LTT pathogen testing

Since about 2004, the method of pathogen-specific lymphocyte transformation tests has been used in specialised laboratories to assess the current T cell reactivity to pathogens in chronic infections.
The scope of application of the test includes above all obligate or facultative intracellular pathogens, such as Borrelia, Yersinia and Chlamydia trachomatis / pneumoniae, but also yeasts such as Candida albicans. With these pathogens, the symptoms of chronic infection and/or chronic immune activation are almost always unspecific, i.e., the diagnosis is primarily based on lab test results.
However, the LTT is rarely indicated to diagnose fresh infections as in such cases the symptoms and standard lab tests are sufficient (e.g. IgM antibody response, direct detection of the pathogen).

To detect a past infection caused by the named (primarily intracellularly) persisting pathogens, the pathogen-specific antibodies in the blood are measured (serological diagnosis using ELISA screening tests or immunoblotting). Direct detection of pathogens using microbiological culture techniques or DNA analysis (e.g. PCR) is of little use, at least from blood samples, because these pathogens do not usually persist in the blood, but in tissue. Tissue samples are typically not available for testing, although there are exceptions (e.g. skin biopsies in borreliosis).

• Serology can be false negative
  Serological testing (antibody measurements using ELISA or immunoblotting) is not without issues. Specific antibodies only appear 2 to 8 weeks, or even later, after an infection. Depending on the type of pathogen, 5-15% of cases can be expected to remain seronegative (primarily in Lyme borreliosis and yersiniosis). In some cases, the poor quality of serological tests is the cause of seronegativity. In Germany, for example, more than 10 different manufacturers of Borrelia ELISA tests are on the market. No standardisation has been established between these companies. As each manufacturer works with its own target antigens and techniques, serological results frequently differ when tests are performed in different laboratories.
• A positive serological result provides no information about the activity of the disease
  It is important to understand that a positive serological result does not in any way indicate an active infection requiring treatment. A positive titre (often IgG, at times also IgM) frequently results from a (possibly unnoticed) earlier infection and does not prove a currently active disease associated with the respective pathogenic microorganism. Pathogen-specific antibodies may persist for years even after the infection has cleared, e.g. after antibiotic treatment. In bacterial serologies, this may apply to IgM as well.
• Conclusion:
  Serological methods can at most provide evidence of past exposure to potentially persisting bacteria. However, no inference can be made as to the present level of activity of the persistent infectious disease. Thus, a pathogen-associated disease can neither be proven nor excluded serologically. In clinical practice, patients all too often undergo, at times prolonged, antibiotic treatment, especially for Borrelia and Yersinia, even though the positive serological result only shows an immune scar. This leads to unnecessary costs.
• The LTT can partially fill this gap
  It has been shown that this gap in serological diagnostic testing can be largely filled with cellular techniques, such as the LTT. The LTT is only positive when an increased number of pathogen-activated lymphocytes (so-called TH1 effector cells) are circulating in the blood. For bacteria with intracellular persistance, this is only the case when at the time of blood collection the immune system is actively attacks the pathogen. It is known that the number of pathogen-specific T cells in the blood decreases significantly after antibiotic treatment. This is easily recognizable when looking at the falling stimulation index. Most experiences with cellular analyses are related to Borreliosis testing. For this, the scientific literature dates back to the 1980ies (see here).
• The validation of the LTT to Borrelia antigens takes several years
  The use of the LTT in pathogen testing, especially to detect T cell reactivity to bacterial antigens, is a challenge for every cell culture laboratory. The quality – especially the specificity – of the method depends crucially on the selection of suitable Borrelia antigens. These must contain all protein structures of relevance for the T-cell immune response on the one hand, but on the other hand, no unspecific proteins capable of inducing a positive reaction in non-infected persons must be present. Even though the selection of antigens during preparation phase is difficult (there are few biotechnology companies in Germany dedicated to the expression of this kind of antigens), testing their suitability is actually quite easy.

This time-consuming validation of the method was undertaken by the Institut für Medizinische Diagnostik between 2002 and 2005. During this time a cell culture medium was developed to which, besides IFN-alpha, polymyxin B was added to suppress any unspecific activation. The validation studies for the LTT used in the Institut für Medizinische Diagnostik Berlin were submitted for publication to the Open Neurology Journal in 2011 and published in 2012 following a stringent peer review process.
The study shows that the LTT, if correctly performed and suitable test antigens are used, has a sensitivity of 89.4% and a specificity of even 98.7% in sero-negative patients. Especially the demonstrated high specificity invalidates the scepticism towards the method which stems from older studies and is based on the misconception that the LTT generates to many false positive results.