Measurement of anti SARS-CoV-2 RBD IgG in saliva: validation of a highly sensitive assay and effects of the sampling collection method and correction by protein.

OBJECTIVES: To develop and evaluate a new highly sensitive assay to detect IgG anti-SARS-CoV-2 RBD in saliva samples. METHODS: A two-step sandwich type immunoassay based on the amplified luminescent proximity homogeneous technology was developed and an analytical validation was performed. As a part of this validation, the influence of factors, such as different sampling conditions (stimulated saliva and passive drool) and the correction of values by total protein content, in the ability of saliva to detect increases in antibodies after an immune stimulus and be an alternative to serum, was evaluated. For this purpose, paired samples of saliva and serum at different times after vaccination were used. RESULTS: Saliva concentrations were lower than serum, but both fluids showed similar kinetics, with higher correlations when saliva was obtained by passive flow and the results were not corrected by protein. CONCLUSIONS: The developed method showed a good analytical performance and can properly measure antibody concentrations in saliva of vaccinated individuals. However, saliva could have a lower sensitivity compared to serum at initial stages of the immune response and also when the antibody response decreased after a stimulus.

Salivary Ferritin Changes in Patients with COVID-19.

High ferritin serum levels can be found in patients with macrophage activation syndrome, and increased serum ferritin due to cytokine storm have been reported in severe COVID-19 patients. Saliva is being increasingly used in COVID-19 tests as a diagnostic sample for virus detection and quantification. This study aimed to evaluate the possible changes in ferritin in saliva in COVID-19 patients. In addition, the effects of different inactivation SARS-CoV-2 treatments in ferritin measurements in saliva, the correlation between ferritin in saliva and serum, and the possible effects of correction of ferritin values by total protein were assessed. Ferritin was measured in saliva from healthy (n = 30) and COVID-19 (n = 65) patients with severe, (n = 18) or mild (n = 47) disease, depending on the need for nasal flow oxygen or assisted respiration. Ferritin was also measured in paired serum and saliva samples (n = 32) from healthy and COVID-19 patients. The evaluated inactivation protocols did not affect the assay's results except the addition of 0.5% SDS. Significantly higher ferritin was found in the saliva of COVID-19 patients (median; 25-75th percentile) (27.75; 9.77-52.2 µg/L), compared with healthy controls (4.21; 2.6-8.08 µg/L). Individuals with severe COVID-19 showed higher ferritin values in saliva (48.7; 18.7-53.9) than mild ones (15.5; 5.28-41.3 µg/L). Significant correlation (r = 0.425; p < 0.001) was found between serum and saliva in ferritin. Ferritin levels were higher in COVID-19 patients in serum and saliva, and the highest values were found in those patients presenting severe symptomatology. In conclusion, ferritin in saliva has the potential to be a biomarker to evaluate severity in patients with COVID-19.

Analytical validation of an automated assay for the measurement of adenosine deaminase (ADA) and its isoenzymes in saliva and a pilot evaluation of their changes in patients with SARS-CoV-2 infection.

OBJECTIVES: The aim of the present study was to validate a commercially available automated assay for the measurement of total adenosine deaminase (tADA) and its isoenzymes (ADA1 and ADA2) in saliva in a fast and accurate way, and evaluate the possible changes of these analytes in individuals with SARS-CoV-2 infection. METHODS: The validation, in addition to the evaluation of precision and accuracy, included the analysis of the effects of the main procedures that are currently being used for SARS-CoV-2 inactivation in saliva and a pilot study to evaluate the possible changes in salivary tADA and isoenzymes in individuals infected with SARS-CoV-2. RESULTS: The automated assay proved to be accurate and precise, with intra- and inter-assay coefficients of variation below 8.2%, linearity under dilution linear regression with R2 close to 1, and recovery percentage between 80 and 120% in all cases. This assay was affected when the sample is treated with heat or SDS for virus inactivation but tolerated Triton X-100 and NP-40. Individuals with SARS-CoV-2 infection (n=71) and who recovered from infection (n=11) had higher mean values of activity of tADA and its isoenzymes than healthy individuals (n=35). CONCLUSIONS: tADA and its isoenzymes ADA1 and ADA2 can be measured accurately and precisely in saliva samples in a rapid, economical, and reproducible way and can be analyzed after chemical inactivation with Triton X-100 and NP-40. Besides, the changes observed in tADA and isoenzymes in individuals with COVID-19 open the possibility of their potential use as non-invasive biomarkers in this disease.