Biomarkers of COVID-19 and technologies to combat SARS-CoV-2.

Due to the unprecedented public health crisis caused by COVID-19, our first contribution to the newly launching journal, Advances in Biomarker Sciences and Technology, has abruptly diverted to focus on the current pandemic. As the number of new COVID-19 cases and deaths continue to rise steadily around the world, the common goal of healthcare providers, scientists, and government officials worldwide has been to identify the best way to detect the novel coronavirus, named SARS-CoV-2, and to treat the viral infection - COVID-19. Accurate detection, timely diagnosis, effective treatment, and future prevention are the vital keys to management of COVID-19, and can help curb the viral spread. Traditionally, biomarkers play a pivotal role in the early detection of disease etiology, diagnosis, treatment and prognosis. To assist myriad ongoing investigations and innovations, we developed this current article to overview known and emerging biomarkers for SARS-CoV-2 detection, COVID-19 diagnostics, treatment and prognosis, and ongoing work to identify and develop more biomarkers for new drugs and vaccines. Moreover, biomarkers of socio-psychological stress, the high-technology quest for new virtual drug screening, and digital applications are described.

Chemically Amplified Multiplex Detection of SARS-CoV-2 and Influenza A and B Viruses via Paint-Programmed Lateral Flow Assays.

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) continues to threaten lives by evolving into new variants with greater transmissibility. Although lateral flow assays (LFAs) are widely used to self-test for coronavirus disease 2019 (COVID-19), these tests suffer from low sensitivity leading to a high rate of false negative results. In this work, a multiplexed lateral flow assay is reported for the detection of SARS-CoV-2 and influenza A and B viruses in human saliva with a built-in chemical amplification of the colorimetric signal for enhanced sensitivity. To automate the amplification process, the paper-based device is integrated with an imprinted flow controller, which coordinates the routing of different reagents and ensures their sequential and timely delivery to run an optimal amplification reaction. Using the assay, SARS-CoV-2 and influenza A and B viruses can be detected with ≈25x higher sensitivity than commercial LFAs, and the device can detect SARS-CoV-2-positive patient saliva samples missed by commercial LFAs. The technology provides an effective and practical solution to enhance the performance of conventional LFAs and will enable sensitive self-testing to prevent virus transmission and future outbreaks of new variants.

Monoclonal Antibodies Specific for SARS-CoV-2 Spike Protein Suitable for Multiple Applications for Current Variants of Concern.

The global coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spawned an ongoing demand for new research reagents and interventions. Herein we describe a panel of monoclonal antibodies raised against SARS-CoV-2. One antibody showed excellent utility for immunohistochemistry, clearly staining infected cells in formalin-fixed and paraffin embedded lungs and brains of mice infected with the original and the omicron variants of SARS-CoV-2. We demonstrate the reactivity to multiple variants of concern using ELISAs and describe the use of the antibodies in indirect immunofluorescence assays, Western blots, and rapid antigen tests. Finally, we illustrate the ability of two antibodies to reduce significantly viral tissue titers in K18-hACE2 transgenic mice infected with the original and an omicron isolate of SARS-CoV-2.

Integration of electrokinetic enrichment of spike protein with a lateral flow assay

Here, we present a SARS-CoV-2 spike (S) protein lateral flow assay enhanced by electrokinetic focusing driven by ion concentration polarization (ICP). In this study, we optimize the configuration of membrane-modified electrodes for a stable location and greatest degree of enrichment for the plug of S-protein. We also show that a test line comprising bioconjugated beads further enhances enrichment. Using this system, we have achieved 40X enrichment of S-protein. We are currently evaluating the impact of increased dwell time over the test line. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.

RAPID, CAPILARY-DRIVEN IMMUNOASSAY FOR SARS-CoV-2 DETECTION

A rapid home-diagnostic test for SARS-CoV-2 was developed that automates the reagent delivery and washing steps required for an enzyme-linked immunosorbent assay (ELISA). The device is made of inexpensive polyethylene film and double-sided adhesive that is patterned, cut, and laminated together to create hollow channels. After sample is added, sample, reagents, and washing buffer are sequentially delivered to and washed from a detection zone on a nitrocellulose test strip, giving the end-user a visual readout in <15 minutes. A smartphone camera was used to capture images, and an analytical limit of detection of 35 PFU/mL was determined. When 22 untrained end-users were asked to visually identify a positive result, 95% correctly identified 150 PFU/mL and above as positive. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.

