Swab, LAMP test, rapid test, serological test, molecular test, antibodies, PCR-RT, IgG, IgM ... words we stumble over daily. But do we know exactly what they mean?"
.First, let's analyze what is the substantial difference between the various types of tests for the identification of Covid 19 or rather for SARS-CoV-2 as it has now been identified by the scientific community.
TAMPON or CLASSIC MOLECULAR TEST
.The pharyngeal or rhino-pharyngeal tampon (rhino = NASO) is the only diagnostic test for SARS-Cov2 infection currently available that is recognized as valid by the World Health Organization:it detects the presence or absence of genetic material of the virus, thus giving confirmation of ongoing infection. In other words, it is used to detect the presence of the virus in the body, regardless of clinical status, that is, to decree so-called positivity.
SYEROLOGICAL TESTING (rapid tests and laboratory tests)
.Also known as IMMUNOLOGICAL TESTS, serological tests allow detection of the presence of antibodies against the SARS CoV-2 virus. In short, they detect the body's response to the infectious agent and are used to tell whether a person has already developed an immune response following Covid 19 infection. Through serological testing, it is indeed possible to go and detect the antibodies produced by our immune system in response to the virus.
HOW IS CLASSIC TAMPON ANALYSIS CARRIED OUT?
Biological material taken with the swab is analyzed in the laboratory using the method known as real-time PCR or quantitative PCR (Italian for reverse polymerase chain reaction test, if followed by the acronym RT it means it is performed in real time). This is a technology used todetect and quantify nucleic acids, in this case of the virus, through the use of special markers that can emit light (fluorescent).
The first step of the analysis is to extract nucleic acids to detect the presence of viral RNA (ribonucleic acid).
Once the RNA has been obtained from the biological sample, it is amplified and the specific genetic markers of SARS-CoV-2, i.e., that part of the viral RNA that characterizes the viral species, are sought and quantified.
This is the most reliable method for detecting even very low concentrations of viral RNA, but if it is performed too early and the virus has not had time to replicate, the analysis may fail and the swab must be repeated. So in a suspected case, when, for example, the patient has symptoms or because he or she came in contact with an infected person, if the first swab gives a negative result, the swab should be repeated for confirmation.
WHAT IS THE LAMP TEST?
LAMP = LOOP-MEDIATED ISOTHERMAL AMPLIFICATION
Loop-mediated isothermal amplification (LAMP) is a process similar to PCR-RT which, instead of using a series of temperature changes to produce copies of the viral RNA, is conducted at the constant temperature of 60-65 °C.
The amount of RNA produced in the LAMP assay is much higher than in PCR-RT and for this reason the positive test result can be "seen" directly without the need to require a machine to read the results.
LAMP is a newer method than PCR-RT and is technically simple and easy to use.
Positive test result:- A positive LAMP result means that the person from whom the sample was taken is currently infected with the virus.
Negative test result: - A negative LAMP result could indicate that the person is not currently infected, that the virus is not present at the site from which the sample was taken, or that it is too early, or too late from the onset of infection to detect the virus. This is why when test results are negative but the patient's symptomatology or history can be traced to possible infection, the test is repeated a few days later to reduce the chance of a false negative.
LAMP tests are unable to detect whether a person has had the virus, and then cleared it after the COVID-19 disease has ended, as they only detect when an active virus is present.
Because it is a newer technology there is less data on its accuracy, but diagnostic companies are currently running clinical trials to be able to validate it.
How do serology tests work? (antibody tests)
.There are two types of serological tests: qualitative and quantitative. The substantial difference lies in the methodology and mode of analysis:
- qualitative tests only determine whether or not a person has developed antibodies, according to a positive/negative logic; qualitative tests are the now well-known rapid tests, in which all that is needed is a drop of capillary blood (obtained with a finger prick) that is examined in a portable kit and from which a match is obtained within minutes, exactly as is the case with the pregnancy test that detects hCG hormone in urine. These are technically "lateral flow immunoassays" (LFIA) where a drop of blood is run on a small plate containing viral proteins conjugated with colored particles and antibodies to human IgM and IgG arranged in two lines, respectively. If the blood contains IgM or IgG against viral proteins, these bind to the viral proteins conjugated with the colored particles on the plates and, as they slide, they remain attached to the antibodies against human IgM and IgG placed on the respective lines, changing their color.
- Quantitative tests, laboratory tests, in addition to detecting the presence of the antibodies in the blood, also allow for the assay of their amounts. Quantitative serological tests require venous blood sampling and are performed the Laboratory using chemiluminescence detection systems (CLIA, CMIA) or enzyme immunoassay systems (ELISA).
