A sensitive gold nanoflower-based lateral flow assay coupled with gold staining technique for the detection of SARS-CoV-2 antigen.

An enhanced lateral flow assay (LFA) is presented for rapid and highly sensitive detection of acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antigens with gold nanoflowers (Au NFs) as signaling markers and gold enhancement to amplify the signal intensities. First, the effect of the morphology of gold nanomaterials on the sensitivity of LFA detection was investigated. The results showed that Au NFs prepared by the seed growth method showed a 5-fold higher detection sensitivity than gold nanoparticles (Au NPs) of the same particle size, which may benefit from the higher extinction coefficient and larger specific surface area of Au NFs. Under the optimized experimental conditions, the Au NFs-based LFA exhibited a detection limit (LOD) of 25 pg mL-1 for N protein using 135 nm Au NFs as the signaling probes. The signal was further amplified by using a gold enhancement strategy, and the LOD for the detection of N protein achieved was 5 pg mL-1. The established LFA also exhibited good repeatability and stability and showed applicability in the diagnosis of SARS-CoV-2 infection.

Potential Usefulness of IgA for the Early Detection of SARS-CoV-2 Infection: Comparison With IgM.

Serological testing can be a powerful complementary approach to achieve timely diagnosis of severe acute respiratory coronavirus 2 (SARS-CoV-2) infection, along with nucleic acid detection. Immunoglobulin (Ig) A antibodies are less frequently utilized to detect SARS-CoV-2 infection than IgM and IgG antibodies, even though IgA antibodies play an important role in protective immunity against SARS-CoV-2. This review discusses the differences in kinetics and assay performance between IgA and IgM antibodies and the factors influencing antibody responses. It highlights the potential usefulness of analyzing IgA antibodies for the early detection of SARS-CoV-2 infection. The early appearance of IgA and the high sensitivity of IgA-based immunoassays can aid in diagnosing coronavirus disease 2019. However, because of cross-reactivity, it is important to recognize the only moderate specificity of the early detection of SARS-CoV-2 IgA antibodies against spike antigens. Either the analysis of antibodies targeting the nucleocapsid antigen or a combination of antibodies against the nucleocapsid and spike antigens may strengthen the accuracy of serological evaluation.

Enhancing Sensitivity in SARS-CoV-2 Rapid Antigen Testing through Integration of a Water-Soluble Polymer Wall.

Lateral flow immunoassays (LFIAs) are recognized for their practicality in homecare and point-of-care testing, owing to their simplicity, cost-efficiency, and rapid visual readouts. Despite these advantages, LFIAs typically fall short in sensitivity, particularly in detecting viruses such as SARS-CoV-2, thus limiting their broader application. In response to this challenge, we have innovated an approach to substantially enhance LFIA sensitivity. This involves the integration of a water-soluble dextran-methacrylate polymer wall with a 15% grafting degree positioned between the test and control lines on the LFIA strip. This novel modification significantly improved the sensitivity of the assay, achieving detection limits as low as 50 pg mL-1 and enhancing the sensitivity by 5-20-fold relative to existing LFIA kits available on the market. Furthermore, our developed LFIA kit (WSPW-LFIA) demonstrated exceptional specificity for SARS-CoV-2. Coupled with a straightforward fabrication process and robust stability, the WSPW-LFIA represents a promising advancement for real-time in vitro diagnosis across a spectrum of diseases.

A Paper-Based Multiplexed Serological Test to Monitor Immunity against SARS-COV-2 Using Machine Learning.

The rapid spread of SARS-CoV-2 caused the COVID-19 pandemic and accelerated vaccine development to prevent the spread of the virus and control the disease. Given the sustained high infectivity and evolution of SARS-CoV-2, there is an ongoing interest in developing COVID-19 serology tests to monitor population-level immunity. To address this critical need, we designed a paper-based multiplexed vertical flow assay (xVFA) using five structural proteins of SARS-CoV-2, detecting IgG and IgM antibodies to monitor changes in COVID-19 immunity levels. Our platform not only tracked longitudinal immunity levels but also categorized COVID-19 immunity into three groups: protected, unprotected, and infected, based on the levels of IgG and IgM antibodies. We operated two xVFAs in parallel to detect IgG and IgM antibodies using a total of 40 µL of human serum sample in <20 min per test. After the assay, images of the paper-based sensor panel were captured using a mobile phone-based custom-designed optical reader and then processed by a neural network-based serodiagnostic algorithm. The serodiagnostic algorithm was trained with 120 measurements/tests and 30 serum samples from 7 randomly selected individuals and was blindly tested with 31 serum samples from 8 different individuals, collected before vaccination as well as after vaccination or infection, achieving an accuracy of 89.5%. The competitive performance of the xVFA, along with its portability, cost-effectiveness, and rapid operation, makes it a promising computational point-of-care (POC) serology test for monitoring COVID-19 immunity, aiding in timely decisions on the administration of booster vaccines and general public health policies to protect vulnerable populations.

Performance of two rapid antigen tests against SARS-CoV-2 in neighborhoods of socioeconomic vulnerability from a middle-income country.

BACKGROUND: As new and improved antigen-detecting rapid diagnostic tests for SARS-CoV-2 infection (Ag-RDT) continue to be developed, assessing their diagnostic performance is necessary to increase test options with accurate and rapid diagnostic capacity especially in resource-constrained settings. This study aimed to assess the performance of two Ag-RDTs in a population-based study. METHODS: We conducted a diagnostic accuracy study in neighborhoods with high socioeconomic vulnerability in Salvador-Brazil, including individuals aged ≥12 years old who attended primary health services, between July and December 2022, with COVID-19 symptoms or who had been in contact with a confirmed case. Two Ag-RDTs were compared in parallel using reverse transcription polymerase chain reaction (RT-PCR) as reference standard, the PanbioTM COVID-19 Ag test (Abbott®) and Immuno-Rapid COVID-19 Ag (WAMA Diagnostic®). Sensitivity, specificity, positive (PPV) and negative predictive values (NPV) were calculated. RESULTS: For the Abbott test the sensitivity was 52.7% (95% CI: 44.3% - 61.0%), specificity 100% (95% CI: 98.7% - 100%), PPV 100% (95% CI: 95.4% - 100%) and NPV 80.4% (95% CI: 75.9% - 84.4%). For the WAMA test, the sensitivity was 53.4% (95% CI: 45.0% - 61.6%), specificity 100% (95% CI: 98.7% - 100%), PPV 100% (95% CI: 95.4% - 100%) and NPV 80.7% (95% CI: 76.2% - 84.6%). Sensitivity for the group with Cycle Threshold (CT) <24 was 82.3% (95%CI: 72.1-90.0, n = 83) for PanbioTM COVID-19 Ag test and 87.3% (95%CI: 77.9-93.8, n = 83) for Immuno-Rapid COVID-19 Ag test. CONCLUSION: Sensitivity for both Ag-RDT was lower than reported by manufacturers. In the stratified analysis, sensitivity was higher among those with lower CT values <24. Specificity was high for both rapid antigen tests. Both Ag-RDT showed to be useful for rapid diagnostic of potential cases of COVID-19. Negative results must be assessed carefully according to clinical and epidemiological information.

A biosensor based on magnetoelastic waves for detection of antibodies in human plasma for COVID-19 serodiagnosis.

This study proposes a new efficient wireless biosensor based on magnetoelastic waves for antibody detection in human plasma, aiming at the serological diagnosis of COVID-19. The biosensor underwent functionalization with the N antigen - nucleocapsid phosphoprotein of the SARS-CoV-2 virus. Validation analyses by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting (WB), atomic force microscopy (AFM), scanning electron microscopy (SEM), energy-dispersive X-ray (EDX) microanalysis and micro-Raman spectroscopy confirmed the selectivity and effective surface functionalization of the biosensor. The research successfully obtained, expressed and purified the recombinant antigen, while plasma samples from COVID-19 positive and negative patients were applied to test the performance of the biosensor. A performance comparison with the enzyme-linked immunosorbent assays (ELISA) method revealed equivalent diagnostic capacity. These results indicate the robustness of the biosensor in reliably differentiating between positive and negative samples, highlighting its potential as an efficient and low-cost tool for the serological diagnosis of COVID-19. In addition to being fast to execute and having the potential for automation in large-scale diagnostic studies, the biosensor fills a significant gap in existing SARS-CoV-2 detection approaches.

Real-world utilisation of SARS-CoV-2 rapid antigen testing to enable face-to-face learning in Australian schools, an ecological study.

