Epstein-Barr virus infection after adolescence and human herpesvirus 6A as risk factors for multiple sclerosis.

BACKGROUND AND PURPOSE: Infections with human herpesvirus 6A (HHV-6A) and Epstein-Barr virus (EBV) have been linked to multiple sclerosis (MS) development. For EBV, late infection has been proposed as a risk factor, but serological support is lacking. The objective of this study was to investigate how age affects the EBV and HHV-6A associated risks of developing MS. METHODS: In this nested case-control study, Swedish biobanks were accessed to find pre-symptomatically collected blood samples from 670 individuals who later developed relapsing MS and 670 matched controls. A bead-based multiplex assay was used to determine serological response against EBV and HHV-6A. Conditional logistic regression was used to calculate odds ratios and 95% confidence intervals. RESULTS: Seropositivity against EBV exhibited a pattern where associations switched from a decreased risk of developing MS in the group below 20 years of age to an increased risk amongst individuals aged 20-29 and 30-39 years (p for trend 0.020). The age of transition was estimated to be 18.8 years. In contrast, HHV-6A was associated with increased MS risk in all age groups (total cohort odds ratio 2.1, 95% confidence interval 1.6-2.7). CONCLUSIONS: This study suggests EBV infection after adolescence and age independent HHV-6A infection as risk factors for MS.

Improved survival of Swedish glioblastoma patients treated according to Stupp.

OBJECTIVES: The median survival in glioblastoma (GBM) patients used to be less than 1 year. Surgical removal of the tumor with subsequent concomitant radiation/temozolomide (the Stupp regimen) has been shown to prolong survival. The Stupp protocol was implemented in the county of Jönköping in 2006. The purpose of this study was to examine if the Stupp treatment has prolonged overall survival, in an unselected patient cohort with histologically verified GBM. MATERIAL AND METHOD: This study includes all patients from the county of Jönköping, with a diagnosis of GBM from January 2001 to December 2012. Patients were divided into 2 cohorts, 2001-2005 and 2006-2012, that is before and after implementation of the Stupp regimen. By reviewing the medical case notes, the dates of the histological diagnosis and of death were identified. The median and mean overall survival and Kaplan-Meier survival analysis were calculated and compared between the 2 cohorts. RESULTS: The mean survival was 110 days longer in the cohort treated according to the Stupp regimen. Four patients in the 2006-2012 cohort and 1 patient in the 2001-2005 cohort are still alive. When comparing survival in patients with radical surgery vs biopsy, those that underwent radical surgery survived longer. The significance was slightly greater in the 2001-2005 cohort (mean 163 vs 344 days, P < .001) than in the 2006-2012 cohort (mean 220 vs 397 days, P = .02). CONCLUSION: Survival significantly improved after the implementation of the Stupp regimen in the study region of Sweden.

Neurofilament light chain in cerebrospinal fluid and prediction of disease activity in clinically isolated syndrome and relapsing-remitting multiple sclerosis.

BACKGROUND AND PURPOSE: Improved biomarkers are needed to facilitate clinical decision-making and as surrogate endpoints in clinical trials in multiple sclerosis (MS). We assessed whether neurodegenerative and neuroinflammatory markers in cerebrospinal fluid (CSF) at initial sampling could predict disease activity during 2 years of follow-up in patients with clinically isolated syndrome (CIS) and relapsing-remitting MS. METHODS: Using multiplex bead array and enzyme-linked immunosorbent assay, CXCL1, CXCL8, CXCL10, CXCL13, CCL20, CCL22, neurofilament light chain (NFL), neurofilament heavy chain, glial fibrillary acidic protein, chitinase-3-like-1, matrix metalloproteinase-9 and osteopontin were analysed in CSF from 41 patients with CIS or relapsing-remitting MS and 22 healthy controls. Disease activity (relapses, magnetic resonance imaging activity or disability worsening) in patients was recorded during 2 years of follow-up in this prospective longitudinal cohort study. RESULTS: In a logistic regression analysis model, NFL in CSF at baseline emerged as the best predictive marker, correctly classifying 93% of patients who showed evidence of disease activity during 2 years of follow-up and 67% of patients who did not, with an overall proportion of 85% (33 of 39 patients) correctly classified. Combining NFL with either neurofilament heavy chain or osteopontin resulted in 87% overall correctly classified patients, whereas combining NFL with a chemokine did not improve results. CONCLUSIONS: This study demonstrates the potential prognostic value of NFL in baseline CSF in CIS and relapsing-remitting MS and supports its use as a predictive biomarker of disease activity.

