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Early detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and diagnosis of coronavirus disease 2019 (COVID-19) are priorities during the pandemic. Symptomatic and suspected asymptomatic individuals should be tested for COVID-19 to confirm infection and to be excluded from social interactions. As molecular testing capacity is overloaded during the pandemic, rapid antigen tests, such as lateral flow immunoassays (LFIAs), can be a useful tool as they allow greater test availability and obtain results in a very short time. This short review aims to present the analytical properties of LFIAs in the detection of SARS-CoV-2 in nasopharyngeal swabs. Lateral flow immunoassay is a method that combines thin-layer chromatography and indirect immunochemical sandwich method and allows the detection of a specific SARS-CoV-2 antigen in nasopharyngeal swabs. Swab specimens should be adequately collected and tested as soon as possible. Users should pay attention to quality control and possible interferences. Antigen tests for SARS-CoV-2 show high sensitivity and specificity in cases with high viral loads, and should be used up to five days after the onset of the first symptoms of COVID-19. False positive results may be obtained when screening large populations with a low prevalence of COVID-19 infection, while false negative results may happen due to improper specimen collection or insufficient amount of antigen in the specimen. So as to achieve reliable results, a diagnostic accuracy study of a specific rapid antigen test should be performed.
Keywords: COVID-19, immunoassay, nasopharynx, SARS-CoV-2 antigen testingSince the outbreak of the coronavirus disease 2019 (COVID-19) pandemic, clinicians and laboratory scientists have been struggling to reveal the most appropriate methods for early diagnosis of the disease. COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a ribonucleic acid (RNA) virus that first appeared in Wuhan, China, in late 2019 (1, 2). SARS-CoV-2 mainly affects the respiratory tract, which can present as mild symptoms or progress into severe complications that can be fatal. For example, it may manifest as acute pulmonary infection, coagulation disorders or gastrointestinal symptoms (most commonly diarrhea and nausea) (3-5). SARS-CoV-2 spreads easily among people who are in close contact. Close contact is defined as being within 1.83 m of an infected person for a cumulative total of 15 minutes or more over a 24-hour period (6, 7). Symptoms (fever, headache, cough, indigestion) may appear as early as 5-6 days and as long as 14 days after possible exposure to the virus (8). Symptomatic and asymptomatic persons who have been in close contact with COVID-19 positive or suspected persons should be tested for COVID-19 to confirm transmission and existing infection (9). Asymptomatic individuals are considered silent transmitters, and those who have had no contact or are unaware that they have been in contact with a COVID-19 positive person are particularly important (10). Given the problem of silent transmission and the lack of conscious contacts, there is a need for screening tests that could indicate positive asymptomatic individuals who should be excluded from social interactions.
Currently, there are two types of diagnostic tests for detection of SARS-CoV-2 in the nasopharynx of infected individuals that indicate COVID-19. First and foremost are molecular tests that detect viral RNA sequences by nucleic acid amplification tests (NAAT), such as real-time reverse transcriptase-polymerase chain reaction (RT-PCR). This method is the gold standard for the detection of SARS-CoV-2, and results can later be confirmed by another NAAT assay or viral sequencing (11, 12). Given the increasing number of COVID-19 cases and the urgent need to expand the capacity for COVID-19 testing during public health emergencies, there is a strong urge for cheaper, faster, and easier-to-use tests that could be used as screening or diagnostic tests for COVID-19 in broader populations. Therefore, another type of diagnostic test has recently been introduced, namely the lateral flow immunoassay (LFIA), which can detect the presence of specific viral antigens, i.e., the spike (S) protein or the nucleocapsid (N) protein (13). These antigen tests aim to rapidly confirm the presence of SARS-CoV-2 in the nasopharyngeal swab of individuals with typical symptoms, as well as to carry out screening in a given population for epidemiological purposes, i.e., to determine how much the virus has spread in a community. In general, LFIAs provide the best information when a person is tested at the time of peak viral load and when exposure to a person with COVID-19 is known (9).
The first antigen test to receive Emergency Use Authorization (EUA) from the US Food and Drug Administration (FDA) on March 21, 2020 was the Cepheid Xpert Xpress SARS-CoV-2 test (14). Currently, more than 100 antigen tests are available for the detection of SARS-CoV-2 (15).
Lateral flow immunoassays have many advantages, such as relative ease of manufacture, ease of use, stability, and use of small sample volumes. One of the most appreciated advantages is that they are relatively inexpensive, making them available in many laboratories (16). On the other hand, there are also limitations in the performance of commonly used LFIAs, especially in terms of analytical sensitivity and test-to-test reproducibility. Test sensitivity may differ in terms of various antigen testing platforms. Laboratories should be aware of which platform is being used and the sensitivity of the test for the population being tested (17). For the interpretation of diagnostic tests, it is mandatory to have data on both their analytical and clinical sensitivity and specificity. Certainly, a prerequisite for diagnostic accuracy and reliability of diagnostic tests is their analytical accuracy and precision. The final clinical interpretation of antigen testing should include information on the presence of symptoms typical of COVID-19 and whether the patient has been in contact with a COVID-19 positive person.
In addition, COVID-19 patients could also be tested serologically for the detection of antibodies to SARS-CoV-2 (11, 18). However, the clinical indications for serological testing and its analytical and clinical performance remain limited (18). Because seroconversion usually occurs two weeks after the onset of symptoms, serological testing should not be used as a diagnostic tool for acute COVID-19. Furthermore, the presence of SARS-CoV-2 antibodies does not indicate immunity to reinfection, but neutralization assays are needed (18).
This review aims to present the basic characteristics of LFIAs in the detection of SARS-CoV-2 in nasopharyngeal swabs during the COVID-19 pandemic from an analytical point of view, including the characteristics and requirements of the preanalytical, analytical, and post-analytical phases of the assay.
The indication for LFIA is made depending on whether the patient tested is a person with severe symptoms of disease, an asymptomatic person who has been in close contact with the patient, or an asymptomatic person who is not aware that he or she has been in contact with an infected person ( Figure 1 ) (9).