Genomic Surveillance for Monitoring Variants of Concern: SARS-CoV-2 Delta, Omicron, and Beyond.

The continuing emergence of new SARS-CoV-2 variants has perpetuated the current pandemic far beyond initial expectations. It is now likely that this virus is here to stay. Thus, a new infrastructure is required for monitoring and tracking of viral outbreaks which includes epidemiological and genomic surveillance. More effective monitoring will support rapid response times required for development of new treatments and vaccines to help manage the spread of the current virus and prepare the platforms required for future pandemics.

A Deep Learning Approach to Identify Chest Computed Tomography Features for Prediction of SARS-CoV-2 Infection Outcomes.

There is still an urgent need to develop effective treatments to help minimize the cases of severe COVID-19. A number of tools have now been developed and applied to address these issues, such as the use of non-contrast chest computed tomography (CT) for evaluation and grading of the associated lung damage. Here we used a deep learning approach for predicting the outcome of 1078 patients admitted into the Baqiyatallah Hospital in Tehran, Iran, suffering from COVID-19 infections in the first wave of the pandemic. These were classified into two groups of non-severe and severe cases according to features on their CT scans with accuracies of approximately 0.90. We suggest that incorporation of molecular and/or clinical features, such as multiplex immunoassay or laboratory findings, will increase accuracy and sensitivity of the model for COVID-19 -related predictions.

Point-of-Care Device for Assessment of Blood Coagulation Status in COVID-19 Patients.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent driving the current COVID-19 pandemic. The acute respiratory distress that occurs in some severe COVID-19 cases has been linked with hypercoagulation or thrombotic events as well as a worse prognosis and increased risk of death. Thus, point-of-care devices that can be used for early detection of coagulation abnormalities would assist in COVID-19 management. This chapter describes the use of the Roche Diagnostics CoaguChek® XS test kit for potential use in COVID-19 personalized medicine approaches.

Multiplex Testing of Oxidative-Reductive Pathway in Patients with COVID-19.

Infection with SARS-CoV-2, the causative agent of COVID-19, causes numerous cellular dysfunctions. The virus enters the host cells and hijacks the cell machinery for its replication, resulting in disturbances of the oxidative, reductive balance, increased production of damaging reactive oxygen species (ROS), and mitochondrial dysfunction. This damaging cycle can make cells less resistant to infection and make the host more likely to experience a severe disease course. Treatment with antioxidants has been tested as a potential approach to reduce the effects of this disorder. Here, we present a protocol to assess the impact of treatment with a mixture of curcuminoids on physiological and molecular biomarkers, focusing on determining total antioxidant capacity. We used a cohort of diabetes patients with an imbalance in redox mechanisms as such patients are more likely to become severely ill from COVID-19 than healthy persons.

Measurement of Mitochondrial Respiration in Cryopreserved Human Peripheral Blood Mononuclear Cells (PBMCs).

Inflammatory diseases caused by infectious agents such as the SARS-CoV-2 virus can lead to impaired reductive-oxidative (REDOX) balance and disrupted mitochondrial function. Peripheral blood mononuclear cells (PBMCs) provide a useful model for studying the effects of inflammatory diseases on mitochondrial function but can be limited by the need to store these cells by cryopreservation prior to assay. Here, we describe a method for improving and determining PBMC viability with normalization of values to number of living cells. The approach can be applied not only to PBMC samples derived from patients with diseases marked by an altered inflammatory response such as viral infections.

COVID-19 Detection Using the NHS Lateral Flow Test Kit.

Approximately one in three people infected with the SARS-CoV-2 virus have mild symptoms or are asymptomatic. However, these individuals can still spread the virus. Regular self-testing can help to detect these individuals and thereby slow the spread and protect the more vulnerable members of society. Here, we present a protocol for use of the COVID-19 rapid antigen test which was made freely available to residents of the United Kingdom in April of this year. This using the lateral flow technique for detection of antigens and is amenable to multiplexing.

Evaluating the Effects of Curcumin on the Cytokine Storm in COVID-19 Using a Chip-Based Multiplex Analysis.

SARS-CoV-2 can stimulate the expression of various inflammatory cytokines and induce the cytokine storm in COVID-19 patients leading to multiple organ failure and death. Curcumin as a polyphenolic compound has been shown to have anti-inflammatory properties and inhibit the release of numerous pro-inflammatory cytokines. We present multiplex analysis using the Evidence Investigator biochip system to determine the effect of curcumin on serum level of cytokines which are typically elevated in cytokine storm events, including tumor necrosis factor (TNF-α), interleukin 6 (IL-6), and IL-10.

Multiplex Testing of the Effect of Statins on Disease Severity Risk in COVID-19 Cases.