Cerebrospinal fluid levels of neurofilament and tau correlate with brain atrophy in natalizumab-treated multiple sclerosis.

BACKGROUND AND PURPOSE: Brain atrophy is related to clinical deterioration in multiple sclerosis (MS) but its association with intrathecal markers of inflammation or neurodegeneration is unclear. Our aim was to investigate whether cerebrospinal fluid (CSF) markers of inflammation or neurodegeneration are associated with brain volume change in natalizumab-treated MS and whether this change is reflected in non-lesional white matter metabolites. METHODS: About 25 patients with natalizumab-treated MS were followed for 3 years with assessment of percentage brain volume change (PBVC) and absolute quantification of metabolites with proton magnetic resonance spectroscopy (1 H MRS). Analyses of inflammatory [interleukin 1ß (IL-1ß), IL-6, C-X-C motif chemokine 8 (CXCL8), CXCL10, CXCL11, C-C motif chemokine 22] and neurodegenerative [neurofilament light protein (NFL), glial fibrillary acidic protein, myelin basic protein, tau proteins] markers were done at baseline and 1-year follow-up. RESULTS: The mean decline in PBVC was 3% at the 3-year follow-up, although mean 1 H MRS metabolite levels in non-lesional white matter were unchanged. CSF levels of NFL and tau at baseline correlated negatively with PBVC over 3 years (r = -0.564, P = 0.012, and r = -0.592, P = 0.010, respectively). CONCLUSIONS: A significant 3-year whole-brain atrophy was not reflected in mean metabolite change of non-lesional white matter. In addition, our results suggest that CSF levels of NFL and tau correlate with brain atrophy development and may be used for evaluating treatment response in inflammatory active MS.

Guidelines for the use of magnetic resonance imaging in diagnosing and monitoring the treatment of multiple sclerosis: recommendations of the Swedish Multiple Sclerosis Association and the Swedish Neuroradiological Society.

Multiple sclerosis (MS) is associated with inflammatory lesions in the brain and spinal cord. The detection of such inflammatory lesions using magnetic resonance imaging (MRI) is important in the consideration of the diagnosis and differential diagnoses of MS, as well as in the monitoring of disease activity and predicting treatment efficacy. Although there is strong evidence supporting the use of MRI for both the diagnosis and monitoring of disease activity, there is a lack of evidence regarding which MRI protocols to use, the frequency of examinations, and in what clinical situations to consider MRI examination. A national workshop to discuss these issues was held in Stockholm, Sweden, in August 2015, which resulted in a Swedish consensus statement regarding the use of MRI in the care of individuals with MS. The aim of this consensus statement is to provide practical advice for the use of MRI in this setting. The recommendations are based on a review of relevant literature and the clinical experience of workshop attendees. It is our hope that these recommendations will benefit individuals with MS and guide healthcare professionals responsible for their care.

Time to secondary progression in patients with multiple sclerosis who were treated with first generation immunomodulating drugs.