OBJECTIVE: The objective of this study was to describe the use of SARS-CoV-2 rapid antigen testing of COVID-19 contacts in New South Wales schools to determine return to in-person school attendance instead of home quarantine, between 6 November and 21 December 2021. METHODS: COVID-19 school contacts were required to quarantine for two weeks postexposure to the case. Students who opted into daily rapid antigen testing logged their results in a database, prior to school attendance, and obtained SARS-CoV-2 nucleic amplification acid testing on day 12-16. Secondary attack rates (SARs) in schools utilising rapid antigen testing (Test-to-Stay schools) and those not utilising rapid antigen testing (non-Test-to-Stay school) were calculated. RESULTS: We identified 9,887 people in 293 schools who reported performing at least one rapid antigen test (RAT). The SAR in RAT schools was 3.4% (95% confidence interval: 2.7-4.1) and non-RAT schools was 2.8% (95% confidence interval: 2.4-3.3). A total of 30,535 school days were preserved through this program. CONCLUSIONS: The use of RATs preserved in-person learning without a significant increase to SAR. IMPLICATION FOR PUBLIC HEALTH: Disruptions in face-to-face learning have long-term detrimental impacts on children and adolescents. Rapid antigen testing has been shown to be beneficial to maintain face-to-face learning in Australian schools and may be a useful method to safeguard from school disruptions in future pandemics.

An indirect ELISA for detecting anti-SARS-CoV-2 antibodies in human sera using a baculovirus-expressed recombinant nucleocapsid antigen.

This study focuses on the development and initial assessment of an indirect IgG enzyme-linked immunosorbent assay (ELISA) specifically designed to detect of anti-SARS-CoV-2 antibodies. The unique aspect of this ELISA method lies in its utilization of a recombinant nucleocapsid (N) antigen, produced through baculovirus expression in insect cells. Our analysis involved 292 RT-qPCR confirmed positive serum samples and 54 pre-pandemic healthy controls. The process encompassed cloning, expression, and purification of the SARS-CoV-2 N gene in insect cells, with the resulted purified protein employed in our ELISA tests. Statistical analysis yielded an Area Under the Curve of 0.979, and the optimized cut-off exhibited 92 % sensitivity and 94 % specificity. These results highlight the ELISA's potential for robust and reliable serological detection of SARS-CoV-2 antibodies. Further assessments, including a larger panel size, reproducibility tests, and application in diverse populations, could enhance its utility as a valuable biotechnological solution for diseases surveillance.

SARS-CoV-2 rapid antigen test sensitivity and viral load in newly symptomatic hospital employees in Berlin, Germany, December, 2020 to February, 2022: an observational study.

BACKGROUND: Evolving SARS-CoV-2 variants and changing levels of pre-existing immunity require re-evaluation of antigen-detecting rapid diagnostic test (Ag-RDT) performance. We investigated possible associations between Ag-RDT sensitivity and various potential influencing factors, such as immunisation status and viral variant, in symptomatic hospital employees. METHODS: In this observational study, RT-PCR, Ag-RDT, and symptom-specific data were collected at three SARS-CoV-2 test centres for employees of the Charité-Universitätsmedizin Berlin hospital (Berlin, Germany). Employees reporting SARS-CoV-2-like symptoms, those at an increased risk of infection (eg, due to contact with an infected person), those testing positive in a previous self-administered Ag-RDT, or those seeking release-testing to return to work at least 7 days after a positive RT-PCR test were eligible for combined testing by RT-PCR and Ag-RDT. Only data from individuals with an ongoing SARS-CoV-2 infection as assessed by RT-PCR were used for further analysis. Bayesian regression analyses were done to evaluate possible differences in viral load and Ag-RDT sensitivity according to viral variant and immunisation status (previous vaccination or recovery from infection), using data from first RT-PCR positive samples in an infection. A comprehensive logistic regression analysis was used to investigate potential concomitant associations between Ag-RDT sensitivity and level of pre-existing immunity, time post symptom onset, viral load, gender, age, and Ag-RDT device. Ag-RDT performance was also compared between supernatants from cell cultures infected with the omicron variant of concern (VOC) or the wild-type strain (pre-VOC). FINDINGS: Between Nov 30, 2020 and Feb 11, 2022, a total of 14 773 samples from 7675 employees were tested for SARS-CoV-2 by both RT-PCR and Ag-RDT. We found a negative association between immunisation status and Ag-RDT sensitivity in symptomatic employees, with an observed sensitivity of 82% (94% highest posterior density interval [HPDI] 78-86) in immunologically naive participants compared with 73% (68-78) in multiply immunised individuals (ie, those with at least two vaccinations or recoveries from infection) and median log10 viral loads of 7·02 (IQR 5·83-8·07) and 8·08 (6·80-8·89), respectively. The dominant viral variant changed several times during the study period, from the pre-VOC period (sensitivity 80% [94% HPDI 75-85] in symptomatic participants) through the alpha variant (82% [70-94]), delta variant (75% [69-82]), and omicron variant (72% [65-79]) waves, concomitantly with a steep increase in vaccination coverage in our dataset. In a comparison of Ag-RDT performance on cell culture supernatants, we found no difference between the wild-type and omicron viral variants. INTERPRETATION: On the basis of our findings and data from other studies, we hypothesise that the observed reduction in clinical Ag-RDT sensitivity, despite higher SARS-CoV-2 RNA loads, is due to shorter incubation times later in our study period resulting from increased population immunity or changes in immune response dynamics caused by later SARS-CoV-2 VOCs. FUNDING: Berlin University Alliance, German Ministry of Education and Research, the EU (Projects EU4Health and ReCoVer), and the Berlin Institute of Health.

Assessment of an in-house IgG ELISA targeting SARS-CoV-2 RBD: Applications in infected and vaccinated individuals.

The study evoluated an in-house Spike Receptor Binding Domain Enzyme-Linked Immunosorbent Assay (RBD-IgG-ELISA) for detecting SARS-CoV-2 IgG antibodies in infected and vaccinated individuals. The assay demonstrated a sensitivity of 91%, specificity of 99.25%, and accuracy of 95.13%. Precision and reproducibility were highly consistent. The RBD-IgG-ELISA was able to detect 96.25% of Polymerase chain reaction (PCR) confirmed cases for SARS-CoV-2 infection, demonstrating positive and negative predictive values of 99,18% and 91,69%, respectively. In an epidemiological survey, ELISA, lateral flow immunochromatographic assay (LFIA), and electrochemiluminescence immunoassay (ECLIA) exhibited diagnostic sensitivities of 68.29%, 63.41%, and 70.73%, respectively, along with specificities of 82.93%, 80.49%, and 80.49%, respectively. Agreement between RBD-IgG-ELISA/PCR was moderate (k index 0.512). However, good agreement between different assays (RBD-IgG-ELISA/LFIA k index 0.875, RBD-IgG-ELISA/ECLIA k index 0.901). Test performance on individuals' samples were inferior due to seroconversion time and chronicity. The IgG-RBD-ELISA assay demonstrated its effectiveness in monitoring antibody levels among healthcare professionals, revealing significant differences both before and after the administration of the third vaccine dose, with heightened protection levels observed following the third dose in five Coronavirus disease (COVID-19) vaccine regimens. In conclusion, the RBD-IgG-ELISA exhibits high reproducibility, specificity, and sensitivity, making it a suitable assay validated for serosurveillance and for obtaining information about COVID-19 infections or vaccinations.

Design and Development of an Antigen Test for SARS-CoV-2 Nucleocapsid Protein to Validate the Viral Quality Assurance Panels.

The continuing mutability of the SARS-CoV-2 virus can result in failures of diagnostic assays. To address this, we describe a generalizable bioinformatics-to-biology pipeline developed for the calibration and quality assurance of inactivated SARS-CoV-2 variant panels provided to Radical Acceleration of Diagnostics programs (RADx)-radical program awardees. A heuristic genetic analysis based on variant-defining mutations demonstrated the lowest genetic variance in the Nucleocapsid protein (Np)-C-terminal domain (CTD) across all SARS-CoV-2 variants. We then employed the Shannon entropy method on (Np) sequences collected from the major variants, verifying the CTD with lower entropy (less prone to mutations) than other Np regions. Polyclonal and monoclonal antibodies were raised against this target CTD antigen and used to develop an Enzyme-linked immunoassay (ELISA) test for SARS-CoV-2. Blinded Viral Quality Assurance (VQA) panels comprised of UV-inactivated SARS-CoV-2 variants (XBB.1.5, BF.7, BA.1, B.1.617.2, and WA1) and distractor respiratory viruses (CoV 229E, CoV OC43, RSV A2, RSV B, IAV H1N1, and IBV) were assembled by the RADx-rad Diagnostics core and tested using the ELISA described here. The assay tested positive for all variants with high sensitivity (limit of detection: 1.72-8.78 ng/mL) and negative for the distractor virus panel. Epitope mapping for the monoclonal antibodies identified a 20 amino acid antigenic peptide on the Np-CTD that an in-silico program also predicted for the highest antigenicity. This work provides a template for a bioinformatics pipeline to select genetic regions with a low propensity for mutation (low Shannon entropy) to develop robust 'pan-variant' antigen-based assays for viruses prone to high mutational rates.

