Methodology: generally measures the immune response to several proteins from a selected Borrelia strain, quantifying them collectively for all proteins. It examines the amount of specific antibodies in the serum produced by blood clotting by detecting their specific binding to proteins (antigens) pre-applied to a surface. The numerical result is not always published. Often, only an IgM and an IgG positive/negative result is reported. In the past, proteins obtained by breaking down Borrelia bacteria were used, but today the use of a mixture of proteins produced artificially on the basis of precise genetics (monoclonal antigens) has become predominant. However, due to the older “fragmentation” method, the medical community still believes in “non-specific binding”, i.e. the idea of “false positive” ELISA results, which is no longer typical today.
What does it show? It measures the level of immune response to Borrelia bacteria. Due to the methodology, it can only detect antibodies that are free in the blood, so if their level is low or the antibodies are bound by large amounts of Borrelia, the result will be negative. Fluctuations in antibodies are a known phenomenon, so different tests may show different results at different times.
What is it suitable for? It measures the recent (IgM) and previous (IgG) immune response to Borrelia infection. In cases where IgM is positive (in approximately 30-50% of patients), the recent immune response indicates an active infection. IgM false positives are now rare, so a positive result definitely indicates an IgM reaction to Borrelia, but not always to a recent infection, rather to a newly developed variant of a previous infection. A positive IgG result may indicate a previous infection, but in chronic cases it may also occur in active infection even if the IgM reaction is negative. The presence of an IgG reaction after successful treatment can help maintain good health, so in treated patients a positive result may even be a good sign, especially if there was no IgG reaction before treatment.
Lyme rapid tests available in pharmacies (Beright, Citest, Alltest, etc.)
Methodology: Indirect test, visually indicates a reaction on a test strip (colour change) that shows the presence of antibodies (immune reaction) against proteins from a selected strain of Borrelia. In principle, it is similar to ELISA tests. In Hungary, combined tests based on the detection of IgM and IgG antibodies are mostly available, and only positive/negative results can be read from the test strip.
What does it show? The presence of an immune reaction against Borrelia bacteria. Due to the methodology, it can only detect antibodies that are free in the blood, so if their levels are low or the antibodies are bound by large amounts of Borrelia, the result will be negative. Fluctuations in antibodies are a known phenomenon, so different tests may show different results at different times.
What is it suitable for? It has a much lower sensitivity than ELISA tests that can be performed in the laboratory, so according to clinical studies, it identifies the disease in 10-20 per cent of cases. Therefore, it is not suitable for either confirming or ruling out the disease, and its use is not recommended!
Western blot, Immunoblot, Virachip, blot, Vibrant serology, etc.
Methodology: Indirect test, usually detects immune reactions against different proteins of several selected Borrelia strains, with a separate reaction occurring for each protein at the specific location of the test strip or multiplex. Each reaction has a separate weighting, and the sum of these weights gives the final result. If the IgM or IgG scores are above the threshold, the result is positive. The details of the individual reactions or the scores are not always published. Often, only an IgM and an IgG positive/negative result is reported. Regardless of their name, modern tests use proteins produced in the laboratory based on specific gene sequences as antigens, so the specificity of the reactions is high and “non-specific binding”, i.e. “false positive” results, are rare. The kits are designed for 100% specificity, but in many cases, negative results are obtained even in infected patients.
What does it show? The presence of an immune response to the Borrelia bacterium. Due to the methodology, it can only detect antibodies that are free in the blood, so if their levels are low or the antibodies are bound by large amounts of Borrelia, the result will be negative. Fluctuations in antibodies are a known phenomenon, so different tests may show different results at different times.
When detailed results are published, the extent of the IgM/IgG reaction can be deduced from the number of proteins against which a reaction is visible. In the event of extensive destruction of the pathogens, a reaction may develop against several proteins. In some cases, the Borrelia strain causing the infection can also be identified, allowing for personalised treatment.
It is expressly contraindicated and a professional error to indicate reactions to individual proteins (antigens, e.g. p41, OspC, VlsE) with the words positive/negative, as this may give the impression that the entire test result is positive. Often, the summary of IgM and IgG results is missing, and only the results of individual antigen-antibody reactions are reported, which is a serious professional error. Most tests only provide separate IgM and IgG results and do not provide combined results for the entire test.
What is it suitable for? It measures the immune response to Borrelia against recent (IgM) and previous (IgG) infections. In cases where IgM is positive (in approximately 30-45% of patients), the recent immune response indicates an active infection. IgM false positives are now rare, so a positive result indicates that an IgM reaction is definitely present, but not always against a recent infection, but against a newly developed variant of a previously contracted infection. A positive IgG result may indicate a previous infection, but in chronic cases it may also occur in active infections even when the IgM reaction is negative. The presence of an IgG reaction after successful treatment can help maintain good health, so in treated patients a positive result may even be a good sign, especially if there was no reaction before treatment. An increase in the breadth of the reaction may be another important indicator of successful treatment.
