Development of receptor binding domain-based double-antigen sandwich lateral flow immunoassay for the detection and evaluation of SARS-CoV-2 neutralizing antibody in clinical sera samples compared with the conventional virus neutralization test.

Vaccination is an effective strategy to fight COVID-19. However, the effectiveness of the vaccine varies among different populations in varying immune effects. Neutralizing antibody (NAb) level is an important indicator to evaluate the protective effect of immune response after vaccination. Lateral flow immunoassay (LFIA) is a rapid, safe and sensitivity detection method, which has great potential in the detection of SARS-CoV-2 NAb. In this study, a fluorescent beads-based lateral flow immunoassay (FBs-LFIA) and a latex beads-based LFIA (LBs-LFIA) using double antigen sandwich (DAS) strategy were established to detect NAbs in the serum of vaccinated people. The limit of detection (LoD) of the FBs-LFIA was 1.13 ng mL- 1 and the LBs-LFIA was 7.11 ng mL- 1. The two LFIAs were no cross-reactive with sera infected by other pathogenic bacteria. Furthermore, the two LFIAs showed a good performance in testing clinical samples. The sensitivity of FBs-LFIA and LBs-LFIA were 97.44% (95%CI: 93.15%-99.18%) and 98.29% (95%CI: 95.84%-99.37%), and the specificity were 98.28% (95%CI: 95.37%-99.45%) and 97.70% (95%CI: 94.82%-99.06%) compared with the conventional virus neutralization test (cVNT), respectively. Notably, the LBs-LFIA was also suitable for whole blood sample, requiring only 3 µL of whole blood, which provided the possibility to detect NAbs at home. To sum up, the two LFIAs based on double antigen sandwich established by us can rapidly, safely, sensitively and accurately detect SARS-CoV-2 NAb in human serum.

COVID-19 serum can be cross-reactive and neutralizing against the dengue virus, as observed by the dengue virus neutralization test.

OBJECTIVES: Observing the serological cross-reactivity between SARS-CoV-2 and dengue virus (DV), we aimed to elucidate its effect on dengue serodiagnosis and infectivity in a highly dengue-endemic city in India. METHODS: A total of 52 COVID-19 (reverse transcription-polymerase chain reaction [RT-PCR] positive) serum samples were tested in rapid lateral flow immunoassays and DV immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) to detect DV or SARS-CoV-2 IgG/immunoglobulin M. The COVID-19 antibody (Ab) positive samples were subjected to a virus neutralization test (Huh7 cells) using DV type 1 (DV1) clinical isolate. RESULTS: Most (93%) of the SARS-CoV-2 Ab-positive serum samples cross-reacted with DV in rapid or ELISA tests. All were DV RNA and nonstructural protein 1 (NS1) antigen-negative. COVID-19 serum samples that were DV cross-reactive neutralized DV1. Of these, 57% had no evidence of DV pre-exposure (DV NS1 Ab-negative). The computational study also supported potential interactions between SARS-CoV-2 Ab and DV1. CONCLUSION: DV serodiagnosis will be inconclusive in areas co-endemic for both viruses. The COVID-19 pandemic appears to impart a protective response against DV in DV-endemic populations.

pyPOCQuant - A tool to automatically quantify Point-Of-Care Tests from images.

Lateral flow Point-Of-Care Tests (POCTs) are a valuable tool for rapidly detecting pathogens and the associated immune response in humans and animals. In the context of the SARS-CoV-2 pandemic, they offer rapid on-site diagnostics and can relieve centralized laboratory testing sites, thus freeing resources that can be focused on especially vulnerable groups. However, visual interpretation of the POCT test lines is subjective, error prone and only qualitative. Here we present pyPOCQuant, an open-source tool implemented in Python 3 that can robustly and reproducibly analyze POCTs from digital images and return an unbiased and quantitative measurement of the POCT test lines.

Summary of the Detection Kits for SARS-CoV-2 Approved by the National Medical Products Administration of China and Their Application for Diagnosis of COVID-19.

The on-going global pandemic of coronavirus disease 2019 (COVID-19) caused by a novel coronavirus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been underway for about 11 months. Through November 20, 2020, 51 detection kits for SARS-CoV-2 nucleic acids (24 kits), antibodies (25 kits), or antigens (2 kits) have been approved by the National Medical Products Administration of China (NMPA). Convenient and reliable SARS-CoV-2 detection assays are urgently needed worldwide for strategic control of the pandemic. In this review, the detection kits approved in China are summarised and the three types of tests, namely nucleic acid, serological and antigen detection, which are available for the detection of COVID-19 are discussed in detail. The development of novel detection kits will lay the foundation for the control and prevention of the COVID-19 pandemic globally.

Laboratory-Based Resources for COVID-19 Diagnostics: Traditional Tools and Novel Technologies. A Perspective of Personalized Medicine.