Wicking in Porous Polymeric Membranes: Determination of an Effective Capillary Radius to Predict the Flow Behavior in Lateral Flow Assays.

The working principle of lateral flow assays, such as the widely used COVID-19 rapid tests, is based on the capillary-driven liquid transport of a sample fluid to a test line using porous polymeric membranes as the conductive medium. In order to predict this wicking process by simplified analytical models, it is essential to determine an effective capillary radius for the highly porous and open-pored membranes. In this work, a parametric study is performed with selected simplified structures, representing the complex microstructure of the membrane. For this, a phase-field approach with a special wetting boundary condition to describe the meniscus formation and the corresponding mean surface curvature for each structure setup is used. As a main result, an analytical correlation between geometric structure parameters and an effective capillary radius, based on a correction factor, are obtained. The resulting correlation is verified by applying image analysis methods on reconstructed computer tomography scans of two different porous polymeric membranes and thus determining the geometric structure parameters. Subsequently, a macroscale flow model that includes the correlated effective pore size and geometrical capillary radius is applied, and the results are compared with wicking experiments. Based on the derived correction function, it is shown that the analytical prediction of the wicking process in highly porous polymeric membranes is possible without the fitting of experimental wicking data. Furthermore, it can be seen that the estimated effective pore radius of the two membranes is 8 to 10 times higher than their geometric mean pore radii.

Point-of-Care Diagnostics for Farm Animal Diseases: From Biosensors to Integrated Lab-on-Chip Devices.

Zoonoses and animal diseases threaten human health and livestock biosecurity and productivity. Currently, laboratory confirmation of animal disease outbreaks requires centralized laboratories and trained personnel; it is expensive and time-consuming, and it often does not coincide with the onset or progress of diseases. Point-of-care (POC) diagnostics are rapid, simple, and cost-effective devices and tests, that can be directly applied on field for the detection of animal pathogens. The development of POC diagnostics for use in human medicine has displayed remarkable progress. Nevertheless, animal POC testing has not yet unfolded its full potential. POC devices and tests for animal diseases face many challenges, such as insufficient validation, simplicity, and portability. Emerging technologies and advanced materials are expected to overcome some of these challenges and could popularize animal POC testing. This review aims to: (i) present the main concepts and formats of POC devices and tests, such as lateral flow assays and lab-on-chip devices; (ii) summarize the mode of operation and recent advances in biosensor and POC devices for the detection of farm animal diseases; (iii) present some of the regulatory aspects of POC commercialization in the EU, USA, and Japan; and (iv) summarize the challenges and future perspectives of animal POC testing.

Customization and testing of a mobile reader app for an open-access SARS-CoV-2 antigen lateral flow assay

Lateral flow assays (LFA’s) are a common diagnostic test form, particularly in low-to-middle income countries (LMIC’s). Visual interpretation of LFA’s can be subjective and inconsistent, especially with faint positive results, and commercial readers are expensive and challenging to implement in LMIC’s. We report a phone-agnostic Android app to acquire images and interpret results of a variety of LFA’s with no additional hardware. Starting from the open-source “rdt-scan” codebase, we integrated new features and revamped the peak detection method. This included improved perspective corrections, phone level check to eliminate shadows, high resolution still-image capture besides existing video frame capture, and new peak detection method. This peak detection incorporated smoothing and baseline removal from the one-dimensional profiles of a given color channel’s intensity averaged across the read window’s width, with location and relative size constraints to correctly report locations and peak heights of control and test lines. The app was tested in a real-world setting in conjunction with an open-access LFA for SARS-CoV-2 antigen developed by GH Labs. The app acquired 155 images of LFA cassettes, and results were compared against both visual interpretation by trained clinical staff and PCR results from the same patients. With an appropriate setting for test line intensity threshold, the app matched visual read for all cases but one missed visual positive. From ROC analyses against PCR, the app outperformed visual read by 1-3% across sensitivity, specificity, and AUC. The app thus demonstrated promise for accurate, consistent interpretation of LFA’s while generating digital records that could also be useful for health surveillance. © 2022 SPIE

Open-Source, Adaptable, All-in-One Smartphone-Based System for Quantitative Analysis of Point-of-Care Diagnostics.

