T-cell response in Lyme disease (TRIL-study)

Lyme is an emerging tick borne disease in the Netherlands with a high impact on
public health caused by Borrelia spp. infected ticks of the Ixodes ricinus
complex and can transmit the Borrelia spp. to humans. In Europe, Borrelia
afzelii and Borrelia garinii are predominant, but B. burgdorferi sensu stricto
can also be encountered. In North America, only infections with B. burgdorferi
sensu stricto have been observed. The most common manifestation of Lyme is
erythema migrans (EM). Other manifestations include radiculitis and arthritis
although many people will clear the infection asymptomatically.

Diagnosis relies on the detection of antibodies against Borrelia spp..
Unfortunately these antibodies can persist a lifetime and therefore do not
discriminate between active disease or cleared infection. Seroprevalence in the
Netherlands has been reported to be as high as 15 % in risk groups such as
owners of hunting dogs and 9% in controls (blood donors), 97% were
asymptomatically infected (Nohlmans et al; 1991). No serological tests are
available for differentiating between active disease or past infection.

The incidence of physician consults for tick bites and erythema migrans has
tripled in the period between 1994 and 2009. It is very likely that the
prevalence of complications of Lyme disease has also increased significantly.
Due to the clinical heterogeneity Lyme is often considered by a clinician, this
combined with the high background seropositivity results in that patients are
often unnecessary treated. A diagnostic test which could differentiate between
active disease and past infection would be very helpful for accurate diagnosis
of active Lyme disease, but could also prevent unnecessary use of antibiotics.

T-cell mediated cellular immunity has a pivotal role in dealing with Borrelia
spp. infection and subsequent clearance or control of the bacterium in the
host. Using ELISpot techniques Borrelia specific circulating activated T-cells
can be measured in the peripheral blood. After clearance of infection activated
T-cells can disappear from the blood since active immune surveillance is no
longer required. No studies are performed on determining T-cell responses in
Lyme which may aid the clinician in accurately diagnosing the outcome of
especially advanced stages of Lyme infection and might be useful in guiding
therapeutic interventions. Using ELISpot techniques, it could be possible to
differentiate patients with active Lyme from patients with a serological scar
after Borrelia infection.

Main objective:
To determine the T cell response to Borrelia specific antigens using an ELISpot
interferon gamma release assay in patients with different clinical
presentations of Lyme and healthy volunteers.

Sub-question 1: Questionnaires
To analyze the answers on the questions of the Lyme-specific questionnaire and
the health-related questionnaire (RAND-36) to study the occurrence of tick
bites, erythema migrans, antibiotic treatment for Lyme, complaints at the start
of the study or during possible earlier episodes of Lyme and to investigate
the quality of life of the study participants.

Sub-question 2: Specificity of the Borrelia ELISpot assay
-) To determine the specificity of the Borrelia ELISpot interferon gamma
release assay by including patients with active disease known to cross-react
with Borrelia such as syphilis, leptospirosis, Epstein-Barr virus (EBV),
cytomegalovirus (CMV), Helicobacter pylori, Mycoplasma pneumonia, rheumatoid
arthritis (RA) or the presence of antinuclear antibodies (ANA) (=study group 5).
-) To test the presence of antibodies against closely related micro organisms
such as leptospirosis, Treponema pallidum and Helicobacter pylori in
participants who have Borrelia specific T cells in their blood (positive
Borrelia ELISpot result). As controls, age and sexmatched study subjects will
be selected who do not have Borrelia specific T cells in their blood (negative
Borrelia ELISpot result).

Sub-question 3: Ganglioside antibodies versus complaints
To determine the presence of antiganglioside antibodies in all study subjects
to see whether an association can be found between those antibodies and the
persistence of complaints in treated Lyme patients.

Sub-question 4: Other cytokines and/or immune cells
To determine whether other factors (cytokines and/or immune cells) could
possibly play a role in the development of Lyme disease and and/or persisting
complaints in order to improve the Borrelia ELISpot technique.

Sub-question 5: Predisposing factors for Lyme neuroborreliosis
For the inclusion of Lyme neuroborreliosis patients the electronic patient
records showed that not all presumed Lyme neuroborreliosis infections were
confirmed and some patients were eventually diagnosed with other diseases such
as neurosyphilis, Guillain-Barre, or had a hernia. Only 11% of all
neuroborreliosis requests at the microbiology lab resulted in a Lyme
neuroborreliosis diagnosis. Since lumbar punctures are very invasive, we want
to investigate whether we can increase the likelihood of a positive Lyme
neuroborreliosis diagnosis by retrospectively analyzing the electronic patient
records of all patients who have had a lumbar puncture for the diagnosis of an
infectious disease. Patient history (anamnesis), supplementary examination by a
physician and other laboratory test outcomes will be analyzed in order to find
predisposing factors. In a prospective study those factors will be tested in
order to increase the likelihood of a positive test outcome and decrease the
number of unnecessary lumbar punctures.

Sub-question 6: Genetic factors vs symptoms
To detect polymorphisms within the Borrelia bacterium and/or human cells (such
as Toll-like receptor polymorphisms, HLA-polymorphisms, etc) which could be
related to persisting symptoms after antibiotic treatment for Lyme borreliosis.

Sub-question 7: Validation of other tests and/or identification of new targets
a) To validate tests other than the Borrelia ELISpot, which detect a response
to the pathogen.
b) To identify new targets for diagnostics and/or immunotherapy for Lyme
disease. These new targets could include e.g. particular DNA sequences,
proteins or lipids that can be used in the development of new diagnostic or
therapeutic tools for Lyme disease.

Descriptive study, evaluation of a new diagnostic test.

All patients with active Lyme disease such as neuroborreliosis or Lyme
arthritis who undergo treatment at the Diakonessen hospital in Utrecht or St
Antonius hospital Nieuwegein/ Leidsche Rijn are elligable. Those patients will
undergo full history and physical examination.

All participants will fill out a standard questionnaire measuring general
health (RAND-36) and a Lyme specific questionaire and will be subjected to a
single blood sampling. No specific risks are associated with any aspect of the
study.

All participants selected for determining the specificity of the Borrelia
ELISpot by testing their blood on the presence of antibodies agaist
leptospirosis, Treponema pallidum and Helicobacter pylori have to sign an
additional informed consent. Positive test results will be reported back to the
participants, which can have an impact on them. They will therefore be offered
more information and/or a consultation with a clinician.

This will be offered in case of:
-) positive Lyme serology in case of healthy volunteers
-) positive Borrelia ELISpot results in case of healthy volunteers and treated
Lyme patients
-) positive serology in case of leptospirosis, Treponema pallidum and
Helicobacter pylori for the selected participants