A Reagent and Virus Benchmarking Panel for a Uniform Analytical Performance Assessment of N Antigen-Based Diagnostic Tests for COVID-19.

Rapid diagnostic tests (RDTs) that detect antigen indicative of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection can help in making quick health care decisions and regularly monitoring groups at risk of infection. With many RDT products entering the market, it is important to rapidly evaluate their relative performance. Comparison of clinical evaluation study results is challenged by protocol design variations and study populations. Laboratory assays were developed to quantify nucleocapsid (N) and spike (S) SARS-CoV-2 antigens. Quantification of the two antigens in nasal eluates confirmed higher abundance of N than S antigen. The median concentration of N antigen was 10 times greater than S per genome equivalent. The N antigen assay was used in combination with quantitative reverse transcription (RT)-PCR to qualify a panel composed of recombinant antigens, inactivated virus, and clinical specimen pools. This benchmarking panel was applied to evaluate the analytical performance of the SD Biosensor Standard Q COVID-19 antigen (Ag) test, Abbott Panbio COVID-19 Ag rapid test, Abbott BinaxNOW COVID-19 Ag test, and the LumiraDx SARS-CoV-2 Ag test. The four tests displayed different sensitivities toward the different panel members, but all performed best with the clinical specimen pool. The concentration for a 90% probability of detection across the four tests ranged from 21 to 102 pg/mL of N antigen in the extracted sample. Benchmarking panels provide a quick way to verify the baseline performance of a diagnostic and enable direct comparisons between diagnostic tests. IMPORTANCE This study reports the results for severe acute respiratory syndrome coronavirus-2 (SARS-COV-2) nucleocapsid (N) and spike (S) antigen quantification assays and their performance against clinical reverse transcription (RT)-PCR results, thus describing an open-access quantification method for two important SARS-CoV-2 protein analytes. Characterized N antigen panels were used to evaluate the limits of detection of four different rapid tests for SARS-CoV-2 against multiple sources of nucleocapsid antigen, demonstrating proof-of-concept materials and methodology to evaluate SARS-CoV-2 rapid antigen detection tests. Quantification of N antigen was used to characterize the relationship between viral count and antigen concentration among clinical samples and panel members of both clinical sample and viral culture origin. This contributes to a deeper understanding of protein antigen and molecular analytes and presents analytical methods complementary to clinical evaluation for characterizing the performance of both laboratory-based and point-of-care rapid diagnostics for SARS-CoV-2.

Rapid SARS-CoV-2 testing with duplexed recombinase polymerase amplification and a bacteriophage internal control

OBJECTIVES/GOALS: Current COVID-19 rapid molecular tests require cartridge-reader detection, expensive circuitry, and complex microfluidics making the most accurate tests unavailable to the masses. Here we present a rapid molecular diagnostic leveraging isothermal amplification and paper-based microfluidics for a low-cost ultra-sensitive COVID-19 assay. METHODS/STUDY POPULATION: We designed a reverse transcription recombinase polymerase amplification (RT-RPA) assay for the detection of SARS-CoV-2 and bacteriophage MS2 RNA. RT-RPA is a sequence specific, ultrasensitive, rapid isothermal DNA amplification technique that is well suited to home based testing due to its rapid assay time, robustness, ease of use, and readout options. RT-RPA reagents are added to a tube and incubated at 39°C in a fluorometer. Realtime fluorometer data gives results in under 15 minutes. This assay also provides visual detection via lateral flow readout with results in 23 minutes. RESULTS/ANTICIPATED RESULTS: We have developed a rapid multiplexed nucleic acid amplification assay with an internal process control for SARS-CoV-2 using single-pot RT-RPA. We screened 21 primer combinations to select primers that demonstrated excellent performance and target specificity against common respiratory viruses. We demonstrate the ability to multiplex SARS-CoV-2 and MS2 detection, utilizing MS2 as an internal process control for lysis, reverse transcription, amplification, and readout. We show duplexed detection using both fluorescence readout and visual readout using lateral flow strips. Duplexed fluorescence detection shows a limit of detection of 25 copies per reaction. Duplexed lateral flow readout shows a limit of detection of 50 copies per reaction DISCUSSION/SIGNIFICANCE: We developed a duplexed RT-RPA assay for SARS-CoV-2 with fluorescence or lateral flow readout. Our assay does not re-quire expensive reader, circuity, or fluid handling. The low material cost, temperature, and robustness make it ideal for a more accurate home-based COVID-19 diagnostic.