Statins have pleiotropic effects on inflammatory responses in addition to their lipid-lowering action, which contributes to their favorable effect on cardiovascular disorders. Statins affect adhesion, migration, antigen presentation, and cytokine generation of immune cells. Pre-clinical and clinical studies suggest that statin intervention targeted early in the infection might help COVID-19 patients to reduce the effects of acute respiratory distress syndrome (ARDS), the cytokine storm, and vascular collapse by modulating harmful pathogenic mechanisms. This chapter presents a protocol for measuring blood-based biomarkers predictive of these responses in COVID-19 patients using two specific multiplex immunoassays that target proteins that differ widely in concentration.

Multiplex Immunoassay for Prediction of Disease Severity Associated with the Cytokine Storm in COVID-19 Cases.

Severe cases of SARS-CoV-2 and other pathogenic virus infections are often associated with the uncontrolled release of proinflammatory cytokines, known as a "cytokine storm." We present a protocol for multiplex analysis of three cytokines, tumor necrosis factor-alpha (TNF-a), interleukin 6 (IL-6), and IL-10, which are typically elevated in cytokine storm events and may be used as a predictive biomarker profile of disease severity or disease course.

Lab-on-a-Chip Immunoassay for Prediction of Severe COVID-19 Disease.

Most people infected by the SARS-CoV-2 virus which causes COVID-19 disease experience mild or no symptoms. Severe forms of the disease are often marked by a hyper-inflammatory response known as a cytokine storm. Thus, biomarker tests which can identify these patients and place them on the appropriate treatment regime at the earliest possible phase would help to improve outcomes. Here we describe an automated microarray-based immunoassay using the Fraunhofer lab-on-a-chip platform for analysis of C-reactive protein due to its role in the hyper-inflammatory response.

Liquid Chromatography-Mass Spectrometry Analysis of Peripheral Blood Mononuclear Cells from SARS-CoV-2 Infected Patients.

COVID-19 disease is caused by infection with the SARS-CoV-2 virus and is associated with a cytokine storm effect in some patients. This can lead to decreased ability of the host to cope with the infection and result in severe disease outcomes. Here, we present a protocol for isolation of peripheral blood mononuclear cells (PBMCs) from COVID-19 patients followed by liquid chromatography-mass spectrometry (LC-MS) profiling to identify the affected molecules and molecular pathways. It is hoped that this will lead to the identification of potential biomarkers for monitoring the disease as well as treatment responses. This approach could also be used in the study of other respiratory viruses.

Antibody-Based Affinity Capture Combined with LC-MS Analysis for Identification of COVID-19 Disease Serum Biomarkers.

Blood serum or plasma proteins are potentially useful in COVID-19 research as biomarkers for risk prediction, diagnosis, stratification, and treatment monitoring. However, serum protein-based biomarker identification and validation is complicated due to the wide concentration range of these proteins, which spans more than ten orders of magnitude. Here we present a combined affinity purification-liquid chromatography mass spectrometry approach which allows identification and quantitation of the most abundant serum proteins along with the nonspecifically bound and interaction proteins. This led to the reproducible identification of more than 100 proteins that were not specifically targeted by the affinity column. Many of these have already been implicated in COVID-19 disease.

A Rapid User-Friendly Lab-on-a-Chip Microarray Platform for Detection of SARS-CoV-2 Variants.

Since the original SARS-CoV-2 virus emerged from Wuhan, China, in late December 2019, a number of variants have arisen with enhanced infectivity, and some may even be capable of escaping the existing vaccines. Here we describe a rapid automated nucleic acid microarray hybridization and readout in less than 15 min using the Fraunhofer lab-on-a-chip platform for identification of bacterial species and antibiotic resistance. This platform allows a fast adaptation of new biomarkers enabling identification of different genes and gene mutations, such as those seen in the case the SARS-CoV-2 variants.

Multiplex Quantitative Polymerase Chain Reaction Test to Identify SARS-CoV-2 Variants.

Quantitative polymerase chain reaction (qPCR) is a routinely used method for detection and quantitation of gene expression in real time. This is achieved through the incorporation and measurement of fluorescent reporter probes in the amplified cDNA strands, since the fluorescent signals increase as the reaction progresses. The availability of multiple probes which fluoresce at different wavelengths allows for multiplexing as this gives rise to amplicons with unique fluorescent signatures. Here we describe a method using the Inhibitor-Tolerant RT-qPCR kit, developed by Meridian Bioscience kit which allows simultaneous real-time quantitation of the UK, South Africa, and Brazil SARS-CoV-2 variants.

Multiplex Quantitative Polymerase Chain Reaction Diagnostic Test for SARS-CoV-2 and Influenza A/B Viruses.

