Dynamics of SARS-CoV-2 seroassay sensitivity: a systematic review and modelling study.

BackgroundSerological surveys have been the gold standard to estimate numbers of SARS-CoV-2 infections, the dynamics of the epidemic, and disease severity. Serological assays have decaying sensitivity with time that can bias their results, but there is a lack of guidelines to account for this phenomenon for SARS-CoV-2.AimOur goal was to assess the sensitivity decay of seroassays for detecting SARS-CoV-2 infections, the dependence of this decay on assay characteristics, and to provide a simple method to correct for this phenomenon.MethodsWe performed a systematic review and meta-analysis of SARS-CoV-2 serology studies. We included studies testing previously diagnosed, unvaccinated individuals, and excluded studies of cohorts highly unrepresentative of the general population (e.g. hospitalised patients).ResultsOf the 488 screened studies, 76 studies reporting on 50 different seroassays were included in the analysis. Sensitivity decay depended strongly on the antigen and the analytic technique used by the assay, with average sensitivities ranging between 26% and 98% at 6 months after infection, depending on assay characteristics. We found that a third of the included assays departed considerably from manufacturer specifications after 6 months.ConclusionsSeroassay sensitivity decay depends on assay characteristics, and for some types of assays, it can make manufacturer specifications highly unreliable. We provide a tool to correct for this phenomenon and to assess the risk of decay for a given assay. Our analysis can guide the design and interpretation of serosurveys for SARS-CoV-2 and other pathogens and quantify systematic biases in the existing serology literature.

Importance, Applications and Features of Assays Measuring SARS-CoV-2 Neutralizing Antibodies.

More than three years ago, the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) caused the unforeseen COVID-19 pandemic with millions of deaths. In the meantime, SARS-CoV-2 has become endemic and is now part of the repertoire of viruses causing seasonal severe respiratory infections. Due to several factors, among them the development of SARS-CoV-2 immunity through natural infection, vaccination and the current dominance of seemingly less pathogenic strains belonging to the omicron lineage, the COVID-19 situation has stabilized. However, several challenges remain and the possible new occurrence of highly pathogenic variants remains a threat. Here we review the development, features and importance of assays measuring SARS-CoV-2 neutralizing antibodies (NAbs). In particular we focus on in vitro infection assays and molecular interaction assays studying the binding of the receptor binding domain (RBD) with its cognate cellular receptor ACE2. These assays, but not the measurement of SARS-CoV-2-specific antibodies per se, can inform us of whether antibodies produced by convalescent or vaccinated subjects may protect against the infection and thus have the potential to predict the risk of becoming newly infected. This information is extremely important given the fact that a considerable number of subjects, in particular vulnerable persons, respond poorly to the vaccination with the production of neutralizing antibodies. Furthermore, these assays allow to determine and evaluate the virus-neutralizing capacity of antibodies induced by vaccines and administration of plasma-, immunoglobulin preparations, monoclonal antibodies, ACE2 variants or synthetic compounds to be used for therapy of COVID-19 and assist in the preclinical evaluation of vaccines. Both types of assays can be relatively quickly adapted to newly emerging virus variants to inform us about the magnitude of cross-neutralization, which may even allow us to estimate the risk of becoming infected by newly appearing virus variants. Given the paramount importance of the infection and interaction assays we discuss their specific features, possible advantages and disadvantages, technical aspects and not yet fully resolved issues, such as cut-off levels predicting the degree of in vivo protection.

Evaluation and comparison of four quantitative SARS-CoV-2 serological assays in COVID-19 patients and immunized healthy individuals, cancer patients, and patients with immunosuppressive therapy.

