During this health crisis, there is much talk about the COVID-19 tests, fundamental tools to diagnose the infection and better understand the behavior of the disease in the face of de-escalation and prevention against possible outbreaks. But you have to be careful: not all tests are the same, nor do they have the same sensitivity, nor do they provide the same information. In addition, we must bear in mind that we are facing a new virus about which relatively little is still known and the urgency of the situation has caused many tests to go on the market without passing all the usual controls, so many of them are not all how reliable that would be desirable.
First, we must differentiate between direct diagnostic tests and indirect diagnostic tests . As its name indicates, in the former what we are looking for is direct evidence of the presence of the virus, either by detecting its genetic material (in this case RNA) by PCR, or by locating some of its proteins (antigen detection test). .
On the other hand, indirect tests look for evidence that our body has been in contact with the virus: they are called serological tests . To do this, antibodies are identified, which are a type of defense proteins generated as part of the immune response to infection. In turn, to identify the antibodies, immunochromatography techniques can be used, which are those commonly used in the so-called rapid tests, or techniques such as ELISA (enzyme-linked immunosorbent assay) or CLIA (chemiluminoassay).
Let’s see what each type of test consists of.
Direct diagnostic genetic test: PCR
As we have already mentioned, this test allows detecting the presence of genetic material of the virus. “It is a reverse transcription PCR: using an enzyme we convert the RNA into DNA and then make the detection. Generally, a gene common to all coronaviruses and one or two specific SARS-CoV-2 genes are amplified ”, explains Guillermo Quindós Andrés, professor of Medical Microbiology at the University of the Basque Country. “For this, what is done is to take samples in a place where the virus can multiply, in this case through the nasopharyngeal or oropharyngeal route (sometimes both are taken, one from the nose and the other from the mouth). A very long swab is used and it is a process that can be quite unpleasant. The US Food and Drug Administration (FDA) is also testing another method that uses saliva, but it is not yet known if it works well ”, explains the expert.
“The key here is the taking and processing of the samples, if it is done well, very good sensitivity is achieved, of 90% or even more,” says Guillermo Martinez De Tejada, professor at the Department of Microbiology and Parasitology at the Faculty. of Medicine of the University of Navarra. The main drawback of this test, precisely, is that specialized personnel and equipment are needed to carry it out, because there is a high risk of contamination of the samples if it is not done accurately. The other problem is time: “It usually takes at least four hours , but also all the administrative work of centralizing and managing the samples has to be added,” explains the expert.
In addition to its high reliability, another advantage of PCR is that it is the first test to test positive: “indirect tests, for example, will be positive later, because you may be infected and have not yet developed antibodies”, Martínez de Tejada tells us. “ We can find RNA from viruses almost from the first day after a person has symptoms, or even in people who are experiencing the infection asymptomatically , because almost five days after being in contact, viruses begin to appear in the nasopharynx. ”, Adds Quindós. Therefore, this technique is the most recommended to make an early diagnosis of a person with symptoms and of all their contacts.
The famous ‘reinfections’ of cured patients
We have all heard news of people who have overcome COVID-19 but continue to test positive. “ There are several interpretations, but it is becoming increasingly clear that it would not be reinfections. One of the hypotheses is that there may be remnants of afunctional RNA, which have not yet fully degraded but no longer have the ability to multiply. It could also be that remains of the virus remain inside the cells that usually digest them, such as phagocytes, ”explains Quindós.
PCR is not useful for mass diagnosis
As we have already said, PCR is the most recommended test to make an early diagnosis of the possible infected and their contacts. “However, it is not good to do it to everyone on a massive scale”, comments the expert from the University of the Basque Country. “Although few, PCR gives those false positives that we mentioned before, and if we did PCR to the entire population, they would increase in absolute value and we would conclude that there are many more infected people than there are in reality. Another added problem is that the PCR gives you a static image: it tells you if you are infected at that moment, but not if you have been infected for a month , for example. We would have to be doing PCR to everyone continuously, that is not feasible ”.
Direct diagnostic test: antigens
There is a second type of direct diagnostic test that is not as widespread in Spain: these are the so-called antigen or protein detection tests. “ Instead of identifying genes, we look for proteins, usually protein S, which is usually the place where the virus binds to the host cell. Other times a nucleocapsid protein (N) is used, which is the envelope that protects the genetic material of the virus ”, explains Quindós.
The sample that is needed is also taken from the nasopharynx, and a technique called immunochromatography or immunofiltration is used. An antibody specific for the selected protein is used and its presence is evidenced by identifying the antigen-antibody reaction. “It works in a similar way to the pregnancy test, and they have the advantage of being very fast and direct, but the sensitivity is lower than the PCR “, indicates Martínez de Tejada.
“Antigen tests have the problem of giving more false negatives, that is, people who have the infection but we do not detect it, and false positives in which the opposite occurs,” explains Quindós. For this expert, the usefulness of these tests is that, because they are faster than PCR, they allow a very fast screening and later, in the cases of people who have tested negative but have significant symptoms, confirm the result with PCR.
