Evaluation Protocol for SARS-CoV-2 Serological Assays.

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has been identified as the causative agent of COVID-19. Accurate detection of SARS-CoV-2 infection is not only important for management of infected individuals but also to break the chain of transmission. Although the polymerase chain reaction (PCR) is the gold standard for diagnosis of acute SARS-CoV-2 infection, there are a number of limitations of these assays, which include the inability to detect past infection and decline in sensitivity 14 days post-symptom onset. There are several serology tests developed for the detection of SARS-CoV-2 antibodies including high-throughput serology platforms and lateral flow immunoassays. These tests should be evaluated for their performance to meet local regulations acceptance criteria. To optimize the diagnostic algorithm for SARS-CoV-2, this protocol describes the evaluation of serological antibody testing using various automated serology platforms and lateral flow immunoassays. This protocol was evaluated in both serum and plasma samples. The sample preparation, procedure, and data analysis are described. The protocol can be adapted for any serological testing.

Performance of the Abbott SARS-CoV-2 IgG serological assay in South African 2 patients.

In late December 2019, pneumonia cases of unknown origin were reported in Wuhan, China. This virus was named SARS-CoV2 and the clinical syndrome was named coronavirus disease 19 (COVID-19). South Africa, despite strict and early lockdown has the highest infection rate in Africa. A key component of South Africa's response to SARSCoV2 was the rapid scale-up of diagnostic testing. The Abbott SARS-CoV2 assay detects IgG antibodies against the Nucleocapsid (N) protein of the SARS-CoV2 virus. This study undertook to validate and evaluate performance criteria of the Abbott assay and to establish whether this assay would show clinical utility in our population. Positive patients (n = 391) and negative controls (n = 139) were included. The Architect-i and Alinity-i systems were analyzers that were used to perform the SARS-CoV-2 IgG assay. In-house ELISA was incorporated into the study as a confirmatory serology test. A total of number of 530 participants was tested, 87% were symptomatic with infection and 13% were asymptomatic. When compared to RT-qPCR, the sensitivity of Architect and Alinity SARS-CoV2 assays was 69.5% and 64.8%, respectively. Specificity for Architect and Alinity assays was 95% and 90.3%, respectively. The Abbott assay was also compared to in house ELISA assay, with sensitivity for the Architect and Alinity assays of 94.7% and 92.5%, respectively. Specificity for Abbott Alinity assays was 91.7% higher than Abbott Architect 88.1%. Based on the current findings testing of IgG after 14 days is recommended in South Africa and supports other studies performed around the world.

Antigen-Based Point of Care Testing (POCT) for Diagnosing SARS-CoV-2: Assessing Performance.

Currently, the most accurate way to diagnose an active SARS-CoV-2 (COVID-19) infection is through detection of viral RNA using reverse transcription polymerase chain reaction (RT-PCR) test. While RT-PCR tests are the most sensitive for identifying infection, there are significant limitations, such as global access to sufficient test kits, turnaround times (TAT) from specimen collection to test result is often greater than 24 h and the need for skilled operators in accredited laboratories requiring specialized equipment. A rapid test performed at the point of care (POC) could provide a result within an approximate time of 30 min post specimen collection, be performed by a health care worker and comprise a simple workflow, improving both turnaround time and potentially decreasing costs (e.g., transport, cold-chain, skilled laboratory staff, complex equipment). Determining the performance of SARS-CoV-2 RT-PCR tests is, however, easier to assess than antigen-based POCT, as residual clinical specimens (swabs in universal transport media [UTM]) are readily available in laboratory environments, and do not require patient informed consent. Evaluating the performance of POCT requires informed-consent driven studies, with patients required to provide a standard of care specimen as well as study evaluation specimens, which is often not acceptable as nasopharyngeal swabbing can be invasive, clinical field trials are costly and time consuming. Many institutions and regulatory bodies also require preliminary data prior to use in field settings. Therefore, we have developed a method to determine the performance of antigen based POCT that can be used by implementers in national healthcare programs, regulators and rapid test developers. The method investigates both quantitative and qualitative parameters, with the latter providing insights into the capability for implementation and national program uptake.

Operational characteristics of 30 lateral flow immunoassays used to identify COVID-19 immune response.