BACKGROUND: Semi-quantitative and quantitative immunoassays are the most commonly used methodology to evaluate immunity post immunization. OBJECTIVES: To compare four quantitative SARS-CoV-2 serological assays in COVID-19 patients and immunized healthy individuals, cancer patients, and patients with immunosuppressive therapy. STUDY DESIGN: 210 serological samples from COVID-19 infection and vaccination cohorts were used to create a serological sample repository. Serological methods from four manufacturers, namely Euroimmun, Roche, Abbott, and DiaSorin, were evaluated for quantitative, semi-quantitative, and qualitative antibody measurements. All four methods measure IgG antibodies against the SARS-CoV-2 spike receptor-binding domain and report the results in Binding Antibody Unit/mL (BAU/mL). A Total Error Allowable (TEa) of ±25% was chosen as the criteria to determine whether two methods are clinically equivalent quantitatively. Semi-quantitative results (titers) were derived using numeric antibody concentration divided by the cut-off value for each method. RESULTS: All paired quantitative comparisons demonstrated unacceptable performance. With ±25% as TEa, the best agreement was 74 (35.2% out of 210 samples) between Euroimmun and DiaSorin, whereas the lowest agreement was 11 (5.2% out of 210 samples) between Euroimmun and Roche. Antibody titers amongst all four methods were significantly different (p < 0.001). The highest titer difference from the same sample is between Roche and DiaSorin with a 1392-fold difference. On qualitative comparison, none of the paired comparison showed acceptable comparison (p < 0.001). CONCLUSIONS: Poor correlation exists between four evaluated assays, quantitatively, semi-quantitatively, and qualitatively. Further harmonization of assays is required to achieve comparable measurements.

A HIGH-THROUGHPUT MULTIPLEXED MICROFLUIDIC DEVICE FOR COVID-19 SEROLOGY ASSAYS

The need to develop high-throughput diagnostic platforms for infectious diseases has never been more evident than with the emergence of SARS-CoV-2 and the ensued COVID-19 pandemic. Microfluidics, in tandem with its multiplexing capabilities, high sensitivity, and potential for automation, provides a unique advantage towards the development of high-throughput serological diagnostic platforms. Here, we present a microfluidic device that detects IgG or IgM raised against four SARS-CoV-2 antigens (spike, S;S1 subunit, S1;the receptor-binding domain, RBD;and nucleocapsid, N) from 50 serum samples in parallel. We validated the platform with a cross-sectional cohort of 66 samples from confirmed COVID-19 patients and a pre-pandemic control of 34 serum samples collected in 2018. The analysis of both antibodies against all four viral antigens provided a sensitivity of 90.4% and a specificity of 94.1%, with both parameters increasing to 100% in late-stage samples (21-30 days after symptoms onset). We expect our device to open the door to massive serological testing, impacting diagnostics, vaccine development, and epidemiological understanding of COVID-19. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.

Comparison of SARS-CoV-2 spike antibody quantitative titer reporting using the World Health Organization International Standard Units by four commercial assays.

The accurate measurement of serological response to SARS-CoV-2 vaccination is needed to correlate responses with effective protective immunity. The World Health Organization (WHO) has created an international standard to allow harmonization of immune response assessment to an arbitrary unit across different commercial assays; however, the accuracy of reporting of SARS-CoV-2 spike antibody titers in international standard units (BAU or IU/mL) from commercial assays is not well studied. Here, we report the performance comparison of four quantitative commercial assays testing for SARS-CoV-2 spike immunoglobins using the WHO's international standard. Sera, EDTA-plasma and heparinized plasma collected from individuals who are vaccine naïve or received BNT162b2 (Pfizer/BioNTech), mRNA-1273 (Moderna) or ChAdOx1-S (Oxford-AstraZeneca) were tested using Abbott Architect AdviseDx SARS-CoV-2 IgG II, DiaSorin LIAISON SARS-CoV-2 TrimericS IgG, Roche Elecsys Anti-SARS-CoV-2 S and GenScript cPass SARS-CoV-2 surrogate virus neutralization assays. The sensitivities ranged from 90% to 100%, and specificities from 88% to 100%. These four assays had excellent agreement (0.79-0.93) and correlation (0.87-0.97); however, Passing-Bablok regression analysis indicated that data generated by these assays were not comparable. Our data suggests that natural SARS-CoV-2 infection elicited a greater antibody response compared to vaccines, evident by a significantly higher neutralizing antibody titer in unvaccinated individuals who seroconverted.

Overview of Neutralization Assays and International Standard for Detecting SARS-CoV-2 Neutralizing Antibody.

