COVID-19 Rapid Test

SARS-CoV-2 is the causative viral agent of the disease COVID-19. It is primarily transmitted through respiratory droplets, and enters into cells via the ACE2 receptor found on alveolar epithelial cells (among other cell types). Symptoms of COVID-19 may appear as early as 2 days after initial infection, or as late as 14 days. There is variation in the severity of symptoms and in their duration and time of onset.

There have been a number of different COVID-19 tests developed to aid in curbing the spread of disease. These either target antigens associated with the SARS-CoV-2 viral molecule, or antibodies produced by the body in response to infection with SARS-CoV-2. These tests include:

• Rapid antigen tests - Point-of-care tests that have the ability to detect antigens found on the surface of the SARS-CoV-2 viral molecule. Must be carried out by trained personnel. Efforts are currently being made to adapt the tests so that they can be used at home.

• PCR-based tests - Currently the most widely used test for diagnosis of COVID-19. PCR-based tests are highly sensitive and can detect extremely low levels of viral particles. However, they require trained health professionals and expensive equipment to carry out the testing.
• Antibody tests - Rather than testing for the presence of SARS-CoV-2 viral molecules, COVID-19 antibody tests establish if a person is/has been effected with the virus by assessing immune response (production of antibodies against SARS-CoV-2 viral molecules). Although strongly suggestive of current or previous COVID-19 infection, this form of test is not suitable for early diagnosis as antibodies cannot be detected for a number of days following initial infection.

COVID-19 rapid tests detect specific proteins, known as antigens, that are expressed on the surface of the SARS-CoV-2 molecule. Typically, the testing procedure begins with a trained healthcare professional taking a nasopharyngeal swab of the suspected infected individual.

Figure 1. Schematic representation of specimen collection

The sample is then placed into a tube containing a liquid (extraction buffer) which lyses (breaks) the virus open, releasing the viral antigens of interest. This mixture is then added to the test cassette. If the tested individual has COVID-19, the viral proteins present in the sample will bind to the antibodies within the strip that are specific to the viral protein of interest. A positive result will be seen as a dark band in the 'test line (T)' zone of the strip.

Figure 2: Schematic representation of how the results of COVID-19 rapid tests are interpreted

Assay Genie Rapid Antigen Test kits have been compared with a leading commercial RT-PCR test. The correlation between these two systems is no less than 97%.

Assay Genie Rapid Antigen Tests have been evaluated with specimens obtained from the patients. RT-PCR is used as the reference method. Specimens were considered positive if RT-PCR indicated a positive result. Specimens were considered negative if RT-PCR indicated a negative result.

*Confidence Intervals

• Results can be retrieved within 15 minutes. There is no need for complex lab procedures to be carried out, such as PCR, that take hours to perform. This is not feasible when high throughput testing is needed to prevent the spread of disease.
• Cheaper than gold-standard testing methods e.g. polymerase chain reaction (PCR) as there is no need for use of expensive lab equipment and reagents.
• Rapid antigen tests allows testing to be carried out in settings where people must be tested quickly e.g. airports.
• Rapid antigen tests allows testing to be carried out in settings that have minimal access to laboratory equipment e.g. schools.
• For individuals that receive a negative result at first, rapid antigen tests are a quick and easy way to re-test people (that have experienced symptoms or have been in close contact with a confirmed case) within a few days of the first test. This is not as easily done with the complex PCR-based testing.

• Rapid antigen tests are less sensitive than the conventional PCR testing currently being used - PCR testing can detect a single SARS-CoV-2 RNA molecule in a microlitre of solution. Rapid antigen tests on the other hand can only detect SARS-CoV-2 viral molecules if there are thousands or even tens of thousands of viral molecules present in the sample, any less and it is likely than infection will go undetected.
• Rapid antigen tests are only effective if the sample is taken from the individual within one week of symptom onset. Sensitivity of test drops to ~75% if sample is taken more than a week following onset of symptoms.
• Rapid antigen tests must be used in conjunction with other forms of testing (clinical findings and other laboratory testing) to confirm positive and negative results.

Positive results - Positive results indicate the presence of viral antigens, but clinical correlation with patient history and other diagnostic information is necessary to determine infection status. Positive results do not rule out bacterial infection or co-infection with other viruses. The agent detected may not be the definite cause of disease. For this reason, rapid antigen tests cannot confirm diagnosis of COVID-19, but can aid with diagnosis of COVID-19.

Negative results - Negative results do not preclude COVID-19 infection and should not be used as the sole basis for treatment or patient management decisions. Negative results should be treated as presumptive and confirmed with a molecular assay, if necessary for patient management. Negative results should be considered in the context of a patient’s recent exposures, history and the presence of clinical signs and symptoms consistent with COVID-19.

COVID-19 point-of-care antibody tests act by detecting antibodies made by the body against the SARS-CoV-2 virus. Firstly, a sample of serum, plasma, fingertip blood or whole blood is taken from a suspected case. If the sample is plasma, fingertip blood, or a whole blood sample, chemicals (Heparin, sodium citrate and EDTA) may be added to prevent coagulation of the sample.

Figure 3: Schematic representation of the COVID-19 antibody test procedure.

Rapid antibody tests come in the form of a small test cassette, similar to the antigen test cassettes. The test cassette is coated with mixed SARS-CoV-2 recombinant antigen proteins (nucleocapsid protein (N protein) and spike protein (S protein)) conjugated with colloidal gold and anti-human IgM and IgG antibody coated on different test lines, respectively.

After the samples has been applied to the test strip, the gold-labelled recombinant antigens bind with anti-SARS-CoV-2 IgM or IgG antibodies in the sample and form marked antigen-antibody complexes. These complexes move to the test card detection zone by capillary action. Marked antigen-antibody complexes will then be captured on different test lines by anti-human IgM and IgG antibody resulting in purplish red streaks on the test lines. The color intensity of each test line increases in proportion to the amount of anti-SARS-CoV-2 IgM and IgG antibodies in the sample.

Figure 4: Schematic representation of how the results of a COVID-19 antibody test are interpreted

Assay Genie antibody test kits have been compared with a leading commercial RT-PCR test. The correlation between these two systems is no less than 93.8%.

Assay Genie antibody tests have been evaluated with specimens obtained from COVID-19 patients. RT-PCR is used as the reference method. Specimens were considered positive if RT-PCR indicated a positive result. Specimens were considered negative if RT-PCR indicated a negative result.

Positive Percent Agreement (PPA) = 120/128 (93.8%), 95% CI: 88.2% to 96.8%

Negative Percent Agreement (NPA) = 240/250 (96%), 95% CI: 92.8% to 97.8%

• Quicker and easier method of COVID-19 detection than conventional PCR testing.
• Cheaper than gold-standard testing methods e.g. PCR as there is no need for use of expensive lab equipment and reagents.
• Can be used to confirm late primary or early secondary SARS-CoV-2 infection.
• Can suggest infection COVID-19 in late stage of infection better than antigen tests (antigen tests are often only reliable within 7 days of onset of symptoms).

• Not suitable for early diagnosis of COVID-19 infection as it takes days for the body to produce antibodies in response to the virus.
• Not as reliable (less sensitive) as the widely used PCR method of testing.
• Rapid antigen tests must be used in conjunction with other forms of testing (clinical findings and other laboratory testing) to confirm positive and negative results. The clinical diagnosis and treatment of patients should be considered in combination with their symptoms/signs, medical history, other laboratory tests, and treatment response.