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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Comparative evaluation of RT-PCR and antigen-based rapid diagnostic tests (Ag-RDTs) for SARS-CoV-2 detection: performance, variant specificity, and clinical implications.

The COVID-19 pandemic has highlighted the critical need for accurate and efficient diagnostic tools for detecting severe acute respiratory coronavirus 2 (SARS-CoV-2) infections. This study presents a comparison of two diagnostic tests: RT-PCR and antigen detection rapid diagnostic tests (Ag-RDTs). This study focused on their performance, variant specificity, and their clinical implications. A simultaneous testing of 268 samples was carried out for SARS-CoV-2 using RT-PCR and Ag-RDTs [flourescence immunoassay (FIA) and lateral flow immunoassay (LFIA)]. Viral load was quantified, and variant identification was performed using a PCR-based assay. The prevalence was found to be 30.2% using reverse transcription PCR (RT-PCR), 26.5% using FIA, and 25% using LFIA. When comparing the FIA and LFIA, the overall diagnostic performance was found to be 80.25% vs 76.54%, 96.79% vs 97.33%, 91.55% vs 90.51%, and 91.88% vs 92.56% for sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV), respectively. Both Ag-RDTs showed a strong agreement with RT-PCR (κ = 0.78-0.80). The overall accuracies of the FIA and LFIA were 92.41% and 92.13%, respectively. The FIA showed higher sensitivity (73.68%) and PPV (92.08%) than the LFIA (65.79% and 90.56%, respectively) in asymptomatic patients. At low Ct values (<25), both Ag-RDTs had 100% sensitivity, but the sensitivity reduced to 31.82% for FIA and 27.27% for LFIA at Ct values > 30. The diagnostic sensitivity of FIA compared to LFIA for detecting the Alpha variant was 78.85% vs. 69.23% and 72.22% vs. 83.33% for the Delta variant. Both Ag-RDTs had 100% sensitivity for detecting Omicron. Both Ag-RDTs performed well in patients with high viral loads and Omicron variant infections compared to those infected with Alpha and Delta variants. This study confirms the comparable performance of RT-PCR and Ag-RDTs, specifically FIA and LFIA, for SARS-CoV-2 detection. The FIA showed higher sensitivity and PPV in asymptomatic cases, while both Ag-RDTs exhibited strong agreement with RT-PCR results. Notably, Ag-RDTs, particularly FIA, proved effective in detecting the Omicron variant and cases with high viral loads, highlighting their potential clinical utility in managing the COVID-19 pandemic.IMPORTANCEThis study is of utmost importance in providing effective responses to manage the COVID-19 pandemic. It rigorously compares the diagnostic accuracy, variant specificity, and practical considerations of reverse transcription PCR (RT-PCR) and antigen detection rapid diagnostic tests (Ag-RDTs) for severe acute respiratory coronavirus 2 (SARS-CoV-2), answering critical questions. The results of this study will help healthcare professionals choose the appropriate testing methods, allocate resources effectively, and enhance public health strategies. Given the evolution of the virus, understanding the performance of these diagnostic tools is crucial to adapting to emerging variants. Additionally, the study provides insights into logistical challenges and accessibility issues, which will contribute to refining testing workflows, particularly in resource-limited settings. Ultimately, the study's impact extends to global healthcare, providing valuable information for policymakers, clinicians, and public health officials as they work together for mitigating the impact of the pandemic.

Comparison of the analytical sensitivity of COVID-19 rapid antigen tests in Australia and Canada.

Rapid testing has become an indispensable strategy to identify the most infectious individuals and prevent the transmission of SARS-CoV-2 in vulnerable populations. As such, COVID-19 rapid antigen tests (RATs) are being manufactured faster than ever yet lack relevant comparative analyses required to inform on absolute analytical sensitivity and performance, limiting end-user ability to accurately compare brands for decision making. To date, more than 1000 different COVID-19 RATs are commercially available in the world, most of which detect the viral nucleocapsid protein (NP). Here, we examine and compare the analytical sensitivity of 26 RATs that are readily available in Canada and/or Australia using two NP reference materials (RMs) - a fluorescent NP-GFP expressed in bacterial cells and NCAP-1 produced in a mammalian expression system. Both RMs generate highly comparable results within each RAT, indicating minimal bias due to differing expression systems and final buffer compositions. However, we demonstrate orders of magnitude differences in analytical sensitivities among distinct RATs, and find little correlation with the median tissue culture infectious dose (TCID50) assay values reported by manufacturers. In addition, two COVID-19/Influenza A&B combination RATs were evaluated with influenza A NP-GFP. Finally, important logistics considerations are discussed regarding the robustness, ease of international shipping and safe use of these reference proteins. Taken together, our data highlight the need for and practicality of readily available, reliable reference proteins for end-users that will ensure that manufacturers maintain batch-to-batch quality and accuracy of RATs. They will aid international public health and government agencies, as well as health and aged care facilities to reliably benchmark and select the best RATs to curb transmission of future SARS-CoV-2 and influenza outbreaks.