We aimed to review the existing literature on the different types of neutralization assays and international standards for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We comprehensively summarized the serological assays for detecting neutralizing antibodies against SARS-CoV-2 and demonstrated the importance of an international standard for calibrating the measurement of neutralizing antibodies. Following the coronavirus disease outbreak in December 2019, there was an urgent demand to detect neutralizing antibodies in patients or vaccinated people to monitor disease outcomes and determine vaccine efficacy. Therefore, many approaches were developed to detect neutralizing antibodies against SARS-CoV-2, such as microneutralization assay, SARS-CoV-2 pseudotype virus assay, enzyme-linked immunosorbent assay (ELISA), and rapid lateral flow assay. Given the many types of serological assays for quantifying the neutralizing antibody titer, the comparison of different assay results is a challenge. In 2020, the World Health Organization proposed the first international standard as a common unit to define neutralizing antibody titer and antibody responses against SARS-CoV-2. These standards are useful for comparing the results of different assays and laboratories.

Limited Correlation between SARS-CoV-2 Serologic Assays for Identification of High-Titer COVID-19 Convalescent Plasma Using FDA Thresholds.

In August 2020, the Food and Drug Administration (FDA) Emergency Use Authorization (EUA) for COVID-19 convalescent plasma (CCP) specified 12 authorized serologic assays and associated assay-specific cutoff values for the selection of high-titer CCP for use in hospitalized patients. The criteria used for establishing these cutoff values remains unclear. Here, we compare the overall agreement and concordance of five serologic assays included in the August 2020 FDA EUA at both the manufacturer-recommended qualitative cutoff thresholds and at the FDA-indicated thresholds for high-titer CCP, using serum samples collected as part of the CCP Expanded Access Program (EAP). The qualitative positive percent agreement (PPA) across assays ranged from 92.3% to 98.8%. However, the high-titer categorization across assays varied significantly, with the PPA ranging from 26.5% to 82.7%. The Roche anti-NC ECLIA provided the lowest agreement compared to all other assays. Efforts to optimize high-titer cutoffs could reduce, although not eliminate, the discordance across assays. The consequences of using nonstandardized assays are apparent in our study, and the high-titer cutoffs chosen for each assay are not directly comparable to each other. The generalized findings in our study will be relevant to any future use of convalescent plasma for either COVID-19 or future pandemics of newly emerged pathogens. IMPORTANCE COVID-19 convalescent plasma (CCP) was one of the first therapeutic options available for the treatment of SARS-CoV-2 infections and continues to be used selectively for immunosuppressed patients. Given the emergence of novel SARS-CoV-2 variants which are resistant to treatment with available monoclonal antibody (MAb) therapy, CCP remains an important therapeutic consideration. The FDA has released several emergency use authorizations (EUA) that have specified which serological assays can be used for qualification of CCP, as well as assay-specific cutoffs that must be used to identify high-titer CCP. In this study, a cohort of donor CCP was assessed across multiple serological assays which received FDA EUA for qualification of CCP. This study indicates a high degree of discordance across the assays used to qualify CCP for clinical use, which may have precluded the optimal use of CCP, including during clinical trials. This study highlights the need for assay standardization early in the development of serological assays for emerging pathogens.

Applying mixture model methods to SARS-CoV-2 serosurvey data from Geneva.

Serosurveys are an important tool to estimate the true extent of the current SARS-CoV-2 pandemic. So far, most serosurvey data have been analyzed with cutoff-based methods, which dichotomize individual measurements into sero-positives or negatives based on a predefined cutoff. However, mixture model methods can gain additional information from the same serosurvey data. Such methods refrain from dichotomizing individual values and instead use the full distribution of the serological measurements from pre-pandemic and COVID-19 controls to estimate the cumulative incidence. This study presents an application of mixture model methods to SARS-CoV-2 serosurvey data from the SEROCoV-POP study from April and May 2020 in Geneva (2766 individuals). Besides estimating the total cumulative incidence in these data (8.1% (95% CI: 6.8%-9.9%)), we applied extended mixture model methods to estimate an indirect indicator of disease severity, which is the fraction of cases with a distribution of antibody levels similar to hospitalized COVID-19 patients. This fraction is 51.2% (95% CI: 15.2%-79.5%) across the full serosurvey, but differs between three age classes: 21.4% (95% CI: 0%-59.6%) for individuals between 5 and 40 years old, 60.2% (95% CI: 21.5%-100%) for individuals between 41 and 65 years old and 100% (95% CI: 20.1%-100%) for individuals between 66 and 90 years old. Additionally, we find a mismatch between the inferred negative distribution of the serosurvey and the validation data of pre-pandemic controls. Overall, this study illustrates that mixture model methods can provide additional insights from serosurvey data.

