DIAGNOSTICS

Lyme disease tests can be divided into two types:

  • serological– defining the response of the immune system to infection
  • direct– looking for bacteria that cause Lyme disease or specific fragments or genetic material.

There is no 100% method for diagnosing Lyme disease.

Serological tests include:  ELISA and Western Blot (WB).

Serological tests check whether the body defends itself against infection. You are not looking for the source of the infection itself, i.e. the presence of bacteria but the antibodies produced by the organism in response to the infection.

Direct test are PCR, PCR RT and LTT, in which the sources of infection are sought, i.e. the bacteria that cause the disease, and more precisely their DNA.

ELISA TEST

Physicians normally recommend an ELISA test. Unfortunately, this test is very unreliable in Lyme disease. The same blood sample sent to several laboratories can give a whole range of results, often completely different: negative, positive, low or high titres. Its credibility therefore remains at the level of 40 – 50%. Nevertheless, the Lyme ELISA test is still being used because it is cheap.The ELISA test is an enzyme-linked immunosorbent assay that involves the identification of specific IgM or IgG antibodies in the test material – serum. These antibodies are created as a result of the body’s immune response to bacterial infection. IgM antibodies are the first line of the immune system response. They usually last several weeks after the bite of a tick infected with Lyme disease. It is only later that IgG antibodies are formed and persist for several months or even years.

Antibodies do not rise immediately after the bite but only after several weeks of delay. Therefore, the ELISA test should be performed at least 2 weeks after, and for some even 6 weeks after tick bite.

In addition, IgG antibodies stay in the blood for a long time during recovery, or convalescence, when there is no active infection anymore.

Standards in the ELISA test are as follows:

  • Negative result – below 9 j. BBU/ml,
  • False positive – 9,1-10,9 j. BBU/ml,
  • Low positive– 11-20 j. BBU/ml,
  • High positive – 21-30 j. BBU/ml,
  • Very high positive – above 30 j. BBU/ml.

 

WESTERN – BLOT TEST

The Western-Blot test involves searching for specific antibodies produced by the Borrelia infection. Antibodies are detected in the test material using special labeled test bands, i.e. labeled Borrelia bacterial protein antigens. In the Western-Blot test, only specific stripes characteristic for Borrelia spirochetes are used – protein bacteria fragments.  If the Western-Blot test result is positive, this indicates active Lyme disease. Its specificity is therefore much higher than in case of ELISA. The credibility of the test is estimated at 70 – 95%.However, the immune system must have time to produce antibodies, so the Western-Blot test can be performed only 4 to 6 weeks after the tick bite. This is more or less the time it takes for the immune system to produce antibodies detected by this test. The test may therefore give a negative result in the initial period of the disease, i.e. for the first few weeks after the bite. The Western Blot test may also give false negative results when in the old, chronic borreliosis, the production of antibodies has been stopped by Borrelia bacteria or if the antibodies have been completely used to fight the disease. If there is a clinical suspicion of the disease, the Western Blot test should be repeated several times, e.g. every few weeks, to find a moment when antibodies are present in the blood.

The Western-Blot examination can be performed at any time, also during antibiotic therapy, however, with antibiotics the chance for a positive result is slightly lower. It is best to perform the test after 6 weeks after discontinuation of antibiotic therapy.

The Western-Blot test is a good test, but its result is always dependent on the presence of free antibodies in the blood produced by the immune system. Unfortunately, some patients with Lyme disease do not have antibodies in the blood (in Lyme disease, the so-called immune complexes blocking antibodies are created). The chance for a positive result in a chronically ill person is 50-60%.

How to interpret results of the Western-Blot test?

Interpretation of the test results is the interpretation of the bands. Some of them are very specific for Borrelia bacteria, and others are less specific, i.e., also found in other bacteria.

Bands:

  • p18 –  surface protein of a bacterial cell (DbpA) — decorin21-binding protein
  • p21 –  antigen specific for the stage phase of infection
  • p22 – surface protein
  • p23–25 – outer membrane protein (OspC),
  • p28 – surface protein
  • p30 –  specific antigen
  • p31- outer membrane protein (OspA),
  • p34 –  outer membrane protein (OspB),
  • p37 –  unknown protein, but related to the presence of Borrelia bacteria

·       p39 – Borrelia membrane protein, highly specific to Borrelia·       p41 – flagellin – a shrinking protein used by many species of bacteria for movement – nonspecific for Borrelia,·       p45 – heat shock protein (HSP45),

  • p58 –  heat shock protein (HSP58),
  • p66 –  heat shock protein (HSP66),
  • p73 –  heat shock protein (HSP73),
  • p83 (p93 or p100) –  membrane protein – recognized indicator of the long duration of Borrelia infection
  • VisE: the lipoprotein of Borrelia’s outer cell membrane, synthesized only in the host organism, e.g. human.

According to Dr. Burrascano in the Western-Blot test, the following bands are important in the diagnosis of Borrelia: p18, p21-24, p31, p34, p37, p39, p83, p93. The presence of the p41 band is generally indicative of spirochete antibodies – these may be other spirochetes, not necessarily Borrelia spirochetes.

With the exception of the p41 band and after the exclusion of other diseases, a single band would suffice to confirm the disease in the Western Blot test. Of course, for some doctors, one band is not enough, because other pathogens can give the so-called cross reactions. It should be assumed, therefore, that the greater the number of bands, the more reliable the recognition.

IgM bands have more diagnostic significance as they suggest active Lyme disease. The level of IgM antibodies is high at the beginning of infection, or contrary to logic, in chronic Lyme disease. This may be because in the chronic Lyme disease the spirochetes are in cells, but when they occasionally get into the bloodstream, the body treats them as a fresh infection and again produces IgM antibodies.

Elevated levels of antibodies in the IgG class can be considered as a residual infection or a chronic, active disease.

Some doctors try to use the Western-Blot test to monitor the effectiveness of treatment. However, the Western-Blot test is not suitable for this. Of course, the presence of bands in the IgM class will indicate further existence of the disease, but any other result, such as “negative in both classes”, “positive or negative IgG” basically does nothing to assess the effectiveness of treatment.

A negative Western-Blot test result does not exclude Lyme disease. Repeated testing after a few months may give a positive result, as the level of antibodies in the patient’s blood changes periodically.

Unfortunately, WB can only be used to detect or confirm Lyme disease; it is not suitable for monitoring the course of treatment.

 

Main causes of discrepancies in the results of serological tests:

  • In the ELISA or Western Blot test, only the antibody titer is determined. IgM antibodies develop only in the 3rd week after the tick bite, and reach its maximal value in 4-6 weeks, and disappear after 8 weeks. If the test is done too early, the result will be negative, even though the body has been infected. Also, too early use of antibiotics may reduce the immune response of the body.
  • IgG antibodies that are formed later remain in the blood for many years. Even if the patient is already cured and the body no longer has Lyme disease, the test result can be positive.
  • A false positive test result may also occur in autoimmune diseases, bacterial infections (especially syphilis) and some viral infections, e.g. Epstein-Barr virus infection.
  • Borreli bacteria can hide in the cells of the human body and in poorly supplied tissues and mask their antigens, so it often happens that the body does not produce antibodies at all, thus the result can be false negative.
  • When antibodies bind to the bacterial antigen to fight the pathogen, the complex can no longer be identified as an antibody. They become part of the antibody / antigen complex, and modern research methods make it impossible to analyze such complexes. Therefore, if there are many antigens, the amount of antibodies may be reduced as a result of research because some of them are already associated with the given antigen to form a specific complex. It may therefore happen that patients with the highest infections have low antibody titres in their tests.
  • The antibody titer is constantly changing and may be low or absent at various stages of the disease. It is impossible to determine whether the infection is still active or has already been cured.

PCR TEST and PCR Real time

PCR (Polymerase Chain Reaction) is a test that directly detects the presence of bacteria in the test material, and in fact its DNA in blood, urine, synovial fluid and cerebrospinal fluid. PCR consists in selective amplification of a bacterial DNA fragment and is characterized by high sensitivity. This study is not related to the production of antibodies by the immune system, so they can be performed right after the tick bite. However, the genetic material of Borrelia is not easy to find in body fluids, due to the fact that it quickly escapes from them and penetrates into other tissues – it lives mainly in the internal tissue. It can be difficult to capture, so if there is no bacterial DNA in the sample, the test, despite its high sensitivity, will come out negative. Therefore, PCR tests are often false negative. The greatest chance for a positive PCR result occurs during the examination of synovial fluid and urine. A positive result is most difficult to obtain from blood or cerebrospinal fluid.

PCR Real time (PCR RT) is a more sensitive and more reliable test. The reaction takes place in a sealed vessel, which practically eliminates accidental contamination of the sample. In addition, PCR RT is able to answer the question of how much DNA chains should be found in the blood sample.