WHAT ARE ANTICORPIES?
An antibody, or immunoglobulin, is a protein with a defensive function against the organism, produced by plasma cells, the final developmental stage of B lymphocytes, in response to the entry of particular substances into the body, called antigens (an antigen is defined as any foreign substance that when introduced into the body is capable of provoking the formation of antibodies and reacting specifically with them by inducing an immune response), to which it combines giving rise to the immune-type inflammatory response. Antigens are glycoproteins present in the structure of microorganisms (viruses, bacteria, protozoa...) that stimulate antibody production.
Currently, immunoglobulins are divided into five general classes: IgG, IgA, IgE, IgM and IgD.
Immunoglobulin A (IgA) are present mainly in external secretions, such as saliva, tears, genitourinary secretions, intestinal and bronchial mucus, colostrum and breast milk, and in all mucous membranes. They represent an important means of defense against local infections, preventing colonization by pathogens and their entry into the body.
TheImmunoglobulin D (IgD) whose role has not yet been fully elucidated.
TheImmunoglobulin E (IgE) present on the surface of particular cells, mast cells, are implicated in allergic reactions and are also extremely important for protection against parasitic infestations.
TheImmunoglobulin G (IgG) are the mainstay of the secondary immune response, meaning they intervene in cases where there has already been a previous encounter with the antigen. During pregnancy, the mother transmits her IgG to the fetus through the placental membrane, conferring immunity to the infant during the first 3-4 months of life.
TheImmunoglobulin M (IgM) are antibodies associated with the primary immune response (initial exposure to foreign antigen) by intervening first upon contact with a new antigen.
WHICH ANTIBODIES DOES THE SIEROLOGY TEST INDICATE? IgM AND IgG?
Turning our focus back to SARS-CoV2 infection (which causes the disease known as COVID19), IgM are the antibodies that first appear in the blood after primary exposure to the virus (usually not earlier than 10 days), and therefore their presence indicates recent infection and disappear after the appearance of IgG.
The IgG begin to form about 15 days after primary exposure to the antigen and persist for a long time. IgG protect the body from new viral replication leading to healing, but it is not yet known whether they protect against new infections a few months later and how long the immunity lasts.
The IgGs protect the body from new viral replication.
Regardless of the mode of detection, serologic tests go to investigate the presence or absence of IgG and IgM antibodies to SARS-CoV-2 virus in the blood.
They are used to investigate the presence or absence of IgG and IgM antibodies to SARS-CoV-2 virus in the blood.
If neither IgM nor IgG is detected, there are probably no antibodies to the virus in our blood. Therefore, it is likely that we have not contracted the infection. However, we may also be at an early stage of infection when the body has not yet produced antibodies (so-called "window period"). In addition, since the real ability of serological tests to detect all cases with the presence of antibodies has not yet been accurately ascertained, we cannot rule out the possibility that in fact the antibodies in the blood are there but the test has not detected them (so-called "false negatives"). It is evident that under the latter two circumstances (serological window and false negatives) people could be infected and even contagious despite having a negative serological test.
These two circumstances are not the same as the ones in which the test was conducted.
If one detects only the presence of IgM, it is likely that our body has produced IgM against the virus and we are at an early stage of the disease. In this case, the swab is generally positive. We have then contracted the infection and probably can transmit it to others. Even in this case, however, it is possible that the test reads the presence of antibodies directed toward proteins of other viruses instead of SARS-COV-2 and thus incorrectly signals the presence of infection in healthy individuals (so-called "false positives").
If both IgM and IgG are detected, it means that our body has probably produced both IgM and IgG against viral proteins and that we are probably in an intermediate stage of infection. In this case, the nasopharyngeal swab may be positive. We have therefore contracted the infection and can probably still transmit it to others.
If the test detects only the presence of IgG it means that our body has produced IgG against SARS-CoV2 and that IgM has already disappeared. We are then probably at a more advanced stage of the infection or we are already cured. In this case, the nasopharyngeal swab may already be negative but, in some cases still positive. We have therefore contracted the infection and we cannot exclude that we may still transmit it to others.
We emphasize that the interpretation of the serological test requires a careful medical history and clinical evaluation by a physician.
ANTIGENIC TEST ON RINO-FARINGEAN TAMPON
This type of test is based on the search in respiratory samples for viral proteins (antigens). An antigen is defined as any foreign substance that when introduced into the body is capable of causing the formation of antibodies and reacting specifically with them, inducing an immune response.
