April 26 2020
Q: Why do we need to test for SARS-CoV-2 (COVID-19)?
A: The tests are performed to:
confirm presence of the virus;
confirm immune response to the virus;
Q: What types of tests for SARS-CoV-2 are available?
A: There are two types of tests in use:
Polymerase Chain Reaction (PCR), which is used to confirm presence of the virus;
Tests for antibodies to the virus, which is used to confirm immune response to the virus. It is also known as serology test;
Note: Recently, massive parallel DNA sequencing has rapidly developed for the identification of both described and new viral pathogens.
Q: How does PCR work?
A: PCR deciphers viral RNA (or DNA in DNA-viruses) by splitting it into small fragments, identifying sequence of nucleotides (building blocks) in each, and then amplifying (multiplying) these fragments and comparing them with a database of known viruses. If virus is “novel” - not present in a database, it is compared against known Species of viruses and further classified to a particular viral Genus, Family, Order and so on, up to a viral Realm. As of 2019, there are 4 known viral Realms.
Q: What are antibodies?
A: Antibodies are protein molecules produced by immune system in response to antigens - substances foreign to an organism (or thought to be foreign, as happens in autoimmune diseases). Antibody traps antigen and therefore neutralizes it. A unique antibody is produced to each unique antigen. Antibodies are also known as immunoglobulins. There are 5 classes of immunoglobulins: A, D, G, E, and M. Normally, Immunoglobulin M is produced first - within few days to 2 or even 4 weeks after encountering new antigen. Immunoglobulin A is normally produced on mucosal surfaces, hence it is known as secretory antibody. Immunoglobulin E is produced against parasites and allergens. immunoglobulin G is produced later - starting at 2 to 4 weeks. Immunoglobulin M production usually wanes by 6 months, while Immunoglobulins G, A, and E persist for much longer - up to a lifetime.
Q: How antibodies are tested?
A: In order to test specific antibody, patient’s serum is mixed with an antigen (virus in our case).
There are two types of antibody tests:
ELISA (enzyme-linked immunosorbent assay) where patient’s serum is gradually diluted to determine antibody titer - the maximum dilution at which reaction between antibodies and antigen occurs, hence higher titer means stronger immune response. High titer indicates that antibody is highly specific to antigen;
Plaque reduction neutralization test (PRN) which determines both the titer and antibody ability to neutralize antigen. This means that besides determining presence of antibody, it measures how well antibody neutralizes (catches and holds) antigen. This test is more expensive and takes longer than ELISA (days vs. hours), which makes it unsuitable for rapid diagnostics.
Q: What about viral culture?
A: Culturing viruses is difficult, long (up to 1 month or longer), expensive and, potentially, dangerous process. Therefore, it has limited use and is not suitable for rapid diagnostics.
Q: How accurate are these tests?
A: The accuracy of the test is characterized by its sensitivity and specificity.
Sensitivity is the probability that a test will indicate 'disease' among those with the disease;
Specificity is the fraction of those without disease who will have a negative test result.
Even if one can hear claims of 100% specificity and sensitivity of some tests, it is not correct. There are no tests which are 100% accurate, because of possibility of errors (both technical and human), difficulty with interpretation, cross-reactivity with similar viruses (antigens), low concentration of antigen or antibody in a sample, and so on. Therefore, sensitivity is always less than 100%, while specificity may approach 100% when antigen/antibody are present in high numbers. The blank estimate is that:
PCR is approximately 80-95% sensitive and up to 95% specific;
ELISA is approximately 70-90% sensitive and 90-95% specific;
Plaque reduction neutralization test is 85-98% sensitive and 60-95% specific;
Q: What is Negative Predictive Value (NPV) and Positive Predictive Value (PPV)?
A: As stated above sensitivity and specificity are characteristics of the test. The population does not affect the results.
A clinician and a patient have a different question: what is the chance that a person with a positive test truly has the disease? Positive and negative predictive values are influenced by the prevalence of disease in the population that is being tested. If we test in a high prevalence setting, it is more likely that persons who test positive truly have disease than if the test is performed in a population with low prevalence.
The PPV tells us how likely it is for someone who tests positive (screen positive) to actually have the disease (true positive). It answers the question, “I tested positive. Does this mean I definitely have the disease?”
Equally, the NPV tells us how likely it is for someone who tests negative (screen negative) to not have the disease (true negative). I.e. it answers the question “I tested negative. Does this mean I definitely don’t have the disease?”
Ying and co-authors (China) published “Diagnostic Indexes of a Rapid IgG/IgM Combined Antibody Test for SARS-CoV-2” on March 30th, 2020. In PCR positive patients, sensitivity of antibody tests was 86%, PPV was 95.1%, NPV was 83%, overall accuracy was 88%.
Q: Am I immune to the virus if I tested antibody positive?
A: No! The positive test, assuming it is accurate (true positive), means that:
You encountered the virus, and
Immune system responded to the virus by making antibodies.
Q: Why presence of antibodies does not always correlate with immunity?
A: The presence of antibodies does not correlate with immunity, because antibodies may not be able to neutralize the virus (ineffective antibodies) or the quantity (titer) of antibodies may not match demand, e.g. there are more viral particles than antibodies.
As noted above, plaque reduction neutralization test, may help to confirm antibodies efficacy, but its use is limited and there is some disagreement regarding interpretation of results.
Also, virus is changing (approximately 30 strains are circulating in the USA as of today), therefore antibodies against one strain may not be fully effective against another. This is exactly what happens with influenza virus, hence vaccination (which triggers antibody production) is only between 35% to 55% effective - depending on the type of virus.
Q: If I do have immunity, will it prevent disease altogether?
A: No! Immunity resulting from past infection or vaccination may not be able to prevent the disease but, typically, results in a lighter form of the disease.
Q: If I have the disease, can I be reinfected by the virus?
A: Yes! The reason for second infection may be waning of immunity, mismatch between viral load (exposure) and antibodies, ineffective antibodies, or changes occurring in the virus, which allow it to overcome defenses.
Q: I tested positive for antibodies. Does this mean I can return to work? Does this mean that I am safe to visit /work with high risk population?
A: No! First, we already discussed the fact that antibody may not confer immunity. Second, immunity may wane with time. Third, it was demonstrated that some people remain carriers of the virus and may shed virus long after recovery.
Q: Is “Immunity Passport” a good idea?
A: No!
First, immunity may neither effective, no lasting;
Second, one may continue shedding virus, while having antibodies;
Third, what are we going to do about other infections, including those we have vaccines for?
Forth, testing for all possible infections and a need for repeat testing to confirm continuing (lasting) immunity is expensive;
Fifth, testing is imprecise;
Sixth, antibody testing evaluates just one of many links in immune response.
Q: What may be a substitute for an “immunity passport”?
A: Currently, the fact of vaccination, as reflected in a vaccination card, is accepted, albeit arbitrarily, as a proof of immunity. This is the only feasible way forward.
Additionally, documentation of the disease in patient’s history may be used as a surrogate for immunity.
Q: Why government is promoting testing as prerequisite to “opening economy”?
A: This is a misguided and wasteful campaign, which is either conducted without understanding of “immunity 101”, or for distraction of public attention. Also, powerful lobby of pharmaceutical companies, diagnostic laboratories, and reagents and equipment manufactures clearly benefits from this campaign.
Q: Why test then?
A: Testing is important for several reasons/purposes:
PCR, PRN and measurement of Immunoglobulin M, especially change in Immunoglobulin levels over time, may be used for diagnosing active infection;
Understanding infection pathogenesis;
Understanding and monitoring of the immune response to infection;
Vaccine development;
Determining who may donate plasma to be used for plasma exchange therapy;
Drugs clinical trials;
Epidemiology, in particular for understanding of the infection scale and spread, which affects calculations of morbidity, mortality, and therefore public health planning, prevention, and allocation of resources;
Medical education;
Piece of mind…even although it might be misleading….
Q: What should be a scope of testing? Who should be tested?
A: The representative sample of population, not more than 5% of total, should be tested and results extrapolated to the rest of population.
For clinical purposes, in addition to disease diagnostics, people with suspected immunodeficiency may benefit from testing.
If and when vaccine becomes available, antibody testing may be used to confirm development of immune response.
Q: There are multiple tests on the market. Which one shall I use?
A: There are no FDA approved tests at this time. To date, only 4 companies received FDA permission to market their test kits under Emergency Authorization Use (EAU). Additionally, approximately 70 laboratories received permission to offer in-house tests with a disclosure that these were neither evaluated no approved by FDA.
Q: Which test is more reliable?
A: The comparative analysis of tests accuracy, NPV and PPV is anecdotal. It is not possible to recommend one test vs. another with confidence at this time.
Q: Are there any tests for home use?
A: Such tests are being developed, but yet to receive FDA approval.
Q: Are there any approved tests for point-of-care (physicians and other provider’s offices)?
A: Not yet.
Q: There are multiple tests (kits) marketed online. Can you recommend one?
A: No, those may not be properly evaluated and may be a part of scams and counterfeit schemes. No tests are approved for online, direct to consumer, marketing to date.