Sensitive Serology Measurements in the Saliva of Individuals with COVID-19 Symptoms Using a Multiplexed Immunoassay.

BACKGROUND: There are numerous benefits to performing salivary serology measurements for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative pathogen for coronavirus disease 2019 (COVID-19). Here, we used a sensitive multiplex serology assay to quantitate salivary IgG against 4 SARS-CoV-2 antigens: nucleocapsid, receptor-binding domain, spike, and N-terminal domain. METHODS: We used single samples from 90 individuals with COVID-19 diagnosis collected at 0 to 42 days postsymptom onset (PSO) and from 15 uninfected control subjects. The infected individuals were segmented in 4 groups (0-7 days, 8-14 days, 15-21 days, and >21 days) based on days PSO, and values were compared to controls. RESULTS: Compared to controls, infected individuals showed higher levels of antibodies against all antigens starting from 8 days PSO. When applying cut-offs with at least 93.3% specificity at every time interval segment, nucleocapsid protein serology had the best sensitivity at 0 to 7 days PSO (60% sensitivity [35.75% to 80.18%], ROC area under the curve [AUC] = 0.73, P = 0.034). Receptor-binding domain serology had the best sensitivity at 8 to 14 days PSO (83.33% sensitivity [66.44%-92.66%], ROC AUC = 0.90, P < 0.0001), and all assays except for N-terminal domain had 92% sensitivity (75.03%-98.58%) at >14 days PSO. CONCLUSIONS: This study shows that our multiplexed immunoassay can distinguish infected from uninfected individuals and reliably (93.3% specificity) detect seroconversion (in 60% of infected individuals) as early as the first week PSO, using easy-to-collect saliva samples.

Patients with severe COVID-19 do not have elevated autoantibodies against common diagnostic autoantigens.

OBJECTIVES: Infection by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative pathogen of coronavirus disease 2019 (COVID-19) presents occasionally with an aberrant autoinflammatory response, including the presence of elevated circulating autoantibodies in some individuals. Whether the development of autoantibodies against self-antigens affects COVID-19 outcomes remains unclear. To better understand the prognostic role of autoantibodies in COVID-19, we quantified autoantibodies against 23 markers that are used for diagnosis of autoimmune disease. To this end, we used serum samples from patients with severe [intensive care unit (ICU)] and moderate (ward) COVID-19, across two to six consecutive time points, and compared autoantibody levels to uninfected healthy and ICU controls. METHODS: Acute and post-acute serum (from 1 to 26 ICU days) was collected from 18 ICU COVID-19-positive patients at three to six time points; 18 ICU COVID-19-negative patients (sampled on ICU day 1 and 3); 21 ward COVID-19-positive patients (sampled on hospital day 1 and 3); and from 59 healthy uninfected controls deriving from two cohorts. Levels of IgG autoantibodies against 23 autoantigens, commonly used for autoimmune disease diagnosis, were measured in serum samples using MSD® U-PLEX electrochemiluminescence technology (MSD division Meso Scale Discovery®), and results were compared between groups. RESULTS: There were no significant elevations of autoantibodies for any of the markers tested in patients with severe COVID-19. CONCLUSIONS: Sample collections at longer time points should be considered in future studies, for assessing the possible development of autoantibody responses following infection with SARS-CoV-2.

Ultrasensitive assay for saliva-based SARS-CoV-2 antigen detection.

OBJECTIVES: Widespread SARS-CoV-2 testing is invaluable for identifying asymptomatic/pre-symptomatic individuals. There remains a technological gap for highly reliable, easy, and quick SARS-CoV-2 diagnostic tests suitable for frequent mass testing. Compared to nasopharyngeal (NP) swab-based tests, saliva-based methods are attractive due to easier and safer sampling. Current saliva-based SARS-CoV-2 rapid antigen tests (RATs) are hindered by limited analytical sensitivity. Here, we report one of the first ultrasensitive, saliva-based SARS-CoV-2 antigen assays with an analytical sensitivity of <0.32 pg/mL, corresponding to four viral RNA copies/µL, which is comparable to that of PCR-based tests. METHODS: Using the novel electrochemiluminescence (ECL)-based immunoassay, we measured the SARS-CoV-2 nucleocapsid (N) antigen concentration in 105 salivas, obtained from non-COVID-19 and COVID-19 patients. We then verified the results with a second, independent cohort of 689 patients (3.8% SARS-CoV-2 positivity rate). We also compared our method with a widely used point-of-care rapid test. RESULTS: In the first cohort, at 100% specificity, the sensitivity was 92%. Our assay correctly identified samples with viral loads up to 35 CT cycles by saliva-based PCR. Paired NP swab-based PCR results were obtained for 86 cases. Our assay showed high concordance with saliva-based and NP swab-based PCR in samples with negative (<0.32 pg/mL) and strongly positive (>2 pg/mL) N antigen concentrations. In the second cohort, at 100% specificity, sensitivity was also 92%. Our assay is about 700-fold more sensitive than the Abbott Panbio Rapid Test. CONCLUSIONS: We demonstrated the ultrasensitivity and specificity assay and its concordance with PCR. This novel assay is especially valuable when compliance to frequent swabbing may be problematic.