Sensitivity and Specificity of Anti-SARS-CoV-2 Detection Kits - Comparison and Agreement between Fifteen Different Assays.

Accurate and rapid diagnosis of coronavirus disease 2019 (COVID-19) is critical for proper care and identification of affected individuals. This led to early availability of many serological assays in the market, but with limited validation. In this study, we aimed to validate the serological assays based on different techniques. We evaluated 15 different assays based on four immunoassay techniques in 235 patients. The most sensitive kits employed were as follows: immunochromatography (Zybio severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2] IgM/IgG Antibody Assay Kit - 83%), ELISA (Aeskulisa SARS-CoV-2 NP IgG -88.1%), chemiluminescence (Alinity SARS-CoV-2 IgG - 82.2%), and immunofluorescence (Lifotronic FA160 (Shenzhen SARS-CoV-2 Assay Kit [IgG]) - 88.9%). The kits by Uniper (Singuway Biotec COVID-19 IgM/IgG Presumptive Kit), Genrui 2019-nCoV IgM/IgG Test Kit, Wondfu SARS-CoV-2 Antibody Test, and Aeskulisa SARS-CoV-2 NP IgG exhibited 100% specificity, whereas IgG assay using Lifotronic FA160 (Shenzhen SARS-CoV-2 Assay Kit) exhibited the lowest specificity at 58%. Maximum agreement was observed between Aeskulisa SARS-CoV-2 NP IgG and Alinity SARS-CoV-2 IgG at 94%. Serological tests are practical alternatives, but their reliability depends on critical validation. The COVID-19 pandemic warranted investment in healthcare research at both the national and international levels.

Serological Assays for Assessing Postvaccination SARS-CoV-2 Antibody Response.

Serological assays for measuring severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies have crucial applications in the control and surveillance of the current COVID-19 pandemic. A large number of such assays have been developed and are now commercially available. However, there are limited studies evaluating the performance of these tests. We evaluated the performances of the following six commercially available serological assays for detecting SARS-CoV-2 antibodies: (i) Genscript cPass surrogate virus neutralization test (Genscript cPass), (ii) Diasorin-SARS-CoV-2 S1/S2 IgG detection (Diasorin-S1/S2 IgG), (iii) Alinity SARS-CoV-2 IgG II (Alinity IgG II), (iv) Diasorin-SARS-CoV-2 TrimericS IgG (Diasorin-TrimericS IgG), (v) Roche Elecsys anti-SARS-CoV-2-cobas (Roche Elecsys), and (vi) AESKU enzyme linked immunosorbent assay (AESKULISA). The results of these tests were compared against the gold standard plaque reduction neutralization test (PRNT). Roche Elecsys had the highest sensitivity, and the Genscript cPass had the highest specificity. Diasorin-TrimericS IgG had the best overall performance with the highest agreement with the PRNT results. Parallel testing of Genscript cPass with Diasorin-TrimericS IgG and Diasorin-S1/S2 IgG had the optimum performance. Based on the receiver operating characteristic (ROC) curve, lowering the cutoff from 30% to 20% in the Genscript cPass significantly increased the sensitivity and the overall agreement with the PRNT results. Commercially available serological assays are good alternatives to the standard PRNT. However, further studies on larger sample numbers are required for optimization of the assay cutoff values and for evaluation of cost effectiveness. IMPORTANCE Commercial serological assays for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are now widely available. This study adds new knowledge regarding the optimization of these assays for evaluating postvaccination antibodies status. It highlights the positive and negative aspects of each assay in terms of sensitivity, specificity, and positive and negative predictive values, compared to the gold standard neutralization test. When using serological assays to assess postvaccine immune status, a balance of all parameters needs to be considered and not simply the high specificity. This balance is particularly relevant in the current situation where countries are aiming to mass vaccinate their populations and bring this pandemic under control. Assays with good sensitivity will have a lower percentage of false negatives and thus provide confidence for vaccination. Understanding the strengths and limitations of commercially available serological assays is important, not only for better application of these tests but also to understand the immune response and the duration of protection postvaccination.