Point-of-care (POC) diagnostics, in particular lateral flow assays (LFA), represent a great opportunity for rapid, precise, low-cost and accessible diagnosis of disease. Especially with the ongoing coronavirus disease 2019 (COVID-19) pandemic, rapid point-of-care tests are becoming everyday tools for identification and prevention. Using smartphones as biosensors can enhance POC devices as portable, low-cost platforms for healthcare and medicine, food and environmental monitoring, improving diagnosis and documentation in remote, low-resource locations. We present an open-source, all-in-one smartphone-based system for quantitative analysis of LFAs. It consists of a 3D-printed photo box, a smartphone for image acquisition, and an R Shiny software package with modular, customizable analysis workflow for image editing, analysis, data extraction, calibration and quantification of the assays. This system is less expensive than commonly used hardware and software, so it could prove very beneficial for diagnostic testing in the context of pandemics, as well as in low-resource countries.

Efficacy of POC Antibody Assays after COVID-19 Infection and Potential Utility for "Immunity Passports".

OBJECTIVE: Numerous manufacturers market lateral flow assays for the detection of SARS-CoV-2 antibodies, but there are many questions about the reliability and efficacy of these tests. MATERIALS AND METHODS: Serum specimens from 60 individuals were analyzed using 2 lateral flow antibody assays, an in-house enzyme-linked immunosorbent assay (ELISA), and the Abbott SARS-CoV-2 IgG chemiluminescent immunoassay. RESULTS: The BioMedomics and Premier Biotech lateral flow assays were positive for IgM in 73.3% and 70% and for IgG in 80% and 73.3% of specimens, respectively. The ELISA assay was positive for IgM and IgG in 73.3% and 86.7% of specimens from infected individuals, whereas the Abbott assay was positive in 80%. The specificities of the 4 assays ranged from 96.7% to 100% for IgM and from 93.3% to 100% for IgG. CONCLUSION: Results of the 2 lateral flow assays were comparable to those of the ELISA and Abbott assays. Assay efficacy depended on length of time after SARS-CoV-2 infection.

Lateral flow assays (LFA) as an alternative medical diagnosis method for detection of virus species: The intertwine of nanotechnology with sensing strategies.

Viruses are responsible for multiple infections in humans that impose huge health burdens on individuals and populations worldwide. Therefore, numerous diagnostic methods and strategies have been developed for prevention, management, and decreasing the burden of viral diseases, each having its advantages and limitations. Viral infections are commonly detected using serological and nucleic acid-based methods. However, these conventional and clinical approaches have some limitations that can be resolved by implementing other detector devices. Therefore, the search for sensitive, selective, portable, and costless approaches as efficient alternative clinical methods for point of care testing (POCT) analysis has gained much attention in recent years. POCT is one of the ultimate goals in virus detection, and thus, the tests need to be rapid, specific, sensitive, accessible, and user-friendly. In this review, after a brief overview of viruses and their characteristics, the conventional viral detection methods, the clinical approaches, and their advantages and shortcomings are firstly explained. Then, LFA systems working principles, benefits, classification are discussed. Furthermore, the studies regarding designing and employing LFAs in diagnosing different types of viruses, especially SARS-CoV-2 as a main concern worldwide and innovations in the LFAs' approaches and designs, are comprehensively discussed here. Furthermore, several strategies addressed in some studies for overcoming LFA limitations like low sensitivity are reviewed. Numerous techniques are adopted to increase sensitivity and perform quantitative detection. Employing several visualization methods, using different labeling reporters, integrating LFAs with other detection methods to benefit from both LFA and the integrated detection device advantages, and designing unique membranes to increase reagent reactivity, are some of the approaches that are highlighted.

SARS-CoV-2 Antibody Rapid Tests: Valuable Epidemiological Tools in Challenging Settings.

