Antigen, Antibody and PCR Tests in COVID-19: Introduction, Importance, Applications and Demerits

Introduction of Antigen, Antibody and PCR Tests in COVID-19

Antigen, antibody, and PCR tests in COVID-19 patients are the most common assays applicable for laboratory diagnosis of COVID-19. Anigen is a foreign substance that induces an immune response. The nucleocapsid (N) is the most common antigen for the SRAS-CoV-2 antigen test and is also called antigen-detecting rapid diagnostic tests (Ag-RDTs). It works in the principle of immunochromatographic (lateral flow) assay. Ag-RDTs for COVID-19 will most often be positive when viral loads are highest and patients are most infectious – typically 1−3 days prior to the onset of symptoms and during the first 5−7 days after the onset of symptoms – and will become negative as the patient clears the infection and recovers. The nasopharyngeal swab is used as a specimen.

The antibody is the immunoglobulin and detectable antibodies in antibody-detecting rapid diagnostic tests (Ab-RDTs) are IgM and IgG but rarely IgA too. SARS-CoV-2 structural components like nucleocapsid (N), spike (S) protein and receptor-binding domain (RBD) has been used as antigens for antibody assay. Among them, nucleocapsid (N) is the most common antigen for the SRAS-CoV-2 antibody test. IgM could be detected as early as 1-day post- symptom onset (PSO) and was detectable in 85% of COVID-19 confirmed patients 7 days PSO.  As for IgG, over 90% of COVID-19 confirmed patients produced this type of antibody 14 days after illness but in rare cases yet fails to produce IgM/ IgG 40 days after symptom onset. Blood is taken as a specimen.

PCR stands for a polymerase chain reaction and PCR test applies to detect genetic material from a specific organism but here for a virus, SARS-Cov-2. The test detects the presence of genes of a virus if you have been infected at the time of the test. The assay is also applicable to detect fragments of the virus even after you have no longer been infected. Test specimens are nasopharyngeal swab,  oropharyngeal swab, bronchoalveolar lavage, sputum, etc.

Importance of Antigen, Antibody, and PCR Tests in COVID-19

Antigen Test

• Antigen test becomes positive typically 1−3 days prior to the onset of symptoms and during the first 5−7 days after the onset of symptoms and therefore known as an early marker of lab diagnosis.
• It is an indicator of active infection.
• It is applicable at the point of care i.e. outside laboratories.
• Simple to perform
• Quick results (within 30 minutes) enabling rapid implementation of infection control measures, including contact tracing

• Less expensive than nucleic acid amplification testing (NAAT), e.g., RT-PCR tests

Antibody Test

• The antibody test is usually an indicator of passive infection since IgM could be detected 7 days post- symptom onset whereas for  14 days after illness.
• It tells your previous infection with SARS-CoV-2.
• It is applicable at the point of care (outside laboratories) or in higher throughput formats in laboratories

• Easy to perform
• Quick results (within 30 minutes)
• Less expensive than nucleic acid amplification testing (NAAT), e.g., RT-PCR tests

PCR test

1. PCR test detects active as well as inactive SARS-CoV-2 infection and thus there is the significance of cycle threshold (Ct) values.
2. Ct < 25 indicates high viral load, Ct 25-30 medium viral load and Ct > 30 low viral load.
3. This test is highly sensitive and specific.
4. It is also a gold standard test for diagnosing COVID-19.

Applications of Antigen, Antibody, and PCR Tests in COVID-19

Antigen Test

• Outbreak investigation/contact tracing: This test is simple, rapid, and easy to conduct in a simple setup and hence easy to screen at-risk individuals and rapidly isolate positive cases.
• Monitoring trends in disease incidence: To evaluate trends in COVID-19 rates in communities as well as health sectors during outbreaks or in regions of widespread community transmission where the positive predictive value and negative predictive value of an Ag-RDT result is sufficient to enable effective infection control.
• Widespread community transmission: This test may be applicable for early detection and isolation of positive cases in various health care facilities.
• Testing of asymptomatic contacts of cases: Antigen negative test result is not sufficient to remove a contact from quarantine requirements whereas a positive antigen result, however, can be useful for targeting isolation procedures and broadening contact-tracing efforts.

Antibody Test

Application of serology (antibody) tests are as follows-

•  To determine seroprevalence
• To find out previous exposure
• To identify highly reactive human donors for the generation of convalescent serum as therapeutic
• Application as supporting approach for contact tracing and screening of health care workers to identify those who are already immune.
• It may also aid as a complementary diagnostic tool for COVID-19.

