Comparison of Rapid and Automated Antigen Detection Tests for the Diagnosis of SARS-CoV-2 Infection

SARS-CoV-2 viral antigen detection may be an interesting alternative to RT-PCR for the diagnosis of SARS-CoV-2 infection as a less laborious or expensive method but requires validation. This study aimed to compare the performance of the DiaSorin™ LiaisonXL automated quantitative antigen test (QAT) and the AAZ™ rapid antigen test (RAT) to the DiaSorin™ MDX RT-PCR assay. A total of 242 nasopharyngeal samples were tested at La Pitié-Salpêtrière University Hospital (Paris, France). Performances for the detection of variants of SARS-CoV-2 were further investigated. RATs were visually read for qualitative results and band intensity was determined. Overall sensitivity was 63.2% for QAT and 58.6% for RAT. For RT-PCR Ct value 25, sensitivity was 89.8% for both tests. Both tests showed comparable sensitivity for detection of variants. There was a strong relationship between antigen concentration and band positivity. On the same set of samples these tests share similar performances.

Phenotyping Polarization Dynamics Of Immune Cells Using A Lipid Droplet - Cell Pairing Microfluidic Platform

The immune synapse is the tight contact zone between a lymphocyte and a cell presenting its cognate antigen. This structure serves as a signaling platform and entails a polarization of intra-cellular components, necessary to the immunological function of the cell. While the surface properties of the presenting cell are known to control the formation of the synapse, their impact on polarization has not yet been studied. Using functional lipid droplets as tunable artificial presenting cells combined with a microfluidic pairing device, we simultaneously observe synchronized synapses and dynamically quantify polarization patterns of individual B cells. By assessing how the ligand concentration, the surface fluidity and the substrate rigidity impact this polarization, we show that its onset depends on the antigen concentration at the synapse, and that the substrate rigidity controls both its onset and its kinetics. Our experimental system enables a fine phenotyping of monoclonal cell populations based on their synaptic readout.

Development of a Double-Antibody Sandwich ELISA for Rapid Detection of the MCP Antigen Concentration in Inactivated ISKNV Vaccines

Infectious spleen and kidney necrosis virus (ISKNV) resulted in severe systemic diseases with high morbidity and mortality in Siniperca chuatsi. Vaccination is the primary method for effective prevention and control of these diseases. The development of inactivated ISKNV vaccines made some progress, but the technique of quality evaluation is scarce. Herein, a measurement of the MCP (major capsid protein) antigen concentration for the inactivated ISKNV vaccine was developed by double-antibody sandwich ELISA. Firstly, mouse monoclonal antibodies against ISKNV particles and MCP were generated. Then, a double-antibody sandwich ELISA was developed using the monoclonal antibody 1C8 1B9 as the capture antibody and Biotin-3B12 6B3 as the detection antibody. A standard curve was generated using the MCP concentration versus OD value with the linear range of concentration of 4.69~300 ng/mL. The assay sensitivity was 0.9 ng/mL. The antigen content of three batches of inactivated ISKNV vaccines was quantitatively detected using the double-antibody sandwich ELISA. The results showed that MCP antigen contents of inactivated ISKNV vaccines were positively correlated with the viral titers. The newly established double-antibody sandwich ELISA provided a useful tool for the detection of antigen quality for ISKNV inactivated vaccines.

Clinical performance of the Roche Elecsys SARS-CoV-2 Antigen fully automated electrochemiluminescence immunoassay

Objective: We assessed the clinical performance of novel Roche Elecsys SARS-CoV-2 Antigen fully automated electrochemiluminescence immunoassay (ECLIA). Design and Methods: We tested 160 subjects, 110 (68.8%) with positive molecular test for SARS-CoV-2 infection in nasopharyngeal samples with Altona Diagnostics RealStar SARS-CoV-2 RT-PCR Kit and Roche Elecsys SARS-CoV-2 Antigen. The local imprecision was validated by analyzing three nasopharyngeal samples with different antigen concentration (1.84, 9.51 and 423.30 TCID50/mL) for 20 consecutive times (intra-assay imprecision) or for 10 consecutive working days (inter-assay imprecision).Results: The local intra- and inter-assay imprecision of Elecsys SARS-CoV-2 Antigen ECLIA was found to be comprised between 2.0-2.0% and 5.8-7.6%, yielding to a total imprecision of 6.2-7.8%. Highly significant correlation was found between Elecsys SARS-CoV-2 Antigen ECLIA and cycle threshold (Ct) values of SARS-CoV-2 S and E genes (both r=-0.91; p<0.001). The area under the curve (AUC), sensitivity and specificity of Elecsys SARS-CoV-2 Antigen ECLIA were 0.83, 0.43 and 1.00 in all samples, 0.99, 0.87 and 0.99 in those with both Ct values <30, as well as 1.00, 1.00 and 0.89 in samples with both Ct values <25. Conclusion: Roche Elecsys SARS-CoV-2 Antigen ECLIA may be a surrogate of molecular testing for identification of super-spreaders.