During the last year, mass screening campaigns have been carried out to identify immunological response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and establish a possible seroprevalence. The obtained results gained new importance with the beginning of the SARS-CoV-2 vaccination campaign, as the lack of doses has persuaded several countries to introduce different policies for individuals who had a history of COVID-19. Lateral flow assays (LFAs) may represent an affordable tool to support population screening in low-middle-income countries, where diagnostic tests are lacking and epidemiology is still widely unknown. However, LFAs have demonstrated a wide range of performance, and the question of which one could be more valuable in these settings still remains. We evaluated the performance of 11 LFAs in detecting SARS-CoV-2 infection, analyzing samples collected from 350 subjects. In addition, samples from 57 health care workers collected at 21 to 24 days after the first dose of the Pfizer-BioNTech vaccine were also evaluated. LFAs demonstrated a wide range of specificity (92.31% to 100%) and sensitivity (50% to 100%). The analysis of postvaccination samples was used to describe the most suitable tests to detect IgG response against S protein receptor binding domain (RBD). Tuberculosis (TB) therapy was identified as a potential factor affecting the specificity of LFAs. This analysis identified which LFAs represent a valuable tool not only for the detection of prior SARS-CoV-2 infection but also for the detection of IgG elicited in response to vaccination. These results demonstrated that different LFAs may have different applications and the possible risks of their use in high-TB-burden settings. IMPORTANCE Our study provides a fresh perspective on the possible employment of SARS-CoV-2 LFA antibody tests. We developed an in-depth, large-scale analysis comparing LFA performance to enzyme-linked immunosorbent assay (ELISA) and electrochemiluminescence immunoassay (ECLIA) and evaluating their sensitivity and specificity in identifying COVID-19 patients at different time points from symptom onset. Moreover, for the first time, we analyzed samples of patients undergoing treatment for endemic poverty-related diseases, especially tuberculosis, and we evaluated the impact of this therapy on test specificity in order to assess possible performance in TB high-burden countries.

Evaluation of serological lateral flow assays for severe acute respiratory syndrome coronavirus-2.

BACKGROUND: COVID-19 has resulted in significant morbidity and mortality worldwide. Lateral flow assays can detect anti-Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) antibodies to monitor transmission. However, standardized evaluation of their accuracy and tools to aid in interpreting results are needed. METHODS: We evaluated 20 IgG and IgM assays selected from available tests in April 2020. We evaluated the assays' performance using 56 pre-pandemic negative and 56 SARS-CoV-2-positive plasma samples, collected 10-40 days after symptom onset, confirmed by a molecular test and analyzed by an ultra-sensitive immunoassay. Finally, we developed a user-friendly web app to extrapolate the positive predictive values based on their accuracy and local prevalence. RESULTS: Combined IgG + IgM sensitivities ranged from 33.9 to 94.6%, while combined specificities ranged from 92.6 to 100%. The highest sensitivities were detected in Lumiquick for IgG (98.2%), BioHit for both IgM (96.4%), and combined IgG + IgM sensitivity (94.6%). Furthermore, 11 LFAs and 8 LFAs showed perfect specificity for IgG and IgM, respectively, with 15 LFAs showing perfect combined IgG + IgM specificity. Lumiquick had the lowest estimated limit-of-detection (LOD) (0.1 µg/mL), followed by a similar LOD of 1.5 µg/mL for CareHealth, Cellex, KHB, and Vivachek. CONCLUSION: We provide a public resource of the accuracy of select lateral flow assays with potential for home testing. The cost-effectiveness, scalable manufacturing process, and suitability for self-testing makes LFAs an attractive option for monitoring disease prevalence and assessing vaccine responsiveness. Our web tool provides an easy-to-use interface to demonstrate the impact of prevalence and test accuracy on the positive predictive values.

Low-Cost Optical Assays for Point-of-Care Diagnosis in Resource-Limited Settings.

Readily deployable, low-cost point-of-care medical devices such as lateral flow assays (LFAs), microfluidic paper-based analytical devices (µPADs), and microfluidic thread-based analytical devices (µTADs) are urgently needed in resource-poor settings. Governed by the ASSURED criteria (affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free, and deliverability) set by the World Health Organization, these reliable platforms can screen a myriad of chemical and biological analytes including viruses, bacteria, proteins, electrolytes, and narcotics. The Ebola epidemic in 2014 and the ongoing pandemic of SARS-CoV-2 have exemplified the ever-increasing importance of timely diagnostics to limit the spread of diseases. This review provides a comprehensive survey of LFAs, µPADs, and µTADs that can be deployed in resource-limited settings. The subsequent commercialization of these technologies will benefit the public health, especially in areas where access to healthcare is limited.

