reaction and requires basic equipment compared to standard qPCR. This method provides an alternative approach to overcome the challenges of detecting SARS-CoV-2 and other rapidly mutating viruses.

Severe Agitation as an Unusual Presentation of Multisystem Inflammatory Syndrome in Children.

Multisystem inflammatory syndrome in children (MIS-C) is a rare hyperinflammatory syndrome that mainly affects children after a primary infection with coronavirus disease 2019 (COVID-19), with the possibility of severe and lethal complications. We report a case of a unique presentation of MIS-C in a four-year-old boy who presented with severe agitation, muscle spasms, and two days of fever. Other findings consistent with MIS-C were revealed later, and he was managed with intravenous immunoglobulin (IVIG) and steroids. He showed a dramatic response of improvement and was discharged. This case report aimed to raise health professionals' awareness about the atypical presentations of MIS-C and the importance of early diagnosis, treatment, and follow-up MIS-C cases to avoid complications affecting children's lives.

Decision Aid in Logistics during COVID-19 Induced Disruption and Significance of a Digital Ecosystem

The COVID-19 outbreak tremendously increased the online purchase behaviour of consumers in most parts of the globe, induced higher demand for the delivery staff, and eventually resulted in a stricter working condition and shortage of delivery staff. Seldom research focuses on this emerging delivery workers' woes. This research embarks on a case study to appraise delivery workers' effectiveness, obstacles, and scenarios as unfolded and encountered during the COVID-19 period. The study analyses the factors that influence the delivery works in various sectors' workplace settings and makes recommendations for the logistics sector's digital transformation, particularly for firms that manage courier services, distribution centres, and e-commerce websites. The Root Cause Analysis (RCA) is utilised to create a proposal to assist the companies in avoiding similar future risks, particularly from parcel delivery staff. © 2022 IEEE.

Prospects of NIR fluorescent nanosensors for green detection of SARS-CoV-2.

The pandemic of the novel coronavirus disease 2019 (COVID-19) is continuously causing hazards for the world. Effective detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can relieve the impact, but various toxic chemicals are also released into the environment. Fluorescence sensors offer a facile analytical strategy. During fluorescence sensing, biological samples such as tissues and body fluids have autofluorescence, giving false-positive/negative results because of the interferences. Fluorescence near-infrared (NIR) nanosensors can be designed from low-toxic materials with insignificant background signals. Although this research is still in its infancy, further developments in this field have the potential for sustainable detection of SARS-CoV-2. Herein, we summarize the reported NIR fluorescent nanosensors with the potential to detect SARS-CoV-2. The green synthesis of NIR fluorescent nanomaterials, environmentally compatible sensing strategies, and possible methods to reduce the testing frequencies are discussed. Further optimization strategies for developing NIR fluorescent nanosensors to facilitate greener diagnostics of SARS-CoV-2 for pandemic control are proposed.

Comparison of Coronary Artery Involvement and Mortality in STEMI Patients With and Without SARS-CoV-2 During the COVID-19 Pandemic: A Systematic Review and Meta-Analysis.

BACKGROUND: Cardiovascular injury with SARS-CoV-2 infection is well known. Several studies have outlined baseline characteristics in patients presenting with STEMI and SARS-CoV-2. Paucity in data exists in selective coronary involvement in patients with STEMI and SARS-CoV-2 during the COVID-19 pandemic. METHODS: A systematic search and meta-analysis of studies meeting the inclusion and exclusion criteria obtained from MEDLINE, Scopus, and Cochrane databases was performed utilizing PRISMA criteria. The main outcome was likelihood of coronary artery involvement among patients with STEMI and SARS-CoV-2 versus without SARS-CoV-2. The primary adverse outcome measured was in-hospital mortality. RESULTS: The final analysis included 5 observational studies with a total of 2,266 patients. There was no statistical significance in LM (OR 1.40; 95% CI: 0.68, 2.90), LAD (OR 1.09; 95% CI 0.83, 1.43), LCX (OR 1.17; 95% CI: 0.75, 1.85), or RCA (OR 0.59; 95% CI: 0.30, 1.17) disease among the 2 groups. LAD disease was the most prevalent coronary involvement among patients with STEMI and SARS-CoV-2 (49.6%). Higher in-hospital mortality was observed in the STEMI and SARS-CoV-2 group (OR 5.24; 95% CI: 3.63, 7.56). CONCLUSIONS: Our analysis demonstrated no statistical significance in selective coronary involvement in patients with STEMI and SARS-CoV-2 during the COVID-19 pandemic. The higher mortality among patients with SARS-CoV-2 and STEMI has been noted in prior studies with concerns being late presentation due to fear of infection, delayed care time, and poor resource allocation. Focus should be placed on identifying and managing comorbidities to reduce mortality.