BACKGROUND: It is currently unknown whether early immunomodulatory treatment in relapsing-remitting MS (RRMS) can delay the transition to secondary progression (SP). OBJECTIVE: To compare the time interval from onset to SP in patients with RRMS between a contemporary cohort, treated with first generation disease modifying drugs (DMDs), and a historical control cohort. METHODS: We included a cohort of contemporary RRMS patients treated with DMDs, obtained from the Swedish National MS Registry (disease onset between 1995-2004, n = 730) and a historical population-based incidence cohort (onset 1950-64, n = 186). We retrospectively analyzed the difference in time to SP, termed the "period effect" within a 12-year survival analysis, using Kaplan-Meier and Cox regression analysis. RESULTS: We found that the "period" affected the entire severity spectrum. After adjusting for onset features, which were weaker in the contemporary material, as well as the therapy initiation time, the DMD-treated patients still exhibited a longer time to SP than the controls (hazard ratios: men, 0.32; women, 0.53). CONCLUSION: Our results showed there was a longer time to SP in the contemporary subjects given DMD. Our analyses suggested that this effect was not solely driven by the inclusion of benign cases, and it was at least partly due to the long-term immunomodulating therapy given.

IgA antibodies against tissue transglutaminase, endomysium and gliadin in idiopathic polyneuropathy.

OBJECTIVES: To study the prevalence of antibodies of IgA class against tissue transglutaminase (tTG), endomysium (EMA) and gliadin (AGA) in patients with chronic idiopathic axonal polyneuropathy (CIAP) and to characterize the patients clinically and neurophysiologically. METHODS: Of 182 patients, 126 patients agreed to blood sampling. Sera were analysed by ELISAs detecting anti-tTG and AGA, whereas EMA was analysed by indirect immunofluorescence (IF) microscopy. Gastrointestinal symptoms were assessed by data from medical records and patient interviews. RESULTS: Nine of 126 patients (7%) were seropositive in at least one test (five with positive anti-tTG and/or EMA and four with positive AGA only). One patient with elevated levels of all specificities had laboratory signs of malabsorption and gastrointestinal complaints with abdominal pain and diarrhoea. CONCLUSIONS: Elevated levels of IgA-AGA were slightly more frequent in patients with CIAP (4%) compared to 2.5% in 1866 healthy blood donors. Highly specific serological markers indicative of coeliac disease (CD) (anti-tTG and EMA) were somewhat more common in our patients with CIAP (4%) than expected from normal reference values and from studies of the prevalence of CD in the general population. Even though these findings may indicate a relationship, the aetiological importance is unclear.

Cytokine mapping in cerebrospinal fluid and blood in multiple sclerosis patients without oligoclonal bands.

OBJECTIVE: Since there are clinical and genetic differences between MS patients with intrathecal oligoclonal bands (OCB+) in the cerebrospinal fluid (CSF) compared with those without (OCB-), the aim was to find out if OCB- patients showed a different pattern of cytokine immune activation compared with OCB+ patients. METHODS: The study included 25 MS patients (10 OCB- and 15 OCB+) and 13 controls. A panel of cytokines was measured; IL-1ß, IL-6, IL-8/CXCL8, IL-10, TNF and GM-CSF in serum, CSF and in supernatants from polyclonally stimulated blood mononuclear cells, where also levels of IL-12p40, IL-13, IL-15, IL-17 and IFN-γ were measured. The concentrations of soluble (s) VCAM-1 and sCD14 were measured in serum and CSF. RESULTS: In general, there were no extensive differences in cytokine concentrations between the OCB- and OCB+ groups. CONCLUSION: OCB- MS patients do not seem to constitute a separate entity concerning inflammatory parameters measured as cytokine concentrations in CSF and blood.

Transcriptional characteristics of CD4+ T cells in multiple sclerosis: relative lack of suppressive populations in blood.

BACKGROUND: Multiple sclerosis (MS) is hypothetically caused by autoreactive Th1 and Th17 cells, whereas Th2 and regulatory T cells may confer protection. The development of Th subpopulations is dependant on the expression of lineage-specific transcription factors. OBJECTIVE: The aim of this study was to assess the balance of CD4(+)T cell populations in relapsing-remitting MS. METHODS: Blood mRNA expression of TBX21, GATA3, RORