Diagnostic Salivary Tests for SARS-CoV-2

Abstract

The diagnosis of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection relies on the detection of viral RNA by real-time reverse transcription polymerase chain reaction (rRT-PCR) performed with respiratory specimens, especially nasopharyngeal swabs. However, this procedure requires specialized medical personnel, centralized laboratory facilities, and time to provide results (from several hours up to 1 d). In addition, there is a non-negligible risk of viral transmission for the operator who performs the procedure. For these reasons, several studies have suggested the use of other body fluids, including saliva, for the detection of SARS-CoV-2. The use of saliva as a diagnostic specimen has numerous advantages: it is easily self-collected by the patient with almost no discomfort, it does not require specialized health care personnel for its management, and it reduces the risks for the operator. In the past few months, several scientific papers, media, and companies have announced the development of new salivary tests to detect SARS-CoV-2 infection. Posterior oropharyngeal saliva should be distinguished from oral saliva, since the former is a part of respiratory secretions, while the latter is produced by the salivary glands, which are outside the respiratory tract. Saliva can be analyzed through standard (rRT-PCR) or rapid molecular biology tests (direct rRT-PCR without extraction), although, in a hospital setting, these procedures may be performed only in addition to nasopharyngeal swabs to minimize the incidence of false-negative results. Conversely, the promising role of saliva in the diagnosis of SARS-CoV-2 infection is highlighted by the emergence of point-of-care technologies and, most important, point-of-need devices. Indeed, these devices can be directly used in workplaces, airports, schools, cinemas, and shopping centers. An example is the recently described Rapid Salivary Test, an antigen test based on the lateral flow assay, which detects the presence of the virus by identifying the spike protein in the saliva within a few minutes.

Keywords: COVID-19; SARS-CoV-2; Severe acute respiratory syndrome-related coronavirus; coronavirus; point-of-care testing; saliva.

Figure 1.

Different salivary samples. Posterior oropharyngeal saliva is the secretion produced when coughing or clearing one’s throat, and it belongs to the respiratory secretions, admixing secretions from both the upper (nasopharynx) and lower (bronchi, lungs) airways (number 1 in the circle). In contrast, oral saliva is produced by the salivary glands and does not belong to the group of respiratory specimens (number 2 in the circle). However, a clear distinction between these 2 kinds of samples is not feasible and does not fall within the aim of laboratory clinical diagnosis of Coronavirus Disease 2019. The saliva produced when coughing will contain oral saliva, while a small quantity of oropharyngeal secretions may be present in oral saliva.

Figure 2.

Coronavirus Disease 2019 (COVID-19) salivary diagnosis procedures. Saliva is collected with the drooling technique, avoiding coughing or expectoration. (a) Real-time reverse transcription polymerase chain reaction (rRT-PCR): this test represents the reference standard for COVID-19 diagnosis and is usually performed on respiratory specimens, but it can be used on saliva. (b) Direct rRT-PCR allows quicker diagnosis because RNA isolation is avoided. (c) Colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a point-of-care technology that allows rapid detection of viral RNA by combining LAMP technology with a colorimetric assay. (d) Antibody detection in saliva can be performed both with enzyme-linked immunosorbent assay (ELISA) or lateral flow assay. (e) Rapid salivary test is an antigen test based on the lateral flow assay, which shows great promise for mass screening.

Figure 3.

Real-time reverse transcription polymerase chain reaction (rRT-PCR) of a salivary positive sample. (a) Schematic illustration of an rRT-PCR result. The amplification curve for positive samples follows a sigmoid trend (i.e., the relative fluorescence intensity increases, with an exponential middle tract, until a plateau phase). No increase in fluorescence is observed when the sample is negative. The threshold is placed so to intersect the amplification curves at the beginning of the exponential tract. The cycle threshold (Ct) represents the cycle number at which the amplification curve intersects the threshold line and is an indicator of the quantity of the amplified target gene. The lower the Ct value, the higher the amount of the target gene and then the viral load. (b) An example of amplification curves in log scale for a salivary sample that tested positive for the presence of all 3 genes associated with SARS-CoV-2 (E, N, and RdRp). The internal control (IC), whose viral load is known, is used as comparison to quantify the viral load of the sample.

Figure 4.

Rapid salivary test (RST) based on lateral flow assay (LFA). (a) The rapid antigen test recognizes the presence of a specific viral antigen, such as the spike protein. Briefly, the salivary sample is applied to a sample pad diluted with a specific buffer, where it runs along the nitrocellulose membrane reaching the absorbent pad placed at the opposite site of the strip. (b) When both the “test-line” (T-line) and the “control line” (C-line) are visible, the test is “positive” (Severe Acute Respiratory Syndrome Coronavirus 2 is present). When only the C-line is visible, the test is “negative.” The test is “invalid” when the C-line is invisible, regardless of the presence of the T-line. This picture represents the proof of concept and the prototype of the diagnostic test published by our group (Azzi, Baj, et al. 2020).

Figure 5.

The suitable setting for each salivary diagnostic procedure. (a) In a hospital or centralized laboratory facility, real-time reverse transcription polymerase chain reaction (rRT-PCR) represents the reference standard. However, this procedure requires an adequate supply of reagents and the presence of specialized personnel. Only selected cases should undergo this procedure, avoiding thus the crowding of laboratories and other health facilities. Direct rRT-PCR without RNA extraction can be used as a preliminary analysis to screen the suspected COVID-19 patients with suggestive symptoms when entering the emergency room. (b) Point-of-care technology represents a valid and useful tool to help physicians who treat patients in a setting outside the hospital, such as a general practitioner’s surgery. (c) Point-of-need devices are suitable for mass screening programs and for the analysis of salivary samples directly on the field where the test is needed, like a school, a cinema or theater, a restaurant, or an airport.