Lyme borreliosis (LB) is caused by spirochetes that are part of the Borrelia burgdorferi sensu lato (s.l.) complex group. The most prevalent manifestation of LB is characterized by a red, migrating... Show moreLyme borreliosis (LB) is caused by spirochetes that are part of the Borrelia burgdorferi sensu lato (s.l.) complex group. The most prevalent manifestation of LB is characterized by a red, migrating skin lesion, also known as erythema migrans. If left untreated, then B. burgdorferi s.l. can disseminate through the body and infect other body parts such as the joints, the nervous system (Lyme neuroborreliosis (LNB)), the heart and/or other parts of the skin, although for the majority of cases LB resolves without treatment. The diagnosis of LB is based on the presence of clinical symptoms and, for most Lyme manifestations, should be supported by laboratory tests. The most widely used laboratory test is based on the detection of Borrelia-specific antibodies. The utility of antibody detection to support the diagnosis of LB, however, is hampered by a low sensitivity of antibody detection early in the infection, and the persistence of antibodies after a cleared infection. The research conducted in this thesis focused on the humoral and the cellular immune response to investigate whether LB diagnostics can be improved. As clear case-definitions are available for active LNB, well-defined patients with LNB were used as a proxy for patients with active LB.The research in this thesis has provided more insight into the added value and the pitfalls of laboratory tests for the diagnosis of LNB. It shows that the interferon-gamma enzyme-linked immunospot assay cannot be used for the diagnosis of LNB. Furthermore, it demonstrates the added value of elevated levels of the B-cell chemokine (C-X-C motif) ligand 13 in the cerebrospinal fluid for the diagnosis of LNB and that more research is necessary to investigate if an international reference standard can be established. Additionally, the research in this thesis underlines that a thorough validation of serological tests is important before its use in diagnostics and it illustrates that antibiotic treatment of an infection with B. burgdorferi s.l. is associated with discordant serological test results. The most important finding of the research conducted in this thesis; however, is the proposal of a diagnostic algorithm for LNB diagnostics. Such an algorithm should include tests that consider both the humoral as well as the cellular immune response as both may contribute to the diagnosis of LNB. By covering different aspects of the immune system, a concrete and feasible tool is provided that can better discriminate between an active and a previous infection with B. burgdorferi s.l., which and this will improve LNB diagnostics. Show less
Gorkom, T. van; Voet, W.; Arkel, G.H.J. van; Heron, M.; Hoeve-Bakker, B.J.A.; Thijsen, S.F.T.; Kremer, K. 2022
The diagnosis of LNB is established by clinical symptoms, pleocytosis, and proof of intrathecal synthesis of Borrelia-specific antibodies. Laboratory diagnosis of LNB is challenging, and validated... Show moreThe diagnosis of LNB is established by clinical symptoms, pleocytosis, and proof of intrathecal synthesis of Borrelia-specific antibodies. Laboratory diagnosis of LNB is challenging, and validated diagnostic algorithms are lacking.Laboratory diagnosis of Lyme neuroborreliosis (LNB) is challenging, and validated diagnostic algorithms are lacking. Therefore, this retrospective cross-sectional study aimed to compare the diagnostic performance of seven commercial antibody assays for LNB diagnosis. Random forest (RF) modeling was conducted to investigate whether the diagnostic performance using the antibody assays could be improved by including several routine cerebrospinal fluid (CSF) parameters (i.e., leukocyte count, total protein, blood-CSF barrier functionality, and intrathecal total antibody synthesis), two-tier serology on serum, the CSF level of the B-cell chemokine (C-X-C motif) ligand 13 (CXCL13), and a Borrelia species PCR on CSF. In total, 156 patients were included who were classified as definite LNB (n = 10), possible LNB (n = 7), or non-LNB patient (n = 139) according to the criteria of the European Federation of Neurological Societies using a consensus strategy for intrathecal Borrelia-specific antibody synthesis. The seven antibody assays showed sensitivities ranging from 47.1% to 100% and specificities ranging from 95.7% to 100%. RF modeling demonstrated that the sensitivities of most antibody assays could be improved by including other parameters to the diagnostic repertoire for diagnosing LNB (range: 94.1% to 100%), although with slightly lower specificities (range: 92.8% to 96.4%). The most important parameters for LNB diagnosis are the detection of intrathecally produced Borrelia-specific antibodies, two-tier serology on serum, CSF-CXCL13, Reibergram classification, and pleocytosis. In conclusion, this study shows that LNB diagnosis is best supported using multiparameter analysis. Furthermore, a collaborative prospective study is proposed to investigate if a standardized diagnostic algorithm can be developed for improved LNB diagnosis. IMPORTANCE The diagnosis of LNB is established by clinical symptoms, pleocytosis, and proof of intrathecal synthesis of Borrelia-specific antibodies. Laboratory diagnosis of LNB is challenging, and validated diagnostic algorithms are lacking. Therefore, this retrospective cross-sectional study aimed to compare the diagnostic performance of seven commercial antibody assays for LNB diagnosis. Multiparameter analysis was conducted to investigate whether the diagnostic performance using the antibody assays could be improved by including several routine (CSF) parameters. The results of this study show that LNB diagnosis is best supported using the detection of intrathecally produced Borrelia-specific antibodies, two-tier serology on serum, CSF-CXCL13, Reibergram classification, and pleocytosis. Furthermore, we propose a collaborative prospective study to investigate the potential role of constructing a diagnostic algorithm using multiparameter analysis for improved LNB diagnosis. Show less
Gorkom, T. van; Voet, W.; Sankatsing, S.U.C.; Nijhuis, C.D.M.; Haak, E. ter; Kremer, K.; Thijsen, S.F.T. 2020
Commercial cellular tests are used to diagnose Lyme borreliosis (LB), but studies on their clinical validation are lacking. This study evaluated the utility of an in-house and a commercial enzyme... Show moreCommercial cellular tests are used to diagnose Lyme borreliosis (LB), but studies on their clinical validation are lacking. This study evaluated the utility of an in-house and a commercial enzyme-linked immunosorbent spot (ELISpot) assay for the diagnosis of Lyme neuroborreliosis (LNB). Prospectively, peripheral blood mononuclear cells (PBMCs) were isolated from patients and controls and analysed using an in-house Borrelia ELISpot assay and the commercial LymeSpot assay. B. burgdorferi B31 whole cell lysate and a mixture of outer surface proteins were used to stimulate the PBMCs and the numbers of interferon-gamma-secreting T cells were measured. Results were evaluated using receiver operating characteristic (ROC) curve analysis. Eighteen active and 12 treated LNB patients, 10 healthy individuals treated for an early (mostly cutaneous) manifestation of LB in the past and 47 untreated healthy individuals were included. Both assays showed a poor diagnostic performance with sensitivities, specificities, positive and negative predictive values ranging from 44.4-66.7%, 42.0-72.5%, 21.8-33.3% and 80.5-87.0%, respectively. The LymeSpot assay performed equally poorly when the calculation method of the manufacturer was used. Both the in-house and the LymeSpot assay are unable to diagnose active LNB or to monitor antibiotic treatment success. Show less
B. burgdorferi has a wide variety of strategies to hide from the host immune system. Complement regulatory binding proteins have been described for almost all complement resistant B. burgdorferi sl... Show moreB. burgdorferi has a wide variety of strategies to hide from the host immune system. Complement regulatory binding proteins have been described for almost all complement resistant B. burgdorferi sl, except for the complement resistant B. bavariensis, one of the species that is known to frequently cause Lyme neuroborreliosis. In chapter 2 it is attempted to identify CRASP-1 proteins in B. bavariensis, formerly known as B. garinii OspA serotype 4. Potential CRASP-1 proteins will be cloned and studied for their ability to interact with host derived fluid phase regulators of complement. The specific role of complement resistance in early effective infection and dissemination of B. burgdorferi sl has not been well investigated. Can complement resistance lead to a better and more effective infection and dissemination? In chapter 3 an in vivo experiment in which the infectivity and dissemination patterns of complement sensitive and complement resistant B. burgdorferi sl in a C3 deficient mouse model is described. After effective transmission from the tick to the host the next challenge in B. burgdorferi infection is rapid and accurate detection of the pathogen. Diagnostics of Lyme disease is often compromised due to specific pathogen properties combined with technical shortcomings of bacterial serology. Two indirect detection methods which can aid in diagnosing patients suffering from Lyme neuroborreliosis were studied. In chapter 4 the performance of the C6-peptide ELISA for detecting antibodies in CSF in Lyme neuroborreliosis patients is studied. While in chapter 5 levels of CXCL13 in several patient populations as a potential biomarker for the diagnosis of Lyme neuroborreliosis is studied. For both indirect markers of presence of B. burgdorferi the specificity in clinically resembling and neuroinfectious diseases is of key importance. Several other infectious and inflammatory diseases that have a clinical presentation that can resemble Lyme disease are included in the analysis. Diagnosing Lyme disease can be difficult in some populations, first because Lyme disease is a relatively rare infection, resembling a large spectrum of other autoimmune and inflammatory diseases. Clinicians could often consider testing for Lyme disease. It is also important to do this in specific preselected populations, because the positive predictive value of a test, but specifically indirect tests such as serology, in a random population, is low.In chapter 6 all patients that present with complaints of arthritis at the early arthritis clinic are tested for Lyme arthritis. The prevalence of B. burgdorferi seropositivity in this population is studied. Another aim is to identify clinical factors which should urge the doctor to test, or explicitely not test, for Lyme disease in a patient presenting with arthritis in Europe. In chapter 7 a case of an HIV positive patient presenting with a meningoencephalitis caused by B. burgdorferi is described. The literature on HIV and Lyme neuroborreliosis co-infections is also reviewed. Show less