Giant Coronary Aneurysms in Multisystem Inflammatory Syndrome in Children Associated With SARS-CoV-2 Infection.

Multisystem inflammatory syndrome in children (MIS-C) can cause a myriad of cardiac manifestations, including coronary dilation and aneurysms; giant aneurysms are infrequent. We describe 3patients with giant coronary aneurysms associated with MIS-C, including the youngest case reported to date, treated with intravenous immunoglobulin, corticosteroids, and biologic agents. (Level of Difficulty: Intermediate.).

Barriers Influencing Vaccine Development Timelines, Identification, Causal Analysis, and Prioritization of Key Barriers by KOLs in General and Covid-19 Vaccine R&D.

A frequently mentioned factor holding back the introduction of new vaccines on the market are their prohibitively long development timelines. These hamper their potential societal benefit and impairs the ability to quickly respond to emerging new pathogens. This is especially worrisome since new pathogens are emerging at all-time high rates of over one per year, and many age-old pathogens are still not vaccine preventable.Through interviews with 20 key-opinion-leaders (KOLs), this study identified innovation barriers that increase vaccine development timelines. These innovation barriers were visualized, and their underlying causes revealed by means of qualitative root cause analysis. Based on a survey the innovation barriers were quantitatively ranked based on their relative impact on both regular, and Covid-19 vaccine development timelines. KOLs identified 20 key innovation barriers, and mapping these barriers onto the Vaccine Innovation Cycle model revealed that all phases of vaccine development were affected. Affected by most barriers is the area between the preclinical studies and the market entry. Difficult hand-off between academia and industry, lack of funding, and lack of knowledge of pathogen targets were often mentioned as causes. Quantitative survey responses from 93 KOLs showed that general vaccine development and Covid-19 vaccine development are impacted by distinct sets of innovation barriers. For the general vaccine development three barriers were perceived of the highest impact; limited ROI for vaccines addressing disease with limited market size, limited ROI for vaccines compared to non-vaccine projects, and academia not being able to progress beyond proof of principle. Of highest impact on Covid-19 vaccine development, are lack of knowledge concerning pathogen target, high risk of upscaling unlicensed vaccines, and proof of principle not meeting late-stage requirements. In conclusion, the current study demonstrates that barriers hampering timelines in vaccine development are present across the Vaccine Innovation Cycle. Prioritizing the impact of barriers in general, and in Covid-19 vaccine development, shows clear differences that can be used to inform policies to speed up development in both war and peace time.

Electrochemical investigations for COVID-19 detection-A comparison with other viral detection methods.

Virus-induced infection such as SARS-CoV-2 is a serious threat to human health and the economic setback of the world. Continued advances in the development of technologies are required before the viruses undergo mutation. The low concentration of viruses in environmental samples makes the detection extremely challenging; simple, accurate and rapid detection methods are in urgent need. Of all the analytical techniques, electrochemical methods have the established capabilities to address the issues. Particularly, the integration of nanotechnology would allow miniature devices to be made available at the point-of-care. This review outlines the capabilities of electrochemical methods in conjunction with nanotechnology for the detection of SARS-CoV-2. Future directions and challenges of the electrochemical biosensors for pathogen detection are covered including wearable and conformal biosensors, detection of plant pathogens, multiplexed detection, and reusable biosensors for on-site monitoring, thereby providing low-cost and disposable biosensors.

Delayed Presentation of STEMI Complicated by Ventricular Septal Rupture in the Era of COVID-19 Pandemic.

A significant concern in current coronavirus disease-2019 (COVID-19) pandemic era is delay in first medical contact in patients with ST-segment elevation myocardial infarction (STEMI), due to reluctance to visit the hospital. We report a case of delayed presentation of STEMI as ventricular septal rupture during the COVID-19 pandemic, a rare presentation in the current age of primary percutaneous coronary intervention. (Level of Difficulty: Beginner.).

Post-MI Ventricular Septal Defect During the COVID-19 Pandemic.

With the COVID-19 pandemic, the fear among patients of contracting it has made them reluctant to seek medical attention on a timely basis even for emergent conditions. We present a case of post infarction ventricular septal rupture due to delayed presentation as a consequence of the fear of COVID-19. (Level of Difficulty: Intermediate.).