PCR  Test

1. It may detect both active and inactive COVID-19.
2. The real-time PCR, cannot find out an initial diagnosis of COVID-19 to determine whether an individual continues to shed infectious SARS-CoV-2, and therefore, a repeat PCR test maybe shows in patients who recover and subsequently develop new COVID-19-related symptoms.
3. Detection of pathogens
4. Genotyping
5. Sequencing
6. Classification of organisms
7. Biomolecular archaeology
8. Mutagenesis
9. Mutation detection

Demerits of Antigen, Antibody, and PCR Tests in COVID-19

Antigen Test

• Variable sensitivity and specificity, generally lower than PCR test.
• Lower sensitivity means negative predictive value (NPV)is lower than for PCR, especially in settings with a high prevalence of COVID-19.
• Confirmatory PCR testing of RDT positives is advised in all low-prevalence settings and for RDT negatives in high-prevalence settings.
• Negative antigen test results cannot be used to remove a contact from quarantine.

Antibody Test

1. The clinical significance of a positive antibody test result is still under investigation
2.  Positive antibody test results do not guarantee the presence of neutralizing antibodies or protective immunity
3.  It is not useful for determining active infections in clinical care or for contact-tracing purposes.
4. Interpretation of these test results depends on the timing of the disease, clinical morbidity, epidemiology and prevalence within the setting, the type of test used, the validation method, and the reliability of the results.

PCR  Test

• Time-consuming test i.e. turnaround time of hours to days.
• Labour intensive test
• It requires laboratory infrastructure and skilled personnel.
• It is more expensive than rapid antigen and antibody tests.

Keynotes on Antigen, Antibody, and PCR Tests in COVID-19

1. The highest percentage of virus detection is from nasopharyngeal sampling between 0 to 4 days post-symptom onset at 89% dropping to 54% after 10 to 14
2. On average, the duration of the detectable virus is longer with lower respiratory tract (LRT) sampling than upper respiratory tract (URT) and even in some patients, the virus was still detectable at 46 days post-symptom onset.
3. In certain areas without access to advanced laboratories, rapid antigen testing, although typically less sensitive than RT-PCR, could also be a relevant alternative e.g. for screening.
4. A small study conducted by Long et al. 2020 found that asymptomatic cases may have a weaker immune response to the virus and that the antibodies may diminish sooner than for symptomatic cases with a reduction in neutralizing antibodies after eight weeks.
5. Reports from Studies conducted by Dahlke et al. 2020 and Ma et al. 2020 pointed out that IgA-specific tests are rare, but a potential use of IgA as an early diagnostic marker.
6. The majority of these tests fall within two categories: either a qualitative, rapid immunochromatographic assay (within 30 minutes) or a slower semi-quantitative enzyme-linked immunoassay (ELISA)/chemiluminescent immunoassay (CLIA) (a few hours). Most commonly, they detect IgM, IgG, or both antibodies, but some detect total antibody or IgA too.
7. Antibody tests are quite accurate around 15−21 days post-infection.
8. The average sensitivity and specificity of FDA-approved antibody tests are 84.9% and 98.6%, respectively.
9. The clinical significance of a positive antibody test is still under investigation. It should be noted that the presence of antibodies that bind to SARS-CoV-2 does not guarantee that they are neutralizing antibodies or that they offer protective immunity. However, antibody testing may be useful for zero surveillance studies to support the investigation of an ongoing outbreak and to support the retrospective assessment of the attack rate or size of an outbreak.
10. COVID-19 confirmed patients with negative antibodies results are also seen.
11. If a candidate with a highly suspicious COVID-19 remains negative by PCR testing and if symptoms are ongoing for at least several days, antibodies may be helpful and enhance diagnostic sensitivity.
12. A cross-sectional study conducted by Bullard 2020 determined PCR positive samples for their ability to infect cell lines. Of 90 samples, only 29% demonstrated viral growth. There was no growth in samples with a Ct > 24 or duration of symptoms > 8 days.
13. The PCR test is the “gold standard” test since it is the most accurate and reliable test.

Further Reading

1. WHO-2019-nCoV-Antigen_Detection-2020.1-eng.pdf
2. https://www.who.int/publications/i/item/antigen-detection-in-the-diagnosis-of-sars-cov-2infection-using-rapid-immunoassays
3. https://covidreference.com/diagnosis
4. https://academic.oup.com/femspd/article/79/1/ftaa069/6123719
5. https://bmcmedicine.biomedcentral.com/articles/10.1186/s12916-020-01810-8
6. https://bmcinfectdis.biomedcentral.com/articles/10.1186/s12879-020-05419-3
7. https://my.clevelandclinic.org/health/diagnostics/21462-covid-19-and-pcr-testing
8. https://apps.who.int/iris/handle/10665/337948
9. https://jcm.asm.org/content/58/11/e01695-20
10. https://link.springer.com/article/10.1007/s00540-020-02875-8