The mode of sample collection is quite similar to that of molecular testing (nasopharyngeal swab), the turnaround time is very short (about 15 minutes), but the sensitivity and specificity of this test are significantly lower than those of molecular testing.
Instead of looking for the genetic material of the coronavirus (tampon with PCR) or antibodies (serologic), an antigenic test goes looking for particular specific proteins of the virus, found on its outer envelope (capsid). However, this involves the possibility of false-negative results in the presence of low viral load, as well as the need to confirm positive results by molecular swabbing.
Antigenic tests are considered a viable alternative to the classical swab test to speed up testing time and reduce costs, but there are still high doubts about their sensitivity and reliability. Many such tests are reliable when the result is positive (that is, when infection is detected), while they have some problems with false negatives, with the risk of an infected individual testing negative.
To do an antigenic test, a swab is usually used, which is, however, rubbed only inside the nostrils, in a less invasive operation than the sampling deep in the nasal septum done for the classic swab. The sample is then treated with a reagent and finally analyzed for antigens, either in the laboratory or through a tester (often a test tube with the reagent inside where the swab just taken is dipped) if a rapid test is performed.
This "rapid swab" has recently been introduced for screening passengers at ports and airports, where it is important to have a response quickly. Even considering the possibility of false-positive results (that is why positive results from the antigenic test are confirmed with the molecular test) and false-negative results (the sensitivity of the test is certainly not 100% and, in addition, the "window period" between the time of risk exposure and the appearance of positivity must be considered), thanks to the use of such rapid tests, a relevant number of infected persons, probably with high viral loads, who would not have been detected otherwise have nevertheless been intercepted.
Antigenic tests exist in POCT versions (i.e., can be performed at the site of collection) and in "laboratory" versions (i.e., require laboratory equipment).
MOLEULAR SALVAGE AND ANTIGENIC TESTS
Recently, tests using saliva as the sample to be tested have been proposed on the market. Saliva sampling is simpler and less invasive than the nasopharyngeal swab, so this type of test could be useful for screening large numbers of people.
As with swabs, there are molecular type tests (i.e., detecting the presence of virus RNA in the sample) and antigenic type tests (detecting viral proteins in the sample).
Saliva generally does not lend itself well to use with highly automated laboratory equipment, which is usually used to process high volumes of molecular samples, because it has variable density and can create problems for highly automated drafting systems. In addition, as far as antigenic testing is concerned, the sensitivity of the test is similar to rapid antigenic testing only in the case where the test is performed in the laboratory, so unless laboratory units are activated at the points where the sample is drawn, it is unlikely to be usable in rapid screening settings.
VARIANCE PROBLEM
.Currently the presence of SARS-Cov2 virus variants has not challenged the sensitivity and specificity of testing for viral detection in nasopharyngeal specimens. This is probably because from a structural point of view the virus has not changed much, rather it has changed its ability to bind to cell receptors that allow it to enter the cell. In biological terms, it is used to say that the virus has more affinity to the entry receptor, so it binds better and faster.
Mutations allow variants of the virus to infect more cells and more rapidly than the original virus. Mutations are the consequence of errors in viral replication within human cells during infection and disease. Some of these "errors" produce "modified" viruses, the SARS-CoV-2 variants precisely, characterized by increased infectivity (the ability to enter cells) and sometimes increased pathogenicity (ability to cause severe disease).
So far we have seen increased infectivity, but it is possible that some variants may also increase pathogenicity.
One point of attention is to understand how the virus will behave under vaccine pressure, that is, whether mutations may be able to evade the antibody response and thus thwart the vaccination process currently underway.
A very recent study, published on March 29, 2021, conducted by the research group of Prof. Crisanti - University of Padua, demonstrates how some of the genetic variants of SARS-CoV-2 may compromise the validity of antigenic testing.
CONCLUSIONS
In light of the above considerations, it can be stated that:
- the molecular RT-PCR test still remains the reference test for the diagnosis of SARS-CoV2;
- rapid antigenic tests on nasopharyngeal swab may be useful in certain contexts, such as rapid screening of numerous people;
- tests using LAMP technology will be useful because of the rapidity of reading results and the absence of machinery required for reporting;
- screening procedures will still and for quite some time be used to monitor for the presence of potentially more pathogenic variants;
- molecular surveillance in RT-PCR will continue to be the "gold-standard" for the detection of variants that will be produced under vaccine pressure.