This allows you to conduct research on the possibility of monitoring Lyme treatment using this test. However, it should be emphasized that so far no test can be used to monitor the course of treatment.

LLT TEST

LTT is a blast lymphocyte transformation test. It involves the study of lymphocytic response of the immune system to Borrelia bacteria antigens. The result of the test is positive only if you have specific Borrelia T cells in the patient’s blood which indicate that the immune system was in the process of combating the pathogen when blood was drawn. In case of effective treatment, the result of the Borrelia LTT study will be negative or there will be a significant regression of the SI (stimulation factor), so the LTT test result can be information about the effectiveness of treatment.

A negative LTT test result does not exclude one hundred percent of active infection. Therefore, monitoring the clinical picture is the basis for the diagnosis of Lyme disease and enables the selection of proper therapy.

This test is considered to be very sensitive, therefore it has indications for use in case of:

  • suspicion of disseminated Lyme disease with uncertain results of serological tests
  • suspicion of Lyme disease survival
  • treatment control after antibiotic therapy
  • suspected relapse.

KKI TEST

Circulating immune complexes (KKI). The KKI test is made of serum from which, by means of special techniques, it isolates and then breaks circulating immune complexes (KKI). The next stage of the study is the detection of anti-monoclonal antibodies released from immune complexes. Both ELISA and Western-Blot techniques can be used to detect the released antibodies.

Polish research indicates that circulating immune complexes in Lyme disease are detected in up to 80% of serum samples of patients suspected of infection, who have failed to detect antibodies with standard serological techniques.

ELISPOT

Elispot is a laboratory test used to detect Borrelia infection and cellular coinfection, and provides information on the cellular activity of the immune system. With ELISpot, a single cell secreting a given protein can be detected.

Advantages of the Elispot test:

  • The study reflects the current activity of Borrelia burgdorferi and chronic Borrelia burgdorferi infections,
  • ELISpot is very sensitive and allows you to detect even one Borrelia burgdorferi cell,
  • ELISpot is between 20 and 200 times more sensitive compared to the conventional ELISA method,
  • ELISpot shows similar sensitivity as RT-PCR (Real Time PCR),
  • ELISpot is helpful when monitoring therapy,
  • ELISpot should usually be negative 4 to 8 weeks after successful therapy.

 

LYMESPOT

LymeSpot, a modified EliSpot study, provides information on the activity of infection and / or inflammation. This test allows distinguishing whether we are dealing with an active or passive infection. Thanks to this research, we can determine whether autoimmune processes, inflammation or patient infection predominate. Compared with the Elispot test, which is based on the production of interferon, Lymespot also determines cytokine IL-2.

CD57 TEST

The CD57 test is used in the diagnosis and therapy of Lyme disease in order to evaluate the effectiveness of Lyme disease treatment in patients undergoing long-term antibiotic therapy (in patients treated with the US ILADS system). Determining the number of CD57 NK can also be useful in distinguishing Lyme disease from other chronic diseases that give similar symptoms.

Active, chronic Lyme disease suppresses the immune system and causes loss of CD57 natural killer subpopulations that are a natural defense of the human body. It is believed that patients with low CD57 NK cells feel worse and are more likely to have neurological Lyme disease. In some patients with Lyme disease, the degree of CD57 lymphocyte depression may be used to show how active Lyme disease is and whether a relapse may occur after treatment. This study can be used as a simple screening test, because it is now believed that only Borrelia reduces the CD57 NK cell count. Determining the number of NK CD57 may also be useful in distinguishing Lyme disease from other chronic diseases that give similar symptoms, such as rheumatoid arthritis, multiple sclerosis or visceral lupus, since NK diseases are at normal levels in these diseases. Thus, a patient with a high number of CD57 NK lymphocytes is likely to have a different disease entity or co-infection.

Course of the study and interpretation of results

Fluorescently labeled antibodies that bind to markers on the surface of cells are added to the patient’s blood. The cells are separated due to their size, the presence of graininess and the type of glow. On this basis, they are grouped and segregated into appropriate populations.

The results include information on sub-populations of CD3-specific lymphocytes: CD3-CD16 + 56 NK cells and CD3-CD16 + 56 / CD57 + NK cells. In many clinical cases, the values obtained reflect the degree of infection. The CD57 titre remains low until the infection is controlled, and the number of NK cells increases.