Wide Real-Life Data Support Reduced Sensitivity of Antigen Tests for Omicron SARS-CoV-2 Infections.

With the continuous spread of new SARS-CoV-2 variants of concern (VOCs), the monitoring of diagnostic test performances is mandatory. We evaluated the changes in antigen diagnostic tests' (ADTs) accuracy along the Delta to Omicron VOCs transition, exploring the N protein mutations possibly affecting ADT sensitivity and assessing the best sampling site for the diagnosis of Omicron infections. In total, 5175 subjects were enrolled from 1 October 2021 to 15 July 2022. The inclusion criteria were SARS-CoV-2 ADT combined with a same-day RT-PCR swab test. For the sampling site analysis, 61 patients were prospectively recruited during the Omicron period for nasal and oral swab analyses by RT-PCR. Next-Generation Sequencing data were obtained to evaluate the different sublineages. Using RT-PCR as a reference, 387 subjects resulted in becoming infected and the overall sensitivity of the ADT decreased from 63% in the Delta period to 33% in the Omicron period. This decrease was highly statistically significant (p < 0.001), and no decrease in viral load was detected at the RNA level. The nasal site presented a significantly higher viral load than the oral site during the Omicron wave. The reduced detection rate of Omicron infections by ADT should be considered in the global testing strategy to preserve accurate diagnoses across the changing SARS-CoV-2 variants.

Diagnostic performance of rapid antigen tests (RAT) for COVID-19 and factors associated with RAT-negative results among RT-PCR-positive individuals during Omicron BA.2, BA.5 and XBB.1 predominance.

BACKGROUND: While numerous studies have evaluated the real-world performance of rapid antigen tests (RATs), data on the effect of Omicron sublineages such as XBB and reinfections on RAT performance is limited. We assessed the performance of RATs and factors associated with RAT-negative results among individuals who tested SARS-CoV-2-positive by reverse transcription-polymerase chain reaction (RT-PCR). METHODS: We conducted a retrospective study among Singapore residents who underwent testing for SARS-CoV-2 with RAT (Acon Flowflex or SD Biosensor) and RT-PCR in the same clinical encounter between 9 May 2022 and 21 November 2022. RT-PCR served as a reference standard for RAT performance. Logistic regression was used to estimate the odds ratios (OR) of factors associated with negative RAT results among RT-PCR-positive cases. RESULTS: Of 8,620 clinical encounters analysed, 3,519 (40.8%) were SARS-CoV-2-positive on RT-PCR. Overall sensitivity and specificity of RAT was 84.6% (95% CI 83.3-85.7%) and 99.4% (95% CI 99.1-99.6%) respectively. Acon Flowflex consistently achieved higher sensitivity and specificity than SD Biosensor test kit. Among RT-PCR-positive cases, individuals who had a previous documented SARS-CoV-2 infection, coinfection with another respiratory pathogen or tested ≥ 6 days from symptom onset had higher odds of testing RAT-negative, but the associations were attenuated after adjustment for cycle threshold values (proxy for viral load). There was no significant difference in RAT performance between Omicron sublineages BA.2, BA.5 and XBB.1. CONCLUSION: Diagnostic performance of RAT was not affected by changes in predominant circulating Omicron sublineages. However, reinfection cases may be under ascertained by RAT. In individuals with a previous SARS-CoV-2 infection episode or symptom onset ≥ 6 days prior to testing, a confirmatory RT-PCR may be considered if there is high clinical suspicion.

Characterization of antibody response to SARS-CoV-2 Orf8 from three waves of COVID-19 outbreak in Thailand.

A dynamic of virus adaptation and a mass vaccination campaign could significantly reduce the severity of clinical manifestations of COVID-19 and transmission. Hence, COVID-19 may become an endemic disease globally. Moreover, mass infection as the COVID-19 pandemic progressed affected the serology of the patients as a result of virus mutation and vaccination. Therefore, a need exists to acquire accurate serological testing to monitor the emergence of new outbreaks of COVID-19 to promptly prevent and control the disease spreading. In this study, the anti-Orf8 antibodies among samples collected in Thailand's first, fourth, and fifth waves of COVID-19 outbreaks compared with pre-epidemic sera were determined by indirect ELISA. The diagnostic sensitivity and specificity of the anti-Orf8 IgG ELISA for COVID-19 samples from the first, fourth, and fifth waves of outbreaks was found to be 100% compared with pre-epidemic sera. However, the diagnostic sensitivity and specificity of the anti-Orf8 IgG ELISA for a larger number of patient samples and controls from the fifth wave of outbreaks which were collected on day 7 and 14 after an RT-PCR positive result were 58.79 and 58.44% and 89.19 and 58.44%, respectively. Our data indicated that some of the controls might have antibodies from natural past infections. Our study highlighted the potential utility of anti-Orf8 IgG antibody testing for seroprevalence surveys but still warrants further investigations.

Accelerated Development of a COVID-19 Lateral Flow Test in an Academic Setting: Lessons Learned.

The COVID-19 pandemic further demonstrated the need for usable, reliable, and cost-effective point-of-care diagnostics that can be broadly deployed, ideally for self-testing at home. Antigen tests using more-detectable reporter labels (usually at the cost of reader complexity) achieve better diagnostic sensitivity, supporting the value of higher-analytical-sensitivity reporter technologies in lateral flow.We developed a new approach to simple, inexpensive lateral flow assays (LFAs) of great sensitivity, based on the glow stick peroxyoxalate chemistry widely used in emergency settings and in children's toys. At the peak of the COVID-19 pandemic, we had the opportunity to participate in the pandemic-driven NIH Rapid Acceleration of Diagnostics (RADx) initiative aiming to develop a deployable lateral flow diagnostic for SARS-CoV-2 nucleoprotein based on our novel glow stick-inspired light-emitting reporter technology. During this project, we screened more than 250 antibody pairs for analytical sensitivity and specificity directly in LFA format, using recombinant nucleoprotein and then gamma-irradiated virions spiked into negative nasal swab extracts. Membranes and other LFA materials and swabs and extraction reagent components also were screened and selected. Optimization of conjugate preparation and spraying as well as pretreatment/conditioning of the sample pad led to the final optimized LFA strip. Technology development also included optimization of excitation liquid enclosed in disposable droppers, design of a custom cartridge and smartphone-based reader, and app development, even a prototype reader usable with any mobile phone. Excellent preclinical performance was first demonstrated with contrived samples and then with leftover clinical samples. Moving beyond traditional academic focus areas, we were able to establish a quality management system (QMS), produce large numbers of customized LFA cassettes by contract injection molding, build in-house facilities to assemble and store thousands of complete tests for verification and validation and usability studies, and source kitting/packaging services and quality standard reagents and build partnerships for clinical translation, regulatory guidance, scale up, and market deployment. We were not able to bring this early stage technology to the point of commercialization within the limited time and resources available, but we did achieve strong proof-of-concept and advance translational aspects of the platform including initial high-performance LFAs, reading by the iPhone app using only a $2 plastic dark box with no lens, and convenient, usable excitation liquid packaging in droppers manufacturable in very large numbers.In this Account, we aim to provide a concise overview of our 18-month sprint toward the practical development of a deployable antigen lateral flow assay under pandemic conditions and the challenges and successes experienced by our team. We highlight what it takes to coach a technically savvy but commercially inexperienced academic team through the accelerated translation of an early stage technology into a useful product. Finally, we provide a guided tutorial and workflow to empower others interested in the rapid development of translatable LFAs.

Clinical accuracy of instrument-based SARS-CoV-2 antigen diagnostic tests: a systematic review and meta-analysis.