A SARS-CoV-2 coronavirus nucleocapsid protein antigen-detecting lateral flow assay.

Inexpensive, simple, rapid diagnostics are necessary for efficient detection, treatment, and mitigation of COVID-19. Assays for SARS-CoV2 using reverse transcription polymerase chain reaction (RT-PCR) offer good sensitivity and excellent specificity, but are expensive, slowed by transport to centralized testing laboratories, and often unavailable. Antigen-based assays are inexpensive and can be rapidly mass-produced and deployed at point-of-care, with lateral flow assays (LFAs) being the most common format. While various manufacturers have produced commercially available SARS-Cov2 antigen LFAs, access to validated tests remains difficult or cost prohibitive in low-and middle-income countries. Herein, we present a visually read open-access LFA (OA-LFA) using commercially-available antibodies and materials for the detection of SARS-CoV-2. The LFA yielded a Limit of Detection (LOD) of 4 TCID50/swab of gamma irradiated SARS-CoV-2 virus, meeting the acceptable analytical sensitivity outlined by in World Health Organization target product profile. The open-source architecture presented in this manuscript provides a template for manufacturers around the globe to rapidly design a SARS-CoV2 antigen test.

Performance of anterior nares and tongue swabs for nucleic acid, Nucleocapsid, and Spike antigen testing for detecting SARS-CoV-2 against nasopharyngeal PCR and viral culture.

OBJECTIVES: This study assesses and compares the performance of different swab types and specimen collection sites for SARS-CoV-2 testing, to reference standard real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and viral culture. METHODS: Symptomatic adults with COVID-19 who visited routine COVID-19 testing sites used spun polyester and FLOQSwabs to self-collect specimens from the anterior nares and tongue. We evaluated the self-collected specimen from anterior nares and tongue swabs for the nucleocapsid (N) or spike (S) antigen of SARS-CoV-2 by RT-PCR and then compared these results with results from RT-PCR and viral cultures from nurse-collected nasopharyngeal swabs. RESULTS: Diagnostic sensitivity was highest for RT-PCR testing conducted using specimens from the anterior nares collected on FLOQSwabs (84%; 95% CI 68-94%) and spun polyester swabs (82%; 95% CI 66-92%), compared to RT-PCR tests conducted using specimens from nasopharyngeal swabs. Relative to viral culture from nasopharyngeal swabs, diagnostic sensitivities were higher for RT-PCR and antigen testing of anterior nares swabs (91-100%) than that of tongue swabs (18-81%). Antigen testing of anterior nares swabs had higher sensitivities against viral culture (91%) than against nasopharyngeal RT-PCR (38-70%). All investigational tests had high specificity compared with nasopharyngeal RT-PCR. Spun polyester swabs are equally effective as FLOQSwabs for anterior nasal RT-PCR testing. CONCLUSIONS: We found that anterior nares specimens were more sensitive than tongue swab specimens or antigen testing for detecting SARS-CoV-2 by RT-PCR. Thus, self-collected anterior nares specimens may represent an alternative method for diagnostic SARS-CoV-2 testing in some settings.

Antibody Screening Results for Anti-Nucleocapsid Antibodies Toward the Development of a Lateral Flow Assay to Detect SARS-CoV-2 Nucleocapsid Protein.

The global COVID-19 pandemic has created an urgent demand for large numbers of inexpensive, accurate, rapid, point-of-care diagnostic tests. Analyte-based assays are suitably rapid and inexpensive and can be rapidly mass-produced, but for sufficiently accurate performance, they require highly optimized antibodies and assay conditions. We used an automated liquid handling system, customized to handle arrays of lateral flow (immuno)assays (LFAs) in a high-throughput screen, to identify anti-nucleocapsid antibodies that will perform optimally in an LFA. We tested 1021 anti-nucleocapsid antibody pairs as LFA capture and detection reagents with the goal of highlighting pairs that have the greatest affinity for the nucleocapsid protein of SARS-CoV-2 within the LFA format. In contrast to traditional antibody screening methods (e.g., ELISA, bio-layer interferometry), the method described here integrates real-time reaction kinetics with transport in, and immobilization directly onto, nitrocellulose. We have identified several candidate antibody pairs that are suitable for further development of an LFA for SARS-CoV-2.

Screening Antibodies Raised against the Spike Glycoprotein of SARS-CoV-2 to Support the Development of Rapid Antigen Assays.