The coronavirus infection 2019 (COVID-19) pandemic, caused by the highly contagious SARS-CoV-2 virus, has provoked a global healthcare and economic crisis. The control over the spread of the disease requires an efficient and scalable laboratory-based strategy for testing the population based on multiple platforms to provide rapid and accurate diagnosis. With the onset of the pandemic, the reverse transcription polymerase chain reaction (RT-PCR) method has become a standard diagnostic tool, which has received wide clinical use. In large-scale and repeated examinations, these tests can identify infected patients with COVID-19, with their accuracy, however, dependent on many factors, while the entire process takes up to 6-8 h. Here we also describe a number of serological systems for detecting antibodies against SARS-CoV-2. These are used to assess the level of population immunity in various categories of people, as well as for retrospective diagnosis of asymptomatic and mild COVID-19 in patients. However, the widespread use of traditional diagnostic tools in the context of the rapid spread of COVID-19 is hampered by a number of limitations. Therefore, the sharp increase in the number of patients with COVID-19 necessitates creation of new rapid, inexpensive, sensitive, and specific tests. In this regard, we focus on new laboratory technologies such as loop mediated isothermal amplification (LAMP) and lateral flow immunoassay (LFIA), which have proven to work well in the COVID-19 diagnostics and can become a worthy alternative to traditional laboratory-based diagnostics resources. To cope with the COVID-19 pandemic, the healthcare system requires a combination of various types of laboratory diagnostic testing techniques, whodse sensitivity and specificity increases with the progress in the SARS-CoV-2 research. The testing strategy should be designed in such a way to provide, depending on the timing of examination and the severity of the infection in patients, large-scale and repeated examinations based on the principle: screening-monitoring-control. The search and development of new methods for rapid diagnostics of COVID-19 in laboratory, based on new analytical platforms, is still a highly important and urgent healthcare issue. In the final part of the review, special emphasis is made on the relevance of the concept of personalized medicine to combat the COVID-19 pandemic in the light of the recent studies carried out to identify the causes of variation in individual susceptibility to SARS-CoV-2 and increase the efficiency and cost-effectiveness of treatment.

Combined anti-SARS-CoV-2 IgA, IgG, and IgM Detection as a Better Strategy to Prevent Second Infection Spreading Waves.

Coronavirus disease (COVID-19) is challenging many health, economic, and social systems. RT-PCR assays are diagnosis gold standard; however, they can lead to false-negative results. Therefore, anti-SARS-CoV-2 IgG, IgM, and IgA investigation can play a complementary role in assessing the individuals immune status. Majority of serological tests focus on IgM and IgG although IgA are the main immunoglobulins involved in mucosal immunity. It has been reported that digestive symptoms may occur in the absence of any typical respiratory symptom. Thus, a complete screening, comprising IgA, IgM, and IgG detection could be more consistent and useful in patients with atypical symptoms or in paucisymptomatic cases. Current literature describes over 200 immunoassays available worldwide, pointing out a great results variability, depending on methodology or antigens' nature. In our study we evaluated anti-SARS-CoV-2 IgA, IgM, and IgG trend on a control group and on two COVID-19 patient groups (early and late infection time) with a lateral-flow combined immunoassay (LFIA) and an enzyme-linked immunosorbent assay (ELISA). Dissimilar antibodies time kinetics have been described in COVID-19 (decreasing IgM concentration with IgA/IgG persistence for a longer time; as well as persistent IgA, IgG, and IgM concentration); our results confirmed both of them depending on the methodology; therefore, it is difficult to compare different studies outcomes, suggesting the importance of a serological tests international standardization. Nevertheless, we propose a flowchart with combined anti-SARS-CoV-2 IgG/IgM/IgA detection as a screening on general population, where serological positivity should be considered as an "alert," to avoid and contain possible new outbreaks.

Review of Current Advances in Serologic Testing for COVID-19.

OBJECTIVES: To examine and summarize the current literature on serologic methods for the detection of antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). METHODS: A literature review was performed using searches in databases including PubMed, medRxiv, and bioRxiv. Thirty-two peer-reviewed papers and 23 preprints were examined. RESULTS: The studies included lateral flow immunoassay, enzyme-linked immunosorbent assay, chemiluminescence immunoassay, and neutralizing antibody assays. The use of all major SARS-CoV-2 antigens was demonstrated to have diagnostic value. Assays measuring total antibody reactivity had the highest sensitivity. In addition, all the methods provided opportunities to characterize the humoral immune response by isotype. The combined use of IgM and IgG detection resulted in a higher sensitivity than that observed when detecting either isotype alone. Although IgA was rarely studied, it was also demonstrated to be a sensitive marker of infection, and levels correlated with disease severity and neutralizing activity. CONCLUSIONS: The use of serologic testing, in conjunction with reverse transcription polymerase chain reaction testing, was demonstrated to significantly increase the sensitivity of detection of patients infected with SARS-CoV-2. There was conflicting evidence regarding whether antibody titers correlated with clinical severity. However, preliminary investigations indicated some immunoassays may be a surrogate for the prediction of neutralizing antibody titers and the selection of recovered patients for convalescent serum donation.

In Vitro Diagnostic Assays for COVID-19: Recent Advances and Emerging Trends.

There have been tremendous advances in in vitro diagnostic (IVD) assays for coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The main IVD assays used for COVID-19 employ real-time reverse transcriptase polymerase chain reaction (RT-PCR) that takes a few hours. But the assay duration has been shortened to 45 min by Cepheid. Of interest is the point-of-care (POC) molecular assay by Abbott that decreased the assay duration to just 5 min. Most molecular tests have been approved by the United States Food and Drug Administration (FDA) under emergency use authorization (EUA) and are Conformité Européenne (CE) marked. A wide range of serology immunoassays (IAs) have also been developed that complement the molecular assays for the diagnosis of COVID-19. The most prominent IAs are automated chemiluminescent IA (CLIA), manual ELISA, and rapid lateral flow IA (LFIA), which detect the immunoglobulin M (IgM) and immunoglobulin G (IgG) produced in persons in response to SARS-CoV-2 infection. The ongoing research efforts and advances in complementary technologies will pave the way to new POC IVD assays in the coming months. However, the performance of IVD assays needs to be critically evaluated before they are employed for the clinical diagnosis of COVID-19.