When are IIFT results declared?

Diagnostic pitfalls

Lyme disease is the most common arthropod-borne disease in the northern hemisphere. Often, due to unspecific symptoms or unclear laboratory findings, there are doubts as to whether there is a disease that requires treatment. This article is intended to help correctly interpret the findings obtained in the laboratory.

Lyme borreliosis is caused by the pathogen Borrelia (B.) burgdorferi. These Borrelia are now called B. burgdorferi sensu lato (see left) - complex and are differentiated from the relapsing fever Borrelia [1].

The symptoms decide

It cannot be stressed enough that Lyme disease in all its disease stages and manifestations is initially a clinical (suspected) diagnosis. Laboratory diagnostics help, depending on the stage of the disease, to confirm or refute this suspicion. It should be noted that the sensitivity of the available methods in the early stages of the disease (e.g. erythema migrans, Borrelia lymphocytoma and acute neuroborreliosis) is limited and a negative test therefore does not rule out Lyme disease.

No test without specific suspicion!

In order to be able to better evaluate the informative value of Borrelia tests, it is necessary to learn something about the performance data of a test. The meaningfulness is usually given with the parameters sensitivity and specificity. However, this is often a bit misleading in practice, hence a calculation example:

Assuming that a test has a sensitivity of 95% and a specificity of 95% (Borrelia tests are mostly significantly worse): The test would now be able to detect a disease with a prevalence in the population of 100/100,000 inhabitants (the prevalence of Lyme -Borreliosis is in this order of magnitude). 1,000 people are now being tested at random. With a sensitivity of 95% he would surely find the one sick person. However, the specificity of 95% will result in 50 people being tested “false positive”. The result is 51 positive test results, of which only one is really sick. In this example, the positive forecast value is just under 2%!

For this reason, it makes no sense to simply do an examination for Borrelia. The probability of false positives is far too high. Only when symptoms and anamnesis indicate a certain likelihood of illness does it make sense to carry out such a test.

serology

Serology is still the main pillar of the diagnosis of Borrelia infections. Today, this is carried out as a two-stage diagnosis, as recommended by specialist societies [2].

In a first stage, IgG (immunoglobulin G) and IgM are now determined by means of enzyme immunoassay (EIA), more rarely by means of indirect immunofluorescence test (IFT). If the result is borderline or positive, an immunoblot is carried out in a second step.

Enzyme Immunoassay (EIA)

The enzyme immunoassay is most commonly used in Borrelia diagnostics. In contrast to the IFT, it can be completely automated and read objectively.

Since Borrelia diagnostics are not standardized, tests differ considerably in terms of the antigen used, the unit and the normal range. It is therefore difficult to compare the results of different tests from different laboratories. Today, EIAs are coated either with lysate antigens (obtained by culturing Borrelia) or with recombinant antigens.

Immunoblots (IB)

With the immunoblots (IB), the individual Borrelia antigens are present separately on a strip. A distinction is made between tests with electrophoretically separated antigens ("Western blots") and so-called line assays or strip tests. In the strip tests, the antigens are sprayed on at certain points with a type of inkjet printer. Strip tests have several advantages. Reading is much easier, the arrangement of the bands can be chosen freely.

In contrast to the EIA, which measures the amount of antibodies, the immunoblot shows which antigens the immune response is directed against. In the early phase of the infection, antibodies are usually only formed against a few antigens (“early bands”), while if the immune response lasts longer, additional bands are added and so-called “late bands” appear (Fig. 1 and 2). A distinction can be made between “specific” and “less specific” antibodies. For these reasons, it is necessary for the laboratory to indicate not only the test result, but also the detected bands (possibly with intensity) on the findings.

Importance of individual antigens

As already mentioned, bands can be classified according to their specificity and according to their appearance during the course of the infection. IgM antibodies against OspC (outer surface protein C) are formed early. They are also the most important antibodies in the early phase and must be well recognized by all test systems. Some tests therefore contain the OspC of several strains. VlsE is also an early and specific marker, especially in IgG test systems. This is why this antigen is included in virtually all current tests. Fig. 1 shows the blot results of five patients with acute Lyme disease.

With increasing duration of the illness (weeks to months) further antibodies are added. Typical “late antibodies” are directed against p100 and DbpA (decorin-binding protein A). Antibodies against p58, p60 and OspA are also only formed in the course of the infection. Fig. 2 shows blot strips from patients with a longer-lasting immune response. The immune response here is directed against a large number of antigens.

Today, blot strips can be objectively read and documented with blot readers. However, the immune response of individual patients is very different and the result is not always as clear as in the examples shown.