SARS-CoV-2 Nucleocapsid Plasma Antigen for Diagnosis and Monitoring of COVID-19

Background Detection of SARS-CoV-2 nucleocapsid antigen in blood has been described, but the diagnostic and prognostic role of antigenemia is not well understood. This study aimed to determine the frequency, duration, and concentration of nucleocapsid antigen in plasma and its association with COVID-19 severity. Methods We utilized an ultrasensitive electrochemiluminescence immunoassay targeting SARS-CoV-2 nucleocapsid antigen to evaluate 777 plasma samples from 104 individuals with COVID-19. We compared plasma antigen to respiratory nucleic acid amplification testing (NAAT) in 74 individuals with COVID-19 from samples collected ± 1 day of diagnostic respiratory NAAT, and in 52 SARS-CoV-2-negative individuals. We used Kruskal-Wallis tests, multivariable logistic regression, and mixed-effects modeling to evaluate whether plasma antigen concentration was associated with disease severity. Results Plasma antigen had 91.9% (95% CI 83.2-97.0%) clinical sensitivity and 94.2% (84.1-98.8%) clinical specificity. Antigen-negative plasma samples belonged to patients with later respiratory cycle thresholds (Ct) when compared with antigen-positive plasma samples. Median plasma antigen concentration (log10 fg/mL) was 5.4 (IQR 3.9-6.0) in outpatients, 6.0 (5.4-6.5) in inpatients, and 6.6 (6.1-7.2) in intensive care unit (ICU) patients. In models adjusted for age, sex, diabetes, and hypertension, plasma antigen concentration at diagnosis was associated with ICU admission (OR 2.8 [95% CI 1.2-6.2], P=.01), but not with non-ICU hospitalization. Rate of antigen decrease was not associated with disease severity. Conclusions SARS-CoV-2 plasma nucleocapsid antigen exhibited comparable diagnostic performance to upper respiratory NAAT, especially among those with late respiratory Ct. In addition to currently available tools, antigenemia may facilitate patient triage to optimize intensive care utilization.

Comparison of the quantitative DiaSorin Liaison antigen test to RT-PCR for the diagnosis of COVID-19 in symptomatic and asymptomatic outpatients

Background:We evaluated the quantitative DiaSorin Liaison SARS-CoV-2 antigen test in symptomatic and asymptomatic individuals consulting their general practioner (GP) during a period of stable intense virus circulation (213/100,000 habitants per day). Methods:Left-over RT-PCR positive (n=204) and negative (n=210) nasopharyngeal samples were randomly selected among fresh routine samples collected from patients consulting their GP. Samples were tested on Liaison XL according to the manufacturer’s instructions. Equivocal results were considered positive. Results:Overall sensitivity and specificity of the Liaison antigen test compared to RT-PCR were 67.7% [95% confidence interval (CI): 60.9%-73.7%] and 100% [CI: 97.8%-100%]. Sensitivity in samples with a viral load ≥105, ≥104 and ≥103 copies/mL was 100% [CI: 96.3%-100.0%], 96.5% [CI: 91.8%-98.7%] and 87.4% [CI: 81.3%-91.5%], respectively. All samples ≤103 copies/mL were antigen negative. The ratio of antigen concentration to viral load in samples ≥103 copies/mL was comparable in symptomatic and asymptomatic individuals (p=0.58). The proportion of RT-PCR positive participants with a high viral load (≥105 copies/mL) was not significantly higher in symptomatic than in asymptomatic participants (63.9% [CI: 54.9%-72.0%] vs. 51.9% [CI: 41.1%-62.6%], p=0.11), but the proportion of participants with a low viral load (<103 copies/mL) was significantly higher in asymptomatic than in symptomatic RT-PCR positive participants (35.4% [CI: 25.8%-46.4%] vs. 14.3% [CI: 9.0%-21.8%], p<0.01). Conclusions:Sensitivity and specificity in samples with a viral load ≥104 copies/mL was 96.5% and 100%. The correlation of antigen concentration with viral load was comparable in symptomatic and asymptomatic individuals.