SARS-CoV-2 Antibody Testing in Health Care Workers: A Comparison of the Clinical Performance of Three Commercially Available Antibody Assays.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies are an excellent indicator of past COVID-19 infection. As the COVID-19 pandemic progresses, retained sensitivity over time is an important quality in an antibody assay that is to be used for the purpose of population seroprevalence studies. We compared 5,788 health care worker (HCW) serum samples by using two serological assays (Abbott SARS-CoV-2 anti-nucleocapsid immunoglobulin G (IgG) and Roche anti-SARS-CoV-2 anti-nucleocapsid total antibody) and a subset of samples (all Abbott assay positive or grayzone, n = 485) on Wantai SARS-CoV-2 anti-spike antibody enzyme-linked immunosorbent assay (ELISA). For 367 samples from HCW with a previous PCR-confirmed SARS-CoV-2 infection, we correlated the timing of infection with assay results. Overall, seroprevalence was 4.2% on Abbott and 9.5% on Roche. Of those with previously confirmed infection, 41% (150/367) and 95% (348/367) tested positive on Abbott and Roche, respectively. At 21 weeks (150 days) after confirmed infection, positivity on Abbott started to decline. Roche positivity was retained for the entire study period (33 weeks). Factors associated (P ≤ 0.050) with Abbott seronegativity in those with previous PCR-confirmed infection included sex (odds ratio [OR], 0.30 male ; 95% confidence interval [CI], 0.15 to 0.60), symptom severity (OR 0.19 severe symptoms; 95% CI, 0.05 to 0.61), ethnicity (OR, 0.28 Asian ethnicity; 95% CI, 0.12 to 0.60), and time since PCR diagnosis (OR, 2.06 for infection 6 months previously; 95% CI, 1.01 to 4.30). Wantai detected all previously confirmed infections. In our population, Roche detected antibodies up to at least 7 months after natural infection with SARS-CoV-2. This finding indicates that the Roche total antibody assay is better suited than Abbott IgG assay to population-based studies. Wantai demonstrated high sensitivity, but sample selection was biased. The relationship between serological response and functional immunity to SARS-CoV-2 infection needs to be delineated. IMPORTANCE As the COVID-19 pandemic progresses, retained sensitivity over time is an important quality in an antibody assay that is to be used for the purpose of population seroprevalence studies. There is a relative paucity of published literature in this field to help guide public health specialists when planning seroprevalence studies. In this study, we compared results of 5,788 health care worker blood samples tested by using two assays (Roche and Elecsys, anti-nucleocapsid antibody) and by testing a subset on a third assay (Wantai enzyme-linked immunosorbent assay [ELISA] anti-spike antibody). We found significant differences in the performance of these assays, especially with distance in time from PCR-confirmed COVID-19 infection, and we feel these results may significantly impact the choice of assay for others conducting similar studies.

Rapid Point-Of-Care Serology and Clinical History Assessment Increase Protection Provided by RT-PCR Screening: A Pilot Study Involving Three Nursing Homes in Brescia, a Hotspot of Lombardy.

Background: COVID-19 outbursts have been registered worldwide within care homes with asymptomatic transmission combined with shortage/inaccuracy of diagnostic tests undermining the efforts at containment of the disease. Nursing facilities in Lombardy (Italy) were left with no, or limited, access to testing for 8 weeks after the outbreak of COVID-19. Methods: This study includes 246 residents and 286 workers of three different nursing homes in Brescia-Lombardy. Clinical questionnaires and rapid serology tests were devised to integrate the data of the first available RT-PCR screening. Follow-up serology after 60-days was performed on 67 of 86 workers with positive serology or clinically suspicious. Findings: Thirty-seven residents and 18 workers had previous positive RT-PCR. Thorough screening disclosed two additional RT-PCR-positive workers. Serology screening revealed antibodies in 59 residents and 48 workers, including 32/37 residents and all workers previously positive at RT-PCR. Follow up serology disclosed antibodies in two additional workers with recent symptoms at the time of screening. The professionals in close contact with residents had more infections (47/226-20.79% vs. 1/60-1.66%; p = 0.00013 Fisher exact-test). A suspicious clinical score was present in 44/64 residents and in 41/50 workers who tested positive with either method with totally asymptomatic disease more frequent among residents 28.1 vs. 10.0% (p = 0.019 Fisher exact-test). Interpretation: Based on the available RT-PCR ± results at the time of symptoms/contacts, our integrated clinical and serological screening demonstrated sensitivity 89% and specificity 87%. This multimodal assessment proved extremely useful in understanding the viral spread in nursing homes, in defining its stage and in implementing protective measures. Rapid serology tests demonstrated efficient and particularly suited for older people less able to move/cooperate.