Pathogen detection

The pathogen detection is reserved for specific questions in the case of Lyme disease. In today's laboratory diagnostics, practically only one nucleic acid amplifying technique (NAT) is used. Usually it is a PCR (polymerase chain reaction). In addition to NAT, cultivation is also available as the “gold standard”. Only with this method can the proof be provided that the pathogens involved are living, capable of reproduction and not Borrelia remains. However, cultivation is complex, takes a long time and is therefore not a routine method.

Unfortunately, both cultivation and PCR have limited sensitivity, which can vary depending on the material. So is z. For example, a PCR from the blood with routine PCR techniques does not make sense, as does the examination of urine [3]. Suitable examination materials are a skin biopsy (e.g. from the edge of an erythema migrans or acrodermatitis chronica atrophicans), synovial fluid or, better, a synovial biopsy and liquor. The sensitivities are between 20 and 80% [4].

Investigation of ticks

For a number of years it has been possible to examine distant ticks for an infection with Borrelia. Various laboratories offer this examination. The usefulness of this examination is controversial, since not every tick is noticed and therefore this procedure does not reliably protect against Lyme disease. In addition, not every bite from a positive tick leads to an infection or to a manifest disease.

Nevertheless, this investigation can be used for risk assessment. In the USA it has been shown that short-term antibiotic prophylaxis significantly reduces the incidence of infection [5]. Examining the tick can significantly reduce the number of people who receive prophylactic treatment. Even if no prophylaxis is carried out, knowledge of an infected tick can influence the further course of action with regard to infection control. This examination of ticks is carried out using NAT. The laboratory must be able to guarantee that the relevant subspecies are recognized with sufficient sensitivity.

Evaluation of the results

In general medical practice, one will mainly be confronted with serological findings. It is important to evaluate this in conjunction with the clinic and anamnesis. The following statements should help the reader to evaluate:

  1. In the early phase of infection, a lack of antibodies does not rule out an infection; with early therapy, antibodies may not be formed. If antibodies are formed, they often belong exclusively to the IgM class.
  2. In cases of acute Lyme disease, isolated IgM antibodies can persist for a long time. If you come across these antibodies later, this can lead to the misdiagnosis of "acute Lyme disease".
  3. A serological follow-up, e.g. B. after three weeks, can help to confirm the diagnosis if the initial findings are unclear. A therapy success control after acute infection is not possible by means of serology.
  4. If IgG antibodies have formed after an acute infection, this does not initially indicate a therapy failure. The few treatment failures are more likely to be found in patients with IgG seroconversion.
  5. The detection of Borrelia-specific antibodies without corresponding symptoms does not constitute an indication for therapy.
  6. A lack of IgG antibodies makes a chronic infection unlikely.
  7. Persistent IgM antibodies without simultaneously detectable IgG antibodies also do not indicate a chronic infection.
  8. At V. a. chronic Lyme disease and detected IgG antibodies, IgM antibodies should not be misunderstood as activity markers. Chronic borreliosis is also possible without IgM antibodies.
  9. In the case of positive IgG antibodies, follow-up controls are only useful at long intervals. Parallel testing of both samples with the same test is optimal.

If the constellation is unclear, the result should be discussed with a microbiologist or laboratory doctor experienced in Borrelia diagnostics. Unfortunately, there are still cases in which a patient is not treated because of incorrectly interpreted laboratory values. The reverse case, namely overtreatment, is more common, however.


1. Stanek G, Reiter M (2011) The expanding Lyme Borrelia complex - clinical significance of genomic species? Clin Microbiol Infect. 17: 487-93
2. Wilske B, Fingerle V, Schulte-Spechtel U. (2007) Microbiological and serological diagnosis of Lyme borreliosis. FEMS Immunol Med Microbiol. 49: 13-21.
3. Wilske B, Zöller L, Brade V, Eiffert H, Göbel UB, Stanek G, and Pfister HW (2000) MIQ 12, Lyme borreliosis. In quality standards in microbiological-infectiological diagnostics. H. Mauch and R. Lütticken, eds. Munich, Germany, Urban & Fischer Verlag, pp. 1-59
4. Aguero-Rosenfeld ME, Wang G, Schwartz I, Wormser GP (2005) Diagnosis of lyme borreliosis. Clin Microbiol Rev 18: 484-509
5. Nadelman RB, Nowakowski J, Fish D, Falco RC, Freeman K, McKenna D, Welch P, Marcus R, Aguero-Rosenfeld ME, Dennis DT, Wormser GP; Tick ​​Bite Study Group. (2001) Prophylaxis with single-dose doxycycline for the prevention of Lyme disease after an Ixodes scapularis tick bite. N Engl J Med. 345: 79-84.




Dr. med. Friedemann Tewald
Specialist in laboratory medicine