BACKGROUND: During the COVID-19 pandemic, antigen diagnostic tests were frequently used for screening, triage, and diagnosis. Novel instrument-based antigen tests (iAg tests) hold the promise of outperforming their instrument-free, visually-read counterparts. Here, we provide a systematic review and meta-analysis of the SARS-CoV-2 iAg tests' clinical accuracy. METHODS: We systematically searched MEDLINE (via PubMed), Web of Science, medRxiv, and bioRxiv for articles published before November 7th, 2022, evaluating the accuracy of iAg tests for SARS-CoV-2 detection. We performed a random effects meta-analysis to estimate sensitivity and specificity and used the QUADAS-2 tool to assess study quality and risk of bias. Sub-group analysis was conducted based on Ct value range, IFU-conformity, age, symptom presence and duration, and the variant of concern. RESULTS: We screened the titles and abstracts of 20,431 articles and included 114 publications that fulfilled the inclusion criteria. Additionally, we incorporated three articles sourced from the FIND website, totaling 117 studies encompassing 95,181 individuals, which evaluated the clinical accuracy of 24 commercial COVID-19 iAg tests. The studies varied in risk of bias but showed high applicability. Of 24 iAg tests from 99 studies assessed in the meta-analysis, the pooled sensitivity and specificity compared to molecular testing of a paired NP swab sample were 76.7% (95% CI 73.5 to 79.7) and 98.4% (95% CI 98.0 to 98.7), respectively. Higher sensitivity was noted in individuals with high viral load (99.6% [95% CI 96.8 to 100] at Ct-level ≤ 20) and within the first week of symptom onset (84.6% [95% CI 78.2 to 89.3]), but did not differ between tests conducted as per manufacturer's instructions and those conducted differently, or between point-of-care and lab-based testing. CONCLUSION: Overall, iAg tests have a high pooled specificity but a moderate pooled sensitivity, according to our analysis. The pooled sensitivity increases with lower Ct-values (a proxy for viral load), or within the first week of symptom onset, enabling reliable identification of most COVID-19 cases and highlighting the importance of context in test selection. The study underscores the need for careful evaluation considering performance variations and operational features of iAg tests.

SARS-CoV-2 immunoassays in a predominantly vaccinated population: Performances and qualitative agreements obtained with two analytical approaches and four immunoassays.

BACKGROUND AND OBJECTIVES: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serosurveys are typically analysed by applying a fixed threshold for seropositivity ('conventional approach'). However, this approach underestimates the seroprevalence of anti-nucleocapsid (N) in vaccinated individuals-who often exhibit a difficult-to-detect anti-N response. This limitation is compounded by delays between the onset of infection and sample collection. To address this issue, we compared the performance of four immunoassays using a new analytical approach ('ratio-based approach'), which determines seropositivity based on an increase in anti-N levels. MATERIALS AND METHODS: Two groups of plasma donors and four immunoassays (Elecsys total anti-N, VITROS total anti-N, Architect anti-N Immunoglobulin G (IgG) and in-house total anti-N) were evaluated. First-group donors (N = 145) had one positive SARS-CoV-2 polymerase chain reaction (PCR) test result and had made two plasma donations, including one before and one after the PCR test (median = 27 days post-PCR). Second-group donors (N = 100) had made two plasma donations early in the Omicron wave. RESULTS: Among first-group donors (97.9% vaccinated), sensitivity estimates ranged from 60.0% to 89.0% with the conventional approach, compared with 94.5% to 98.6% with the ratio-based approach. Among second-group donors, Fleiss's κ ranged from 0.56 to 0.83 with the conventional approach, compared with 0.90 to 1.00 with the ratio-based approach. CONCLUSION: With the conventional approach, the sensitivity of four immunoassays-measured in a predominantly vaccinated population based on samples collected ~1 month after a positive test result-fell below regulatory agencies requirement of ≥95%. The ratio-based approach significantly improved the sensitivities and qualitative agreement among immunoassays, to the point where all would meet this requirement.

A flow cytometry-based assay to measure neutralizing antibodies against SARS-CoV-2 virus.

The COVID-19 pandemic caused by the SARS-CoV-2 virus has highlighted the need for serological assays that can accurately evaluate the neutralizing efficiency of antibodies produced during infection or induced by vaccines. However, conventional assays often require the manipulation of live viruses on a level-three biosafety (BSL3) facility, which presents practical and safety challenges. Here, we present a novel, alternative assay that measures neutralizing antibodies (NAbs) against SARS-CoV-2 in plasma using flow cytometry. This assay is based on antibody binding to the S protein and has demonstrated precision in both intra- and inter-assay measurements at a dilution of 1:50. The cut-off was determined using Receiver Operating Characteristic (ROC) analysis and the value of 36.01% has shown high sensitivity and specificity in distinguishing between pre-pandemic sera, COVID-19 patients, and vaccinated individuals. The efficiency significantly correlates with the gold standard test, PRNT. Our new assay offers a safe and efficient alternative to conventional assays for evaluating NAbs against SARS-CoV-2.