Methodological approaches to optimize multiplex oral fluid SARS-CoV-2 IgG assay performance and correlation with serologic and neutralizing antibody responses.

BACKGROUND: Oral fluid (hereafter, saliva) is a non-invasive and attractive alternative to blood for SARS-CoV-2 IgG testing; however, the heterogeneity of saliva as a matrix poses challenges for immunoassay performance. OBJECTIVES: To optimize performance of a magnetic microparticle-based multiplex immunoassay (MIA) for SARS-CoV-2 IgG measurement in saliva, with consideration of: i) threshold setting and validation across different MIA bead batches; ii) sample qualification based on salivary total IgG concentration; iii) calibration to U.S. SARS-CoV-2 serological standard binding antibody units (BAU); and iv) correlations with blood-based SARS-CoV-2 serological and neutralizing antibody (nAb) assays. METHODS: The salivary SARS-CoV-2 IgG MIA included 2 nucleocapsid (N), 3 receptor-binding domain (RBD), and 2 spike protein (S) antigens. Gingival crevicular fluid (GCF) swab saliva samples were collected before December 2019 (n = 555) and after molecular test-confirmed SARS-CoV-2 infection from 113 individuals (providing up to 5 repeated-measures; n = 398) and used to optimize and validate MIA performance (total n = 953). Combinations of IgG responses to N, RBD and S and total salivary IgG concentration (µg/mL) as a qualifier of nonreactive samples were optimized and validated, calibrated to the U.S. SARS-CoV-2 serological standard, and correlated with blood-based SARS-CoV-2 IgG ELISA and nAb assays. RESULTS: The sum of signal to cutoff (S/Co) to all seven MIA SARS-CoV-2 antigens and disqualification of nonreactive saliva samples with ≤15 µg/mL total IgG led to correct classification of 62/62 positives (sensitivity [Se] = 100.0%; 95% confidence interval [CI] = 94.8%, 100.0%) and 108/109 negatives (specificity [Sp] = 99.1%; 95% CI = 97.3%, 100.0%) at 8-million beads coupling scale and 80/81 positives (Se = 98.8%; 95% CI = 93.3%, 100.0%] and 127/127 negatives (Sp = 100%; 95% CI = 97.1%, 100.0%) at 20-million beads coupling scale. Salivary SARS-CoV-2 IgG crossed the MIA cutoff of 0.1 BAU/mL on average 9 days post-COVID-19 symptom onset and peaked around day 30. Among n = 30 matched saliva and plasma samples, salivary SARS-CoV-2 MIA IgG levels correlated with corresponding-antigen plasma ELISA IgG (N: ρ = 0.76, RBD: ρ = 0.83, S: ρ = 0.82; all p < 0.001). Correlations of plasma SARS-CoV-2 nAb assay area under the curve (AUC) with salivary MIA IgG (N: ρ = 0.68, RBD: ρ = 0.78, S: ρ = 0.79; all p < 0.001) and with plasma ELISA IgG (N: ρ = 0.76, RBD: ρ = 0.79, S: ρ = 0.76; p < 0.001) were similar. CONCLUSIONS: A salivary SARS-CoV-2 IgG MIA produced consistently high Se (> 98.8%) and Sp (> 99.1%) across two bead coupling scales and correlations with nAb responses that were similar to blood-based SARS-CoV-2 IgG ELISA data. This non-invasive salivary SARS-CoV-2 IgG MIA could increase engagement of vulnerable populations and improve broad understanding of humoral immunity (kinetics and gaps) within the evolving context of booster vaccination, viral variants and waning immunity.

Self-Collected Oral Fluid Saliva Is Insensitive Compared With Nasal-Oropharyngeal Swabs in the Detection of Severe Acute Respiratory Syndrome Coronavirus 2 in Outpatients.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic control will require widespread access to accurate diagnostics. Salivary sampling circumvents swab supply chain bottlenecks, is amenable to self-collection, and is less likely to create an aerosol during collection compared with the nasopharyngeal swab. METHODS: We compared real-time reverse-transcription polymerase chain reaction Abbott m2000 results from matched salivary oral fluid (gingival crevicular fluid collected in an Oracol device) and nasal-oropharyngeal (OP) self-collected specimens in viral transport media from a nonhospitalized, ambulatory cohort of coronavirus disease 2019 (COVID-19) patients at multiple time points. These 2 sentences should be at the beginning of the results. RESULTS: There were 171 matched specimen pairs. Compared with nasal-OP swabs, 41.6% of the oral fluid samples were positive. Adding spit to the oral fluid percent collection device increased the percent positive agreement from 37.2% (16 of 43) to 44.6% (29 of 65). The positive percent agreement was highest in the first 5 days after symptoms and decreased thereafter. All of the infectious nasal-OP samples (culture positive on VeroE6 TMPRSS2 cells) had a matched SARS-CoV-2 positive oral fluid sample. CONCLUSIONS: In this study of nonhospitalized SARS-CoV-2-infected persons, we demonstrate lower diagnostic sensitivity of self-collected oral fluid compared with nasal-OP specimens, a difference that was especially prominent more than 5 days from symptom onset. These data do not justify the routine use of oral fluid collection for diagnosis of SARS-CoV-2 despite the greater ease of collection. It also underscores the importance of considering the method of saliva specimen collection and the time from symptom onset especially in outpatient populations.

COVID-19 Serology at Population Scale: SARS-CoV-2-Specific Antibody Responses in Saliva.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of an ongoing pandemic that has infected over 36 million and killed over 1 million people. Informed implementation of government public health policies depends on accurate data on SARS-CoV-2 immunity at a population scale. We hypothesized that detection of SARS-CoV-2 salivary antibodies could serve as a noninvasive alternative to serological testing for monitoring of SARS-CoV-2 infection and seropositivity at a population scale. We developed a multiplex SARS-CoV-2 antibody immunoassay based on Luminex technology that comprised 12 CoV antigens, mostly derived from SARS-CoV-2 nucleocapsid (N) and spike (S). Saliva and sera collected from confirmed coronavirus disease 2019 (COVID-19) cases and from the pre-COVID-19 era were tested for IgG, IgA, and IgM to the antigen panel. Matched saliva and serum IgG responses (n = 28) were significantly correlated. The salivary anti-N IgG response resulted in the highest sensitivity (100%), exhibiting a positive response in 24/24 reverse transcription-PCR (RT-PCR)-confirmed COVID-19 cases sampled at >14 days post-symptom onset (DPSO), whereas the salivary anti-receptor binding domain (RBD) IgG response yielded 100% specificity. Temporal kinetics of IgG in saliva were consistent with those observed in blood and indicated that most individuals seroconvert at around 10 DPSO. Algorithms employing a combination of the IgG responses to N and S antigens result in high diagnostic accuracy (100%) by as early as 10 DPSO. These results support the use of saliva-based antibody testing as a noninvasive and scalable alternative to blood-based antibody testing.