Long-Term Longitudinal Evaluation of Six Commercial Immunoassays for the Detection of IgM and IgG Antibodies against SARS CoV-2.

The number of serological assays for SARS-CoV-2 has skyrocketed in the past year. Concerns have been raised regarding their performance characteristics, depending on the disease severity and the time of the analysis post-symptom onset (PSO). Thus, independent validations using an unbiased sample selection are required for meaningful serology data interpretation. We aimed to assess the clinical performance of six commercially available assays, the seroconversion, and the dynamics of the humoral response to SARS-CoV-2 infection. The study included 528 serum samples from 156 patients with follow-up visits up to six months PSO and 161 serum samples from healthy people. The IgG/total antibodies positive percentage increased and remained above 95% after six months when chemiluminescent immunoassay (CLIA) IgG antiS1/S2 and electro-chemiluminescent assay (ECLIA) total antiNP were used. At early time points PSO, chemiluminescent microparticle immunoassay (CMIA) IgM antiS achieved the best sensitivity. IgM and IgG appear simultaneously in most circumstances, and when performed in parallel the sensitivity increases. The severe and the moderate clinical forms were significantly associated with higher seropositivity percentage and antibody levels. High specificity was found in all evaluated assays, but the sensitivity was variable depending on the time PSO, severity of disease, detection method and targeted antigen.

Longitudinal Analysis and Comparison of Six Serological Assays up to Eight Months Post-COVID-19 Diagnosis.

BACKGROUND: There is much data available concerning the initiation of the immune response after SARS-CoV-2 infection, but long-term data are scarce. METHODS: We thus longitudinally evaluated and compared the total and neutralizing immune response of 61 patients to SARS-CoV-2 infection up to eight months after diagnosis by RT-PCR using several commercial assays. RESULTS: Among the 208 samples tested, the percentage of seropositivity was comparable between assays up to four months after diagnosis and then tended to be more heterogeneous between assays (p < 0.05). The percentage of patients with a neutralizing titer decreased from 82% before two months postdiagnosis to 57% after six months. This decrease appeared to be more marked for patients under 65 years old and those not requiring hospitalization. The percentage of serology reversion at 6 months was from 11% with the WANTAI total assay to over 39% with the ABBOTT IgG assay. The neutralizing antibody titers decreased in parallel with the decrease of total antibody titers, with important heterogeneity between assays. CONCLUSIONS: In conclusion, serological tests show equivalent sensitivity in the first months after the diagnosis of SARS-CoV-2 infection, but their performance later, postinfection, must be considered when interpreting the results.

The Utility of Specific Antibodies Against SARS-CoV-2 in Laboratory Diagnosis.

The Coronavirus Disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has now become a global pandemic due to its high transmissibility. The unavoidable shortcomings of traditional diagnostic assay, including nucleic acid testing, diverse serological assays characterized by high-throughput and less workload, are playing a more and more crucial role to supplement the nucleic acid test. In this review, we summarize the dynamic change of the specific IgM, IgG, and IgA antibodies against SARS-CoV-2 as well as neutralizing antibodies and discuss the clinical utility and limitations of the different serological assays. SARS-CoV-2, a newly discovered virus, shows some unique pathogenetic and epidemiological characteristics that have not been completely understood so far. Currently, studies about the antibody responses against SARS-CoV-2 and the clinical utility of serological testing are increasing. It's well suggested that the combination of serological tests and nucleic acid tests can cohesively improve the testing efficiency for identifying COVID-19 suspected patients.

Humoral Responses and Serological Assays in SARS-CoV-2 Infections.