Severe acute respiratory coronavirus-2 (SARS-CoV-2) is a novel viral pathogen and therefore a challenge to accurately diagnose infection. Asymptomatic cases are common and so it is difficult to accurately identify infected cases to support surveillance and case detection. Diagnostic test developers are working to meet the global demand for accurate and rapid diagnostic tests to support disease management. However, the focus of many of these has been on molecular diagnostic tests, and more recently serologic tests, for use in primarily high-income countries. Low- and middle-income countries typically have very limited access to molecular diagnostic testing due to fewer resources. Serologic testing is an inappropriate surrogate as the early stages of infection are not detected and misdiagnosis will promote continued transmission. Detection of infection via direct antigen testing may allow for earlier diagnosis provided such a method is sensitive. Leading SARS-CoV-2 biomarkers include spike protein, nucleocapsid protein, envelope protein, and membrane protein. This research focuses on antibodies to SARS-CoV-2 spike protein due to the number of monoclonal antibodies that have been developed for therapeutic research but also have potential diagnostic value. In this study, we assessed the performance of antibodies to the spike glycoprotein, acquired from both commercial and private groups in multiplexed liquid immunoassays, with concurrent testing via a half-strip lateral flow assays (LFA) to indicate antibodies with potential in LFA development. These processes allow for the selection of pairs of high-affinity antispike antibodies that are suitable for liquid immunoassays and LFA, some of which with sensitivity into the low picogram range with the liquid immunoassay formats with no cross-reactivity to other coronavirus S antigens. Discrepancies in optimal ranking were observed with the top pairs used in the liquid and LFA formats. These findings can support the development of SARS-CoV-2 LFAs and diagnostic tools.

Current state of commercial point-of-care nucleic acid tests for infectious diseases.

The COVID-19 pandemic has put the spotlight on the urgent need for integrated nucleic acid tests (NATs) for infectious diseases, especially those that can be used near patient ("point-of-care", POC), with rapid results and low cost, but without sacrificing sensitivity or specificity of gold standard PCR tests. In the US, the Clinical Laboratory Improvement Amendments Certificate of Waiver (CLIA-waiver) is mandated by the Food and Drug Administration (FDA) and designated to any laboratory testing with high simplicity and low risk for error, suitable for application in the POC. Since the first issuance of CLIA-waiver to Abbot's ID NOW Influenza A&B in 2015, many more NAT systems have been developed, received the CLIA-waiver in the US or World Health Organization (WHO)'s pre-qualification, and deployed to the front line of infectious disease detection. This review highlights the regulatory process for FDA and WHO in evaluating these NATs and the technology innovation of existing CLIA-waived systems. Understanding the technical advancement and challenges, unmet needs, and the trends of commercialization facilitated through the regulatory processes will help pave the foundation for future development and technology transfer from research to the market place.

SARS-CoV-2 Coronavirus Nucleocapsid Antigen-Detecting Half-Strip Lateral Flow Assay Toward the Development of Point of Care Tests Using Commercially Available Reagents.

The SARS-CoV-2 pandemic has created an unprecedented need for rapid diagnostic testing to enable the efficient treatment and mitigation of COVID-19. The primary diagnostic tool currently employed is reverse transcription polymerase chain reaction (RT-PCR), which can have good sensitivity and excellent specificity. Unfortunately, implementation costs and logistical problems with reagents during the global SARS-CoV-2 pandemic have hindered its universal on demand adoption. Lateral flow assays (LFAs) represent a class of diagnostic that, if sufficiently clinically sensitive, may fill many of the gaps in the current RT-PCR testing regime, especially in low- and middle-income countries (LMICs). To date, many serology LFAs have been developed, though none meet the performance requirements necessary for diagnostic use cases, primarily due to the relatively long delay between infection and seroconversion. However, on the basis of previously reported results from SARS-CoV-1, antigen-based SARS-CoV-2 assays may have significantly better clinical sensitivity than serology assays. To date, only a very small number of antigen-detecting LFAs have been developed. Development of a half-strip LFA is a useful first step in the development of any LFA format. In this work, we present a half-strip LFA using commercially available antibodies for the detection of SARS-CoV-2. We have tested this LFA in buffer and measured an LOD of 0.65 ng/mL (95% CI of 0.53 to 0.77 ng/mL) ng/mL with recombinant antigen using an optical reader with sensitivity equivalent to a visual read. Further development, including evaluating the appropriate sample matrix, will be required for this assay approach to be made useful in a point of care setting, though this half-strip LFA may serve as a useful starting point for others developing similar tests.