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.

Effect of thermal and chemical treatments used for SARS-COV-2 inactivation in the measurement of saliva analytes.

The present study aims to assess the effects of thermal and chemical inactivating procedures, that can be used for SARS-CoV-2 inactivation, on different salivary analytes. SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) protein profile and a panel of 25 specific biomarkers of oxidative status, stress, metabolism and tissue damage were evaluated in samples subjected to different treatments: thermal (65 °C or 92 °C) and chemical with detergents [sodium dodecyl sulphate (SDS), Triton X-100 or NP-40]. Salivary SDS-PAGE profile was most affected by heating at 92 °C, with three and two protein bands decreasing and increasing their expression levels, respectively. This treatment also affected the results of several enzymes, with some of them being also affected by heating at 65 °C and incubation with SDS. The use of Triton X-100 or NP-40 resulted in increased values of cortisol, triglycerides and glucose, not affecting the other tested biomarkers. The present results will help researchers and clinicians to select the best protocols to work in safe conditions with saliva, taking into account the target analyte planned to be measured.

Evaluation of sample treatments in a safe and straightforward procedure for the detection of SARS-CoV-2 in saliva.

OBJECTIVES: To evaluate four sample treatments in a safe and straightforward procedure to detect SARS-CoV-2 in saliva. METHODS: Four sample treatments were evaluated in a 3-step procedure to detect SARS-CoV-2 in saliva: 1) heating at 95 °C for 5 min for sample inactivation; 2) sample treatment; 3) analysis by reverse-transcription loop-mediated isothermal amplification (LAMP). Saliva samples used were from infected individuals or were spiked with known quantities of viral particles. RESULTS: Three treatments had a limit of detection (LOD) of 500.000 viral particles per ml of saliva and could be used to detect individuals with potential to transmit the disease. The treatment of phosphate buffer, dithiothreitol, ethylenediaminetetraacetic acid and proteinase K, with an additional 95 °C heating step, yielded a lower LOD of 95; its sensitivity ranged from 100% in patients with nasopharyngeal swab reverse-transcriptase polymerase chain reaction cycle threshold values <20 to 47.8% for values >30. CONCLUSIONS: This report highlights the importance of an adequate sample treatment for saliva to detect SARS-CoV-2 and describes a flexible procedure that can be adapted to point-of-care. Although its sensitivity when LAMP is used is lower than reverse-transcriptase polymerase chain reaction, this procedure can contribute to COVID-19 control by detecting individuals able to transmit the disease.

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.