Ultrasensitive measurement of both SARS-CoV2 RNA and serology from saliva

Tests for COVID-19 generally measure SARS-CoV2 viral RNA from nasal swabs or antibodies against the virus from blood. It has been shown, however, that both viral particles and antibodies against those particles are present in saliva, which is more accessible than both swabs and blood. We present methods for highly sensitive measurements of both viral RNA and serology from the same saliva sample. We developed an efficient saliva RNA extraction method and combined it with an ultrasensitive serology test based on Single Molecule Array (Simoa) technology. We apply our test to the saliva of patients who presented to the hospital with COVID-19 symptoms, some of whom tested positive with a conventional RT-qPCR nasopharyngeal swab test. We demonstrate that combining viral RNA detection by RT-qPCR with serology identifies more patients as infected than either method alone. Our results suggest the utility of combining viral RNA and serology testing from saliva, a single easily accessible biofluid.

Evaluation of serological lateral flow assays for severe acute respiratory syndrome coronavirus-2

BackgroundCOVID-19 has resulted in significant morbidity and mortality worldwide. Lateral flow assays can detect anti-Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) antibodies to monitor transmission. However, standardized evaluation of their accuracy and tools to aid in interpreting results are needed. MethodsWe evaluated 20 IgG and IgM assays selected from available tests in April 2020. We evaluated the assays performance using 56 pre-pandemic negative and 56 SARS-CoV-2-positive plasma samples, collected 10-40 days after symptom onset, confirmed by a molecular test and analyzed by an ultra-sensitive immunoassay. Finally, we developed a user-friendly web app to extrapolate the positive predictive values based on their accuracy and local prevalence. ResultsCombined IgG+IgM sensitivities ranged from 33.9% to 94.6%, while combined specificities ranged from 92.6% to 100%. The highest sensitivities were detected in Lumiquick for IgG (98.2%), BioHit for both IgM (96.4%), and combined IgG+IgM sensitivity (94.6%). Furthermore, 11 LFAs and 8 LFAs showed perfect specificity for IgG and IgM, respectively, with 15 LFAs showing perfect combined IgG+IgM specificity. Lumiquick had the lowest estimated limit-of-detection (LOD) (0.1 g/mL), followed by a similar LOD of 1.5 g/mL for CareHealth, Cellex, KHB, and Vivachek. ConclusionWe provide a public resource of the accuracy of select lateral flow assays with potential for home testing. The cost-effectiveness, scalable manufacturing process, and suitability for self-testing makes LFAs an attractive option for monitoring disease prevalence and assessing vaccine responsiveness. Our web tool provides an easy-to-use interface to demonstrate the impact of prevalence and test accuracy on the positive predictive values.

Serial Profiling of SARS-CoV-2 Antigens and Antibodies in COVID-19 Patient Plasma

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected millions of people worldwide. PCR tests are currently the gold standard for diagnosis of the current coronavirus disease (COVID-19) and serology tests are used to detect seroconversion in infected patients. However, there is a lack of quantitative and ultra-sensitive viral antigen tests for COVID-19. Here we show that Single Molecule Array (Simoa) assays can quantitatively detect SARS-CoV-2 spike, S1 subunit, and nucleocapsid antigens in the plasma of COVID-19 patients. Combined with Simoa anti-SARS-CoV-2 serological assays, we show correlation between production of antibodies and clearance of viral antigens from serial plasma samples from COVID-19 patients. Furthermore, we demonstrate the presence of viral antigens in blood correlates with disease severity in hospitalized COVID-19 patients. These data suggest that SARS-CoV-2 viral antigens in the blood could be a marker for severe COVID-19 cases. One Sentence SummarySARS-CoV-2 antigens S1, spike, and nucleocapsid and anti-SARS-Cov-2 antibodies were measured in longitudinal plasma samples from COVID-19 patients using Single Molecule Array (Simoa) assays.

Evaluation of two commercial and two non-commercial immunoassays for the detection of prior infection to SARS-CoV-2

BackgroundSeroepidemiology is an important tool to characterize the epidemiology and immunobiology of SARS-CoV-2 but many immunoassays have not been externally validated raising questions about reliability of study findings. To ensure meaningful data, particularly in a low seroprevalence population, assays need to be rigorously characterized with high specificity. MethodsWe evaluated two commercial (Roche Diagnostics and Epitope Diagnostics IgM/IgG) and two non-commercial (Simoa and Ragon/MGH IgG) immunoassays against 68 confirmed positive and 232 pre-pandemic negative controls. Sensitivity was stratified by time from symptom onset. The Simoa multiplex assay applied three pre-defined algorithm models to determine sample result. ResultsThe Roche and Ragon/MGH IgG assays each registered 1/232 false positive, the primary Simoa model registered 2/232 false positives, and the Epitope registered 2/230 and 3/230 false positives for the IgG and IgM assays respectively. Sensitivity >21 days post symptom-onset was 100% for all assays except Epitope IgM, but lower and/or with greater variability between assays for samples collected 9-14 days (67-100%) and 15-21 days (69-100%) post-symptom onset. The Simoa and Epitope IgG assays demonstrated excellent sensitivity earlier in the disease course. The Roche and Ragon/MGH assays were less sensitive during early disease, particularly among immunosuppressed individuals. ConclusionsThe Epitope IgG demonstrated good sensitivity and specificity. The Roche and Ragon/MGH IgG assays registered rare false positives with lower early sensitivity. The Simoa assay primary model had excellent sensitivity and few false positives. SummarySARS-CoV-2 immunoassays can be valuable tools for informing the global response, but many currently available assays have not been independently validated. We conducted a performance assessment of four assays including the Roche Diagnostics and Epitope Diagnostics immunoassays.

Ultra-Sensitive High-Resolution Profiling of Anti-SARS-CoV-2 Antibodies for Detecting Early Seroconversion in COVID-19 Patients

The COVID-19 pandemic continues to infect millions of people worldwide. In order to curb its spread and reduce morbidity and mortality, it is essential to develop sensitive and quantitative methods that identify infected individuals and enable accurate population-wide screening of both past and present infection. Here we show that Single Molecule Array assays detect seroconversion in COVID-19 patients as soon as one day after symptom onset using less than a microliter of blood. This multiplexed assay format allows us to quantitate IgG, IgM and IgA immunoglobulins against four SARS-CoV-2 targets, thereby interrogating 12 antibody isotype-viral protein interactions to give a high resolution profile of the immune response. Using a cohort of samples collected prior to the outbreak as well as samples collected during the pandemic, we demonstrate a sensitivity of 86% and a specificity of 100% during the first week of infection, and 100% sensitivity and specificity thereafter. This assay should become the gold standard for COVID19 serological profiling and will be a valuable tool for answering important questions about the heterogeneity of clinical presentation seen in the ongoing pandemic.