COVID-19 disease caused by the novel SARS-CoV-2 virus represents a new challenge for healthcare systems. The molecular confirmation of infection is crucial to guide public health decision-making. This task could be made more difficult during the next influenza season. Thus, a rapid and user-friendly diagnostic test to discriminate SARS-CoV-2 from influenza viruses is urgently needed. Here, we present a multiplex quantitative polymerase chain reaction (qPCR) assay capable of distinguishing SARS-CoV-2 from influenza A and B cases. This assay benefits from the use of an inhibitor tolerant PCR mix which obviates the need for the rate-limiting extraction step, allowing for a more rapid and accurate analysis.

Challenges of Multiplex Assays for COVID-19 Research: A Machine Learning Perspective.

Multiplex assays that provide simultaneous measurement of multiple analytes in biological samples have now developed into widely used technologies in the study of diseases, drug discovery, and other medical areas. These approaches span multiple assay systems and can provide readouts of specific assay components with similar accuracy as the respective single assay measurements. Multiplexing allows the consumption of lower sample volumes, lower costs, and higher throughput compared with carrying out single assays. A number of recent studies have demonstrated the impact of multiplex assays in the study of the SARS-CoV-2 virus, the infectious agent responsible for the current COVID-19 pandemic. In this respect, machine learning techniques have proven to be highly valuable in capturing complex disease phenotypes and converting these insights into models which can be applied in real-world settings. This chapter gives an overview of opportunities and challenges of multiplexed biomarker analysis, with a focus on the use of machine learning aimed at identification of biological signatures for increasing our understanding of COVID-19 disease, and for improved diagnostics and prediction of disease outcomes.

Multivalent Vaccine Strategies in Battling the Emergence of COVID-19 Variants.

The emergence of new SARS-CoV-2 variants has led to increased transmission and more severe cases of COVID-19, with some having the ability to escape the existing vaccines. This review discusses the importance of developing new vaccine strategies to keep pace with these variants to more effectively manage the pandemic. Many of the new vaccine approaches include multivalent display of the most highly mutated regions in the SARS-CoV-2 spike protein such that they resemble a virus particle and can stimulate an effective neutralization response. It is hoped that such approaches help to manage the existing pandemic and provide a robust infrastructure toward fast tracking responses across the world in case of future pandemics.

Multiplex Technologies in COVID-19 Research, Diagnostics, and Prognostics: Battling the Pandemic.

Due to continuous technical developments and new insights into the high complexity of infectious diseases such as COVID-19, there is an increasing need for multiplex biomarkers to aid clinical management and support the development of new drugs and vaccines. COVID-19 disease requires rapid diagnosis and stratification to enable the most appropriate treatment course for the best possible outcomes for patients. In addition, these tests should be rapid, specific, and sensitive. They should rule out other potential causes of illness with simultaneous testing for other diseases. Elevated levels of specific biomarkers can be used to establish severity risks of chronic diseases so that patients can be provided the proper medication at the right time. This review describes the state-of-the-art technologies in proteomics, transcriptomics, and metabolomics, for multiplex biomarker approaches in COVID-19 research.

Effectiveness of Curcumin on Outcomes of Hospitalized COVID-19 Patients: A Systematic Review of Clinical Trials.

Despite the ongoing vaccination efforts, there is still an urgent need for safe and effective treatments to help curb the debilitating effects of COVID-19 disease. This systematic review aimed to investigate the efficacy of supplemental curcumin treatment on clinical outcomes and inflammation-related biomarker profiles in COVID-19 patients. We searched PubMed, Scopus, Web of Science, EMBASE, ProQuest, and Ovid databases up to 30 June 2021 to find studies that assessed the effects of curcumin-related compounds in mild to severe COVID-19 patients. Six studies were identified which showed that curcumin supplementation led to a significant decrease in common symptoms, duration of hospitalization and deaths. In addition, all of these studies showed that the intervention led to amelioration of cytokine storm effects thought to be a driving force in severe COVID-19 cases. This was seen as a significant (p < 0.05) decrease in proinflammatory cytokines such as IL1ß and IL6, with a concomitant significant (p < 0.05) increase in anti-inflammatory cytokines, including IL-10, IL-35 and TGF-α. Taken together, these findings suggested that curcumin exerts its beneficial effects through at least partial restoration of pro-inflammatory/anti-inflammatory balance. In conclusion, curcumin supplementation may offer an efficacious and safe option for improving COVID-19 disease outcomes. We highlight the point that future clinical studies of COVID-19 disease should employ larger cohorts of patients in different clinical settings with standardized preparations of curcumin-related compounds.

Resveratrol as a Probable Multiheaded Treatment Approach for COVID-19.

The COVID-19 pandemic has plagued the world for more than 1 year now and has resulted in over 77 million cases and 1.7 million related deaths. While we await the rollout of the vaccines, new treatments are urgently needed to reduce the effects of this devastating virus. Here, we describe a number of preclinical studies which show promising effects of the polyphenol resveratrol.