Serology or antibody tests for COVID-19 are designed to detect antibodies (mainly Immunoglobulin M (IgM) and Immunoglobulin G (IgG) produced in response to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS CoV-2) infection. In this study, 30 lateral flow immunoassays were tested using serum or plasma from patients with confirmed SARS CoV-2 infection. Negative serological controls were accessed from a well-characterised bank of sera which were stored prior to February 2020. Operational characteristics and ease of use of the assays are reported. 4/30 (13%) of kits (Zheihang Orient Gene COVID-19 IgG/IgM, Genrui Novel Coronavirus (2019-nCoV) IgG/IgM, Biosynex COVID-19 BSS IgG/IgM, Boson Biotech 2019-nCoV IgG/IgM) were recommended for SAHPRA approval based on kit sensitivity. Of these, only the Orientgene was recommended by SAHPRA in August 2020 for use within the approved national testing algorithm while the remaining three received limited authorization for evaluation. All kits evaluated work on the same basic principle of immunochromatography with minor differences noted in the shape and colour of cartridges, the amount of specimen volume required and the test duration. Performance of the lateral flow tests were similar to sensitivities and specificities reported in other studies. The cassettes of the majority of kits evaluated (90%) detected both IgG and IgM. Only 23% of kits evaluated contained all consumables required for point-of-care testing. The study highlights the need for thorough investigation of kits prior to implementation.

Performance of the EUROIMMUN Anti-SARS-CoV-2 ELISA Assay for detection of IgA and IgG antibodies in South Africa.

Severe Acute Respiratory Syndrome-Coronavirus 2 (SARS-CoV-2) has been identified as the causative agent for causing the clinical syndrome of COVID -19. Accurate detection of SARS-CoV-2 infection is not only important for management of infected individuals but also to break the chain of transmission. South Africa is the current epicenter of SARS-CoV-2 infection in Africa. To optimize the diagnostic algorithm for SARS-CoV-2 in the South African setting, the study aims to evaluate the diagnostic performance of the EUROIMMUN Anti-SARS-CoV-2 assays. This study reported the performance of EUROIMMUN enzyme-linked immunosorbent assay (ELISA) for semi-quantitative detection of IgA and IgG antibodies in serum and plasma samples targeting the recombinant S1 domain of the SARS-CoV-2 spike protein as antigen. Samples were collected from 391 individuals who had tested positive for SARS-CoV-2 and 139 SARS CoV-2 negative controls. Samples were stratified by number of days' post-PCR diagnosis and symptoms. The sensitivity of EUROIMMUN IgG was 64.1% (95% CI: 59.1-69.0%) and 74.3% (95% CI: 69.6-78.6%) for IgA and the specificity was lower for IgA [84.2% (95% CI: 77-89.2%)] than IgG [95.2% (95% CI: 90.8-98.4%)]. The EUROIMMUN Anti-SARS-CoV-2 ELISA Assay sensitivity was higher for IgA but low for IgG and improved for both assays in symptomatic individuals and at later timepoints post PCR diagnosis.

SARS-CoV-2 Antigens Expressed in Plants Detect Antibody Responses in COVID-19 Patients.

Background: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has swept the world and poses a significant global threat to lives and livelihoods, with 115 million confirmed cases and at least 2.5 million deaths from Coronavirus disease 2019 (COVID-19) in the first year of the pandemic. Developing tools to measure seroprevalence and understand protective immunity to SARS-CoV-2 is a priority. We aimed to develop a serological assay using plant-derived recombinant viral proteins, which represent important tools in less-resourced settings. Methods: We established an indirect ELISA using the S1 and receptor-binding domain (RBD) portions of the spike protein from SARS-CoV-2, expressed in Nicotiana benthamiana. We measured antibody responses in sera from South African patients (n = 77) who had tested positive by PCR for SARS-CoV-2. Samples were taken a median of 6 weeks after the diagnosis, and the majority of participants had mild and moderate COVID-19 disease. In addition, we tested the reactivity of pre-pandemic plasma (n = 58) and compared the performance of our in-house ELISA with a commercial assay. We also determined whether our assay could detect SARS-CoV-2-specific IgG and IgA in saliva. Results: We demonstrate that SARS-CoV-2-specific immunoglobulins are readily detectable using recombinant plant-derived viral proteins, in patients who tested positive for SARS-CoV-2 by PCR. Reactivity to S1 and RBD was detected in 51 (66%) and 48 (62%) of participants, respectively. Notably, we detected 100% of samples identified as having S1-specific antibodies by a validated, high sensitivity commercial ELISA, and optical density (OD) values were strongly and significantly correlated between the two assays. For the pre-pandemic plasma, 1/58 (1.7%) of samples were positive, indicating a high specificity for SARS-CoV-2 in our ELISA. SARS-CoV-2-specific IgG correlated significantly with IgA and IgM responses. Endpoint titers of S1- and RBD-specific immunoglobulins ranged from 1:50 to 1:3,200. S1-specific IgG and IgA were found in saliva samples from convalescent volunteers. Conclusion: We demonstrate that recombinant SARS-CoV-2 proteins produced in plants enable robust detection of SARS-CoV-2 humoral responses. This assay can be used for seroepidemiological studies and to measure the strength and durability of antibody responses to SARS-CoV-2 in infected patients in our setting.