High-Surety Isothermal Amplification and Detection of SARS-CoV-2.

Isothermal nucleic acid amplification tests (iNATs), such as loop-mediated isothermal amplification (LAMP), are good alternatives to PCR-based amplification assays, especially for point-of-care and low-resource use, in part because they can be carried out with relatively simple instrumentation. However, iNATs can often generate spurious amplicons, especially in the absence of target sequences, resulting in false-positive results. This is especially true if signals are based on non-sequence-specific probes, such as intercalating dyes or pH changes. In addition, pathogens often prove to be moving, evolving targets and can accumulate mutations that will lead to inefficient primer binding and thus false-negative results. Multiplex assays targeting different regions of the analyte and logical signal readout using sequence-specific probes can help to reduce both false negatives and false positives. Here, we describe rapid conversion of three previously described SARS-CoV-2 LAMP assays that relied on a non-sequence-specific readout into individual and multiplex one-pot assays that can be visually read using sequence-specific oligonucleotide strand exchange (OSD) probes. We describe both fluorescence-based and Boolean logic-gated colorimetric lateral flow readout methods and demonstrate detection of SARS-CoV-2 virions in crude human saliva.IMPORTANCE One of the key approaches to treatment and control of infectious diseases, such as COVID-19, is accurate and rapid diagnostics that is widely deployable in a timely and scalable manner. To achieve this, it is essential to go beyond the traditional gold standard of quantitative PCR (qPCR) that is often faced with difficulties in scaling due to the complexity of infrastructure and human resource requirements. Isothermal nucleic acid amplification methods, such as loop-mediated isothermal amplification (LAMP), have been long pursued as ideal, low-tech alternatives for rapid, portable testing. However, isothermal approaches often suffer from false signals due to employment of nonspecific readout methods. We describe general principles for rapidly converting nonspecifically read LAMP assays into assays that are read in a sequence-specific manner by using oligonucleotide strand displacement (OSD) probes. We also demonstrate that inclusion of OSD probes in LAMP assays maintains the simplicity of one-pot assays and a visual yes/no readout by using fluorescence or colorimetric lateral-flow dipsticks while providing accurate sequence-specific readout and the ability to logically query multiplex amplicons for redundancy or copresence. These principles not only yielded high-surety isothermal assays for SARS-CoV-2 but might also aid in the design of more sophisticated molecular assays for other analytes.

Evaluation of two automated and three rapid lateral flow immunoassays for the detection of anti-SARS-CoV-2 antibodies.

INTRODUCTION: Several SARS-CoV-2 immunoassays have been developed recently. The purpose of this study was to assess the performance of five immunoassays for the detection of SARS-CoV-2 antibodies. METHODS: Two quantitative automated immunoassays (Maglumi™2019-n-Cov IgG and IgM and Euroimmun Anti-SARS-CoV-2 IgG and IgA assays) and three lateral flow rapid tests were performed. This retrospective study included 200 residual sera from patients and healthy volunteers. Case serum samples (n = 128) were obtained from COVID-19 patients confirmed by RT-qPCR and CT-scan. Days since onset of symptoms was collected from their medical records. Control non-SARS-CoV-2 samples (n = 72) were obtained from anonymous stored residual serum samples. RESULTS: Maglumi™ IgG/IgM tests showed overall less sensitivity than Euroimmun IgG/IgA test (84.4 % versus 64.3 %). Both tests showed similar specificities of IgG at 99 % and 100 %, respectively. The results from the lateral flow assays were easily interpretable with unambiguous coloured reading bands. The overall sensitivity of the three tests was similar (around 70 %) without any significant differences. The sensitivity of the three lateral flow assays and also of the serological quantitative assays increased during the second week after symptom onset and all reached similar values (91 %-94 %) after 14 days. CONCLUSION: This study shows accurate and equivalent performance of the five serological antibody assays (ELISA, CLIA and three lateral flow tests) in detecting SARS-CoV-2 antibodies 14 days after the onset of COVID-19 symptoms. This is compatible with their application in specific clinical contexts and in determining epidemiological strategies for the COVID-19 pandemic.