Feline Leukemia Virus p27 Antigen Concentration and Proviral DNA Load Are Associated with Survival in Naturally Infected Cats

Longitudinal studies of cats naturally infected with feline leukemia virus (FeLV) are important for understanding disease outcomes. Levels of p27 antigen and copy numbers of proviral DNA have been associated with FeLV-infection courses. The purpose of this prospective study was to establish cutoff values for p27 antigen concentration and proviral DNA load that distinguished high positive from low positive groups of cats and to evaluate an association with survival. At enrollment, 254 cats were tested by point-of-care and microtiter plate enzyme-linked immunosorbent assays (ELISAs) for p27 antigen and real-time polymerase chain reaction (PCR) for proviral DNA. The 127 positive cats were retested monthly for six months and monitored for survival over the four-year study. A receiver operating characteristic-based analysis of samples with concordant or discordant qualitative results for p27 antigen and proviral DNA was used to establish cutoff values, and when applied to test results at enrollment for classifying cats as high positive or low positive, a significant difference in survival was observed. High positive cats had a median survival of 1.37 years (95% CI 0.83–2.02) from time of enrollment, while most low positive cats were still alive (93.1% survival). Quantitative results for p27 antigen concentration and proviral DNA load were highly correlated with survival times in FeLV-infected cats.

Development of Printable Graphene Electrochemical Biosensor for Environmental Monitoring and Medical Applications

Algal bloom is a type of harmful water pollution, which is mainly caused by the cyanobacteria or dinoflagellate that releases a variety of algal toxins into a water source. Among them, microcystins are often detected, of which microcystin-leucine-arginine (MC-LR) is known as one of the most toxic variants that has received a great amount of attention due to its serious consequences after ingestion such as irreversible organ damage or even death. Human cytomegalovirus (HCMV) is a type of herpes virus that can widely spread via mucous contact, resulting in many severe symptoms or even death especially for infants, pregnant women and immunocompromised patients if there is no timely diagnosis. Following these reasons, there is an urgent need to develop a commercially viable and sensitive monitoring system to reach a rapid identification on water quality or human health. This work mainly focuses on the development of vertically aligned graphene (VAG) electrodes through the novel use of flexographic printing and photonic annealing techniques for highly sensitive detection of biological targets using non-Faradaic electrochemical impedance spectroscopy (EIS). For the detection of MC-LR, the biosensor achieved an low limit of detection (LOD) of 1.2 ng/L via baseline method. In the baseline method, measurement was first performed using PBS. After that, measurement was then performed on antigen solution drop-casted on the biosensor. The biosensing response between PBS and antigen acquired at a specific frequency was dependent on the target concentration. The biosensor also exhibited excellent selectivity with high percentage of recovery (i.e., 91.8 %) and stability (i.e., 108.8 % and 99.4 % after one and three weeks, respectively). Moreover, similar good performance (i.e., 98.4%) was observed in tap water spiked with the antigen. As for the detection of CMV pp65-antigen, biosensing results showed a good linearity when tested on the control group (i.e., 0 ng/mL) up to 38,500 ng/mL of the antigen concentration using the same baseline measurement. The VAG biosensor showed a dynamic range of between 3.85 and 38,500 ng/mL for the detection of HCMV pp65-antigen, which matches with the clinically relevant range of 102 ~ 106 genomes/mL based on measurement performed on viral loaded urine samples using PCR technique. Measurements on the target concentration using the biosensor were also performed using a non-baseline method. In this method, only the antigen solution was used throughout the measurement, where the biosensing result was determined by the difference in the response recorded at the start of measurement and after a certain incubation duration at a specific frequency. In particular, the change in phase showed a strong correlation against the target concentration. The biosensing response for the control group (i.e., 0.38°±0.191°) up to 38,500 ng/mL (i.e., 2.26°±0.543°) antigen concentration was highly comparable to those (i.e., 0.16°±0.0854° for the control group and 2.21°±0.105° for 38,500 ng/mL) derived from the baseline method, implying the strong feasibility of the non-baseline testing.