Lymphocyte Transformation Test (LTT), Elispot, Lymphocyte Activation Test
Methodology: An indirect test based on the principle that in one type of white blood cell, lymphocytes, activation and cell division only occur when they encounter a specific, i.e. precisely “matching” protein (their specific antigen). A positive LTT reaction indicates the presence of antigen-specific lymphocytes (memory cells) in the patient’s blood, which were most likely produced upon encountering the antigen being tested (e.g. a protein). In the case of Lyme disease, the test is based on the activation and/or proliferation of memory T or B cells specific to Borrelia proteins after the patient’s peripheral mononuclear cells (PMBCs) have been incubated together with Borrelia culture and/or proteins derived from Borrelia in a cell culture. The Elispot method specifically incubates T cells with multiple antigens and Borrelia from culture and examines their activation. Activation is indicated by the production of certain messenger substances (cytokines or chemokines), which are detected after the cells are removed using an ELISA test based on the specific molecule. A newer version of this is Fluorspot, which directly detects the messenger substances produced instead of using the ELISA method. The process is multi-step: first, an immune response based on T cells must already be established and maintained in the body, viable T cells must remain in the sample during processing, they must respond to the antigens presented to them (e.g. even in the case of a new variant, but only respond to relevant pathogens), and the production of messenger substances must be detectable.
What does it show? A successful test shows how many more T cells or messenger molecules are produced when incubated with specific proteins than without proteins. In a positive test, the amount produced in the induced sample is usually 2 or 3 times that of the non-induced sample, which is indicated by the “Stimulation Index”, SI.
What is it used for? It shows with a high degree of probability whether there is a T-cell immune response to Borrelia proteins, which is different from the immune response based on specific antibodies. If the reaction is positive, there is a good chance that this immune response will aid in the healing process. However, as shown in summary publications (meta-analyses) published in the last decade, the Elispot method is not suitable for confirming the actual presence of infection in a patient and is therefore not recommended for diagnostic purposes. The rate of false positive and false negative results is too high for it to be used on its own to detect an existing infection, but it does provide important information about the state of the immune system.
PCR, Polymerase Chain Reaction, genetic diagnostics, NGS (Next Generation Sequencing), Full Genome Sequencing (full genome sequencing)
Methodology: A direct test that detects the genome, i.e. the genetic material, DNA, of pathogens present in blood or tissue samples (e.g. joint fluid, cerebrospinal fluid, skin biopsy). The PCR test attempts to amplify the pathogen DNA from the sample using starting sequences containing certain parts of the DNA of the pathogen being sought. If the pathogen DNA is not present, the starting sequence (primer) will not bind to it and thus the amplification of the sought-after genome fragment will not start. The genome fragment being sought must be general enough to find all pathogen variants, but specific enough that it is not present in the DNA of another pathogen or the host organism. The test cannot eliminate close matches, and numerous false reactions may occur.
Recently, it has become possible to determine the entire genome of a pathogen, which is then compared to the genomes of known pathogens, but this is mainly used for scientific purposes (NGS, FGS). Genetic tests can have many sources of error: failure to isolate DNA, failure to remove other contaminating molecules from the sample, failure to find the desired genetic variant of the pathogen in the sample, false binding due to inappropriate reaction temperature, contamination of the sample by a few copies of DNA in the laboratory or in another sample.
What does it show? It is assumed that the presence of the pathogen’s genetic material clearly indicates the presence of the pathogen, as its DNA is likely to be eliminated from the body within weeks or months after the pathogen’s destruction. The detection limit of the tests is very low, so they can usually detect the presence of 1-5 pathogens per millilitre. This also means that a randomly present pathogen can be detected, even though it does not cause disease. A well-designed test (proper isolation, primers, temperature, time, etc.) clearly confirms the presence of a given genome sequence in the sample. This can confirm that the diagnosis made by other tests is correct, i.e. that the specific pathogen being sought is present.
What is it suitable for? Its sensitivity is less than 10%. A positive test confirms the random or pathogenic presence of the bacterium – the two cannot be distinguished, so other tests with diagnostic value must be performed. In cases where the test is negative, it is highly likely that the pathogen with the DNA variant sought is not present. However, given the high variability of Borrelia burgdorferi, the bacterium that causes Lyme disease, a negative result does not in any way rule out the presence of another variant of the pathogen, as the pathogen significantly changes its genome in 3-4 week cycles (development of new generations). Its diagnostic value is low, which is due to genetic variability and not to the “disappearance” of the pathogen.
Microscopic tests from finger prick blood, a drop of blood, untreated (native) venous blood, Enderlein method
Methodology: The drop of blood obtained by pricking the fingertip is placed directly on a microscope slide and examined immediately or after incubation at a higher temperature using a dark-field or phase-contrast microscope. In the case of venous blood, the native untreated blood is usually centrifuged and certain parts or fractions of it, e.g. plasma, are examined under a microscope. Staining procedures (e.g. Giemsa, immunofluorescence) are also used in rare cases. The Enderlein test was originally developed to observe the decomposition of blood during incubation at 30°C for several hours, and the rate of decomposition the shapes are characteristic of the patient’s healthy or sick state – thus it is not suitable for detecting specific pathogens, as the decomposition products are not pathogens but artificial products.
What does it show? If the sample is not taken in a properly designed liquid, the formation of artificial products begins immediately. These include fine filaments formed from platelets and red blood cells, which appear to be alive due to the flow of fluid. Not only do they obscure pathogens that are otherwise very rarely visible, but they also give rise to false diagnoses. However, the confirmed presence of a single pathogen does not confirm the level of infection or the presence of clinical symptoms of the disease.
What is it suitable for? Specific staining methods (immunofluorescence) are suitable for confirming the presence of a specific pathogen, but this can only be confirmatory in nature alongside a test of diagnostic value (verified in clinical research). The use of inadequately prepared, single-drop blood-based microscopic methods is not permitted and is professionally questionable. The images and videos most likely show artificial products. In the case of Lyme disease and its co-infections, neither a positive nor a negative result is suitable for drawing any conclusions or making a diagnosis. The latest studies confirm that these native blood tests lead to misdiagnosis.
DualDur DD-LYME 4.0 automatic dark field microscopy (CE IVD certified, clinically validated)
Methodology: Blood samples or other body fluid samples (joint fluid, cerebrospinal fluid) collected using a special blood collection (cell technology) fluid are stored in a cool place and prepared using a special method. The sample is examined by an automatic microscope for live and characteristically motile spirochete bacteria (Borrelia) concentrated more than a hundred times. Artificial intelligence helps to select the appropriate shapes, calculate statistics, and simplifies human control. It automatically suggests diagnostic categories.
What does it show? The special fluid prevents artefacts from forming from the very first second, and the preparation concentrates the smaller bacteria present in the blood plasma, allowing the quantity, density and other qualitative characteristics of the Borrelia bacteria to be determined. The presence of Borrelia above a certain concentration clearly indicates the presence of the disease. The test measures the current level of infection and is not dependent on the development of an immune response, ongoing treatment or the stage of the disease.
According to the latest publications, once Borrelia burgdorferi enters the body, it is continuously present in the blood, but always with changing genetic variants that cannot be detected by other tests. PCR tests cannot detect variants with altered genetics after the initial stage, which is why many studies in the past concluded that Borrelia disappears from the blood (even though only the PCR test becomes negative). Borrelia benefits from its continuous presence in the blood, as this ensures its spread by ticks. The blood of an infected person is a safe environment for Borrelia, as the immune response that has already developed has no effect on new generations of Borrelia, and they can escape from phagocytes due to their greater speed. However, the shape and movement of Borrelia are constant, so DualDur automated microscopy can detect the pathogen.
DualDur immunofluorescence microscopy specifically stains Borrelia burgdorferi bacteria present in the prepared sample with antibodies that only bind to Borrelia burgdorferi bacteria, causing them to glow blue-green, indicating the presence of the pathogen.
What is it suitable for? The test not only shows the level of Borrelia infection (++, +, +/-, -), but also establishes a diagnostically correct positive or negative category (P, N). This is based on the largest clinical study conducted in Europe in the field of Lyme disease, which confirmed that DualDur automated microscopy was by far the most sensitive of the tests and also reliably specific. The probability of a positive result in a patient who actually has Lyme disease is 96% (positive predictive value, PPV).
Bacterial culture
Methodology: Blood or other tissue samples (primarily skin biopsies from the area of erythema migrans) are placed in a special liquid culture medium. Under appropriate conditions, they are cultured for several weeks at around 33°C in an oxygen-poor environment, with nutrients being replenished during this time. Finally, the cultured pathogen is identified using a microscope, immunofluorescence microscopy or genetic testing (PCR).
What does it show? The presence of Borrelia burgdorferi in the tissue sample. Unfortunately, it is very complicated to perform, there is a high possibility of error, and the chance of a positive result in a patient with Lyme disease is between 10 and 20%. However, a positive result is sufficiently reliable evidence that the pathogen is present – but it does not confirm the patient’s clinical condition (sick or healthy). A negative result does not rule out infection.
What is it suitable for? It is only used in experimental settings and is not suitable for diagnosis due to its complexity and low sensitivity.
