NMDA Journal Winter 2020-21

20 New Mexico Dental Journal, Winter 2020-21  continued from page 18 3. Enough synthesized progeny population is available and researchers are able to see the virus within the sample. In rRT-PCR machines, the amplified material gives off a fluorescent form as it continues to build up after several amplification cycles (J. Hopkins University). The key to the molecular test is the amplifica- tion. Without it, the researchers would not be able to visualize the virus within the sample. In the past, researchers had to clone a DNA sequence into a vector and replicate it in a living cell, which would take days or weeks. With PCR technology, the test now can be done in a matter of hours. With current PCR COVID-19 tests, people are obtaining their test results in 2–5 days, depending on the number of tests the lab have to complete. The rRT-PCR test is highly specific and highly sensitive but requires a lab to complete the complex process. The high specificity and high sensitivity are why the molecular test is the “Gold Standard” for COVID-19 testing. Antigen Test Antigen (point-of-care) testing is used to detect surface markers, viral proteins, on the outside of the virus in a matter of 15 min- utes. Antigen testing eliminates the need for primers, reverse transcriptase processes, amplification, and specialized lab equip- ment. They can be completed conveniently in a health professional’s office. The FDA has stated that these tests can have a higher probability of false negatives, but they are highly accurate for positive results. Of course, instructing patients to obtain a PCR test would be recommended if a patient tests positive, or if the patient is symptomatic and has a negative result. There are a few antigen tests for COVID-19 that the FDA has authorized as emergency use only (EUA: Emergency Use Authorized). They all work in detecting the nucleocapsid protein (N protein/antigen) of the virus. The N protein “plays an important role in both SARS-CoV and SARS-CoV-2 infection by pack- aging viral RNA and aiding in the release of additional viral particles from infected cells.” The N protein can be detected with a few techniques: lateral flow immunoassay, chro- matogenic digital immunoassay, and lateral flow immunofluorescent sandwich assay (Prinzi 2020). Lateral flow immunoassay is used by Abbott BinaxNowTM COVID-19 Ag Card, which uses nasal swabs on a credit-card-sized card. The test functions like a pregnancy test with a control line and a test line. Droplets of a reagent are added to a well on the card. The nasal swab is inserted into a second well and rotated a few times. The card is then sealed and in 15 minutes the results are displayed on the window. The card has conjugated anti- bodies that are tagged with a substrate (label) that will attach to the N protein, if present. The nasal sample and reagent flow across the card by capillary action so the tagged antibodies will adhere to the test line antibodies, thus showing a positive. In addition to the rapid results, Abbott also has designed a phone app called NAVICATM that can be downloaded at no charge. This app links the test card to the patient, who can keep an electronic record of their tests. Abbott sees this as possibly being used as a “digital pass” each time a patient gets a neg- ative result. Currently, it is reported that this test has a sensitivity of 97.1 percent and speci- ficity of 98.5 percent. Becton Dickinson VeritorTM Plus System uses the chromatogenic digital immunoassay technique, which is direct and qualitative in the detection of N antigens. Again, this is a lateral flow test strip that is covered with tagged antibodies that adhere to the N pro- tein. The antibodies are also conjugated with detector particles that allow the binding to be visualized using a BD instrument. Becton Dickinson also has antigen tests for strep and flu, and they used that technology to develop the SARS-CoV-2 antigen test. To complete a test, a nasal swab sample is taken and transferred to a processing tube containing a liquid reagent. This sample/ reagent is then placed on a test device that looks like a USB device. After 15 minutes, the test device is inserted into the BD Analyzer that seems to be no bigger than a credit card machine. The results read-out on whether it is positive or negative for SARS- CoV-2. This test has 98–100 percent specific- ity and sensitivity of 84 percent. Lastly, there is the Quidel Sofia 2 SARS Anti- gen System and Sofia 2 Flu + SARS Antigen FIA that uses a lateral flow immunofluores- cent sandwich assay (FIA-Fluorescent Immu- noassays). A fluorescent compound is used as the detector reagent that absorbs light and energy (excitation energy) at a specific wavelength. The fluorescent compound then emits the light at a different wavelength. The difference in light absorption wavelength and light emission wavelength is called the Stokes shift. A wide Stokes Shift is desirable; to there will be no risks of misinterpretation of the assay data. Both Quidel tests use an analyzer and the technique is similar to the Becton Dickinson System. The sample/reagent is placed on a testing device and after 15 minutes it is inserted into the Sofia Analyzer for the results. The “Sofia 2 Flu + SARS Antigen FIA” detects the N protein from influenza A, influenza B, and SARS-CoV-2. “Sofia 2 SARS” test has a sensitivity of 96.7 percent and a specificity of 100 percent. Sensitivity for “Sofia 2 Flu + SARS” test is 95.2 percent and specificity is 100 per- cent per their product specifications. It is advisable to read the specifications from any company that develops an antigen test for detecting SARS-CoV-2. Also, be sure that the SARS-CoV-2 virus in asymptomatic patients can be detected in the antigen COVID-19 test your office chooses to use for rapid testing. Saliva Sample A rapid COVID-19 antigen test that uses saliva samples, rather than a nasal swab, is still to be developed. Arizona State University has two researchers with the School of Molecular Sciences, Dr. Mark Hayes and Dr. Alexander Green, and one researcher from the School of Electrical, Computer and Energy Engineering, Dr. Jennifer Blain Chris- ten, who are tasked with developing a saliva sample test. ASU received a $6 million grant to develop this test. Dr. Hayes stated, “A key aspect of detecting COVID-19 is to be able to isolate and identify coronavirus away from all the other bioparticles in saliva, which we have the ability to do.” Once the detection technology is developed, the plan is to use it in a disposable microfluidic chip (Klemasze- wski 2020). It is hopeful that this will come to fruition, as most of us do not like obtaining the uncomfortable nasal swab. Furthermore, swabs, PPE, and the chemicals used in labs can be on short supply, thus making the cur- rent test processes expensive. There is a FDA EUA molecular (PCR) test, Sal- ivaDirectTM, which uses saliva as a sample. It was developed and validated by research- ers with the Yale School of Public Health. This test is unique in that it does not require spe- cialized reagents that a lab would normally need to process a PCR test, it skips the step of RNA extraction, and they are working to ensure that all labs can process the test with- out buying new equipment. Yale researchers note that SalivaDirectTM is not a kit, but a protocol on how to process saliva-based RT- qPCR testing inexpensively. Currently, there are 12 designated labs, the closest to New Mexico being Paratus Diagnostics near San Antonio, Texas.