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The diagnosis of Lyme neuroborreliosis (LNB) is based on neurological symptoms, cerebrospinal fluid (CSF) pleocytosis, and intrathecally produced Borrelia-specific antibodies. In most cases, the presence of intrathecally produced Borrelia-specific antibodies is determined by using an enzyme-linked immunosorbent assay (ELISA). The edge effect is a known phenomenon in ELISAs and can negatively influence the assay reproducibility and repeatability, as well as index calculations of sample pairs which are tested in the same run. For LNB diagnostics, an index calculation is used for which the relative amounts of Borrelia-specific antibodies in CSF and serum are measured to calculate a CSF/serum quotient, which is needed to calculate the Borrelia-specific antibody index (AI). The presence of an edge effect in an ELISA used for LNB diagnostics may thus have implications. In this study, we investigated the intra-assay variation of the commercial Enzygnost Lyme link VlsE/IgG ELISA used for LNB diagnostics and showed the presence of an edge effect. Minor adaptations in the ELISA protocol decreased this effect. The adapted protocol was subsequently used to test 149 CSF-serum pairs of consecutive patients received in a routine diagnostic laboratory. By simulation, we showed that, if the standard protocol would have been used, then the edge effect for this study population could have resulted in 15 (10.1%) false-pathological and two (1.3%) false-normal Borrelia-specific IgG AIs. Thus, the observed edge effect can lead to inaccurate LNB diagnoses. Our study underlines that the edge effect should be investigated when ELISAs are implemented in routine diagnostics, as this phenomenon can occur in any ELISA.

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-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.

Dutch laboratories are currently changing their breakpoint criteria from mostly Clinical Laboratory and Standards Institute (CLSI) breakpoints to European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints. To evaluate the impact of these changes, we studied antimicrobial resistance trends of Escherichia coli in blood specimens from January 2008 to January 2012 using CLSI and EUCAST breakpoints and compared them with the antimicrobial susceptibility test (AST) interpretations reported by Dutch laboratories participating in the Infectious Disease Surveillance Information System for Antibiotic Resistance (ISIS-AR). ISIS-AR collects AST interpretations, including underlying minimal inhibitory concentrations (MICs) of routinely cultured bacterial species on a monthly basis from Dutch laboratories. MICs of Etests or automated systems were reinterpreted according to the CLSI 2009 and EUCAST 2010 guidelines. Trends in non-susceptibility (i.e. intermediate resistant and resistant) over time were analysed by the Cochran–Armitage test for trend. The effects of the change from CLSI to EUCAST breakpoints on non-susceptibility were small. There were no differences in non-susceptibility to amoxicillin, amoxicillin/clavulanic acid, cefuroxim, gentamicin and co-trimoxazol and only small differences (1–1.5%) for ciprofloxacin between AST interpretations by CLSI or EUCAST. However, for ceftazidime, and cefotaxime/ceftriaxone the proportion of non-susceptibility was substantially higher when EUCAST breakpoints were used (2–3%). The effects on time trends of the change in guidelines were limited, with only substantial differences for the oxymino-cephalosporins. Our study shows that the implementation of EUCAST breakpoints has a limited effect on the proportion of non-susceptible isolates and time trends in E. coli for most, but not all, antimicrobial agents.

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 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.