Taqman PACMAN: a simple molecular approach for positive rapid antigen test confirmation during periods of low prevalence.

Antigen-based rapid diagnostic tests (Ag-RDTs) were widely deployed to enhance SARS-CoV-2 testing capacity during the COVID-19 pandemic. Consistent with national guidance for low prevalence settings, positive Ag-RDTs were confirmed using nucleic acid amplification tests (NAATs) to avoid false positive results. However, increasing demands for positive Ag-RDT confirmation competed with other testing priorities in clinical laboratories. This work hypothesized that real-time RT-PCR without nucleic acid extraction (NAE) would be sufficiently sensitive to support positive Ag-RDT confirmation. Ag-RDT and NAAT results from community-based asymptomatic testing sites prior to the omicron variant wave were compared to calculate the weekly false positive rate (FPR) and false detection rate (FDR). Real-time RT-PCR was compared with and without NAE using 752 specimens previously tested positive for SARS-CoV-2 using commercial NAATs and 344 specimens from Ag-RDT-positive individuals. The impact of SARS-CoV-2 prevalence on laboratory resources required to sustain Ag-RDT confirmation was modeled for the RT-PCR with and without NAE. Overall, FPR was low [0.07% (222/330,763)] in asymptomatic testing sites, but FDR was high [30.7% (222/724)]. When RT-PCR was compared with and without NAE, 100% concordance was obtained with NAAT-positive specimens, including those from Ag-RDT-positive individuals. NAE-free RT-PCR significantly reduced time to results, human resources, and overall costs. A 30.7% FDR reaffirms the need for NAAT-based confirmation of positive Ag-RDT results during low SARS-CoV-2 prevalence. NAE-free RT-PCR was shown to be a simple and cost-sparing NAAT-based solution for positive Ag-RDT confirmation, and its implementation supported data-driven broader Ag-RDT deployment into communities, workplaces, and households. IMPORTANCE: Rapid antigen testing for SARS-CoV-2 was widely deployed during the COVID-19 pandemic. In settings of low prevalence, national guidance recommends that positive antigen test results be confirmed with molecular testing. Given the high testing burden on clinical laboratories during the COVID-19 pandemic, the high volume of positive antigen tests submitted for confirmatory testing posed challenges for laboratory workflow. This study demonstrated that a simple PCR method without prior nucleic acid purification is an accurate and cost-effective solution for positive rapid antigen test confirmation. Implementing this method allowed molecular confirmatory testing for positive antigen tests to be sustained as antigen testing was expanded into large populations such as workplaces, schools, and households.

Development of Detection Antibody Targeting the Linear Epitope in SARS-CoV-2 Nucleocapsid Protein with Ultra-High Sensitivity.

The COVID-19 pandemic caused by SARS-CoV-2 highlighted the importance of reliable detection methods for disease control and surveillance. Optimizing detection antibodies by rational screening antigens would improve the sensitivity and specificity of antibody-based detection methods such as colloidal gold immunochromatography. In this study, we screened three peptide antigens with conserved sequences in the N protein of SARS-CoV-2 using bioinformatical and structural biological analyses. Antibodies that specifically recognize these peptides were prepared. The epitope of the peptide that had the highest binding affinity with its antibody was located on the surface of the N protein, which was favorable for antibody binding. Using the optimal antibody that can recognize this epitope, we developed colloidal gold immunochromatography, which can detect the N protein at 10 pg/mL. Importantly, this antibody could effectively recognize both the natural peptide antigen and mutated peptide antigen in the N protein, showing the feasibility of being applied in the large-scale population testing of SARS-CoV-2. Our study provides a platform with reference significance for the rational screening of detection antibodies with high sensitivity, specificity, and reliability for SARS-CoV-2 and other pathogens.

Estimating SARS-CoV-2 infection probabilities with serological data and a Bayesian mixture model.

The individual results of SARS-CoV-2 serological tests measured after the first pandemic wave of 2020 cannot be directly interpreted as a probability of having been infected. Plus, these results are usually returned as a binary or ternary variable, relying on predefined cut-offs. We propose a Bayesian mixture model to estimate individual infection probabilities, based on 81,797 continuous anti-spike IgG tests from Euroimmun collected in France after the first wave. This approach used serological results as a continuous variable, and was therefore not based on diagnostic cut-offs. Cumulative incidence, which is necessary to compute infection probabilities, was estimated according to age and administrative region. In France, we found that a "negative" or a "positive" test, as classified by the manufacturer, could correspond to a probability of infection as high as 61.8% or as low as 67.7%, respectively. "Indeterminate" tests encompassed probabilities of infection ranging from 10.8 to 96.6%. Our model estimated tailored individual probabilities of SARS-CoV-2 infection based on age, region, and serological result. It can be applied in other contexts, if estimates of cumulative incidence are available.