In December 2019, the novel betacoronavirus Severe Acute Respiratory Disease Coronavirus 2 (SARS-CoV-2) was first detected in Wuhan, China. SARS-CoV-2 has since become a pandemic virus resulting in hundreds of thousands of deaths and deep socioeconomic implications worldwide. In recent months, efforts have been directed towards detecting, tracking, and better understanding human humoral responses to SARS-CoV-2 infection. It has become critical to develop robust and reliable serological assays to characterize the abundance, neutralization efficiency, and duration of antibodies in virus-exposed individuals. Here we review the latest knowledge on humoral immune responses to SARS-CoV-2 infection, along with the benefits and limitations of currently available commercial and laboratory-based serological assays. We also highlight important serological considerations, such as antibody expression levels, stability and neutralization dynamics, as well as cross-reactivity and possible immunological back-boosting by seasonal coronaviruses. The ability to accurately detect, measure and characterize the various antibodies specific to SARS-CoV-2 is necessary for vaccine development, manage risk and exposure for healthcare and at-risk workers, and for monitoring reinfections with genetic variants and new strains of the virus. Having a thorough understanding of the benefits and cautions of standardized serological testing at a community level remains critically important in the design and implementation of future vaccination campaigns, epidemiological models of immunity, and public health measures that rely heavily on up-to-date knowledge of transmission dynamics.

COVID-19: Insight into the asymptomatic SARS-COV-2 infection and transmission.

The existence of a substantial but unclear number of asymptomatic SARS-COV-2 patients worldwide has raised concerns among global public health authorities. In this review, according to the published literature, we provided the evidence that asymptomatic infections can result in person-to-person transmission. Four studies suggested that the virus can be transmitted by asymptomatic patients for at least two consecutive generations, indicating its strong infectivity. Asymptomatic infection tends to be, but is not only, identified among young people (<20 years old). The majority of asymptomatic patients appear to have a milder clinical course during hospitalization, but the severity of the symptoms of the secondary patients infected by SARS-COV-2 from asymptomatic patients varies with their physical constitution. The proportion of asymptomatic individuals among all confirmed cases widely differed (from 1.95% to 87.9%) according to the study setting and the populations studied. The increasing large-scale tests are expected to give more information about the true number of asymptomatic infections in the population. In China and other countries, various guidelines for management of asymptomatic cases have been issued. Importantly, early detection, early reporting, early isolation and early treatment of asymptomatic patients require the joint efforts of policy makers, clinicians, technicians, epidemiologists, virologists and patients.

Future developments in biosensors for field-ready SARS-CoV-2 virus diagnostics.

According to the evidence, the coronavirus disease 19 (COVID-19) is caused by a zoonotic pathogen named respiratory syndrome coronavirus 2 (SARS-CoV-2). This virus can spread through personal contact, respiratory droplets, and also through airborne transmission. A rapid, low-cost, and effective biosensor platform is essential to diagnose patients with COVID-19 infection, predominantly the asymptomatic individuals, and prevent the spread of the SARS-CoV-2 via transmission routes. The objective of this review is to provide a comparative view among current diagnostic methods, focusing on recently suggested biosensors for the detection of SARS-CoV2 in clinical samples. A capable SARS-CoV-2 biosensor can be designed by the holistic insights of various biosensor studies.

Characterisation of SARS-CoV-2 Lentiviral Pseudotypes and Correlation between Pseudotype-Based Neutralisation Assays and Live Virus-Based Micro Neutralisation Assays.

The recent outbreak of a novel Coronavirus (SARS-CoV-2) and its rapid spread across the continents has generated an urgent need for assays to detect the neutralising activity of human sera or human monoclonal antibodies against SARS-CoV-2 spike protein and to evaluate the serological immunity in humans. Since the accessibility of live virus microneutralisation (MN) assays with SARS-CoV-2 is limited and requires enhanced bio-containment, the approach based on "pseudotyping" can be considered a useful complement to other serological assays. After fully characterising lentiviral pseudotypes bearing the SARS-CoV-2 spike protein, we employed them in pseudotype-based neutralisation assays in order to profile the neutralising activity of human serum samples from an Italian sero-epidemiological study. The results obtained with pseudotype-based neutralisation assays mirrored those obtained when the same panel of sera was tested against the wild type virus, showing an evident convergence of the pseudotype-based neutralisation and MN results. The overall results lead to the conclusion that the pseudotype-based neutralisation assay is a valid alternative to using the wild-type strain, and although this system needs to be optimised and standardised, it can not only complement the classical serological methods, but also allows serological assessments to be made when other methods cannot be employed, especially in a human pandemic context.