Indirect diagnostic test: antibodies
As we have already mentioned, in these diagnostic tests what is sought is the response of the human body to the virus, in this case through the detection of antibodies. “It is known that both protein S and nucleoprotein N induce antibody responses,” explains Quindós. “To detect them, the sample used is blood, either by pricking the finger or with venous blood, depending on the method. Rapid tests usually use capillary blood taken from the finger and ELISA and CLIA venous blood ”.
In the case of rapid tests, the technique used is usually immunochromatography, the same one that is used in the antigen test . However, you have to be careful, because as we already mentioned, the urgency of the situation has led to the release of numerous rapid tests that may not be entirely reliable. “Almost all the trials indicate in their commercial information a specificity of 100%. However, this figure could not be correct since the validation studies are methodologically inadequate and with a very small number of samples ”, is explained in a document published by the Spanish Society of Infectious Diseases (SEIMC) with recommendations for the use of antibody detection tests. The tests that analyze by ELISA or CLIA are more sensitive and specific, although they have the disadvantage of taking longer than the quick ones.
Some tests detect total antibodies and others are capable of differentiating immunoglobulins A (IgA), immunoglobulins M (IgM) and immunoglobulins G (IgG). Regarding IgA, the SEIMC document indicates that at this time there are no published reliable studies on its behavior, so IgM and IgG provide more information.
“IgMs appear earlier: they are a grosser response, the first that the body produces. IgGs are already part of a finer and stronger response, in which they intervene by coordinating T lymphocytes. Furthermore, the immune memory, if it is established, will be mainly with a response with IgG antibodies ”, clarifies Quindós. “It could also give you an idea of what phase of the infection you are in, although it is true that many times the two IgM and IgG responses overlap. Usually, if you detect IgM you can think that the patient is in an acute phase, if you detect both it would already be a subacute phase, and if only IgG appears you could think of the recovery phase, or that the infection has even been overcome ”.
¿Quick test the ELISA / CLIA test?
“The good thing about the quick test is that in a few minutes you have a result. In addition, it could theoretically be done anywhere (a health center, the dentist, a pharmacy), but it has the disadvantage that we have mentioned before, its current low reliability ”, explains Quindós.
“ These tests should not be interpreted in isolation: we should complete it with a description of the symptoms and, if possible, perform a PCR to cover all the possibilities: to know if the patient is infected right now or if the disease has already passed. For example, imagine that in a test you only get positive for IgG. You do not have IgM but you still have quite clear clinical symptoms, you have not fully recovered. A PCR should be done to see if the virus continues to replicate in the nasopharynx or not. That is to say, all this must be taken as one more tool that will help you in the clinical diagnosis ”, reflects the expert. ” If you want to detect antibodies and you want to do it very well, it is better to use ELISA or CLIA, which are also recommended for doing seroprevalence studies .”
Antibodies are only part of the immune response
“Let’s say that the test works perfectly and no antibodies are detected,” explains Martínez de Tejada. “This does not mean that you have not passed the infection. We are seeing that there are people, especially the younger ones, with a positive PCR and who after a month do not have antibodies , or a very weak positive is obtained. This is due to the fact that the most effective defense against the virus is actually cellular defense, via T lymphocytes, instead of the humoral response, which is mediated by antibodies ”. That is to say: It can happen that a person gets rid of the infection without the need to release antibodies. “In older people it happens the other way around, in people 50 years and up it is more common to find antibodies and, in addition, at very high levels”, continues the expert. “And the same happens with immunity, that young person who has passed the virus and does not have antibodies could also be immunized because their T lymphocytes are going to be able to remember and have memory. Establishing a link between antibodies and immunity is dangerous ”.
What is the antibody test NOT for?
As we have already mentioned, these tests are not the most recommended for early detection of infected people and their contacts. “For the use of the test in the Hospital-Emergency setting, the test would have to be applied in patients with an evolution of at least 7 days , since the available scientific information indicates that before that time the host’s immune response is non-existent or very low. . Without this temporal nuance, there is a risk of a very low sensitivity of the assay ”, explains the SEIMC document. Nor is its use recommended for the follow-up of symptomatic cases in social and residential centers, since it is precisely in this group where more false positives (due to autoimmune diseases, rheumatoid arthritis; …) false negatives, (due to treatment with immunosuppressants, immunosenescence ) are produced.
What is the antibody test for?
“Serological tests are useful at the population level and for epidemiological studies,” explains Martínez de Tejada. That would be its most important application, since it will allow us to understand the epidemiology of the disease and learn more about the role that asymptomatic people may have played in the spread of the epidemic.
Other applications cited by the SEIMC report would be its use in research and research for vaccines, and to “define previous exposure and identify highly reactive human donors for the generation of hyperimmune serum as a therapeutic approach.” Quindós also points out that, like antigen tests, rapid serological tests can also allow rapid screening to later confirm negative tests with clinical suspicion with PCR.
What about immunity? As we have already mentioned, antibodies are only part of the body’s immune defense and, in any case, it is currently not known whether having antibodies implies being immune to a second infection or how long that supposed immunological memory would last. It is dangerous at this time to assume that having a positive antibody implies being protected against COVID-19, as would be done in the case of the controversial immunity passports, as it can make us relax the social distancing and self-protection measures that we do. we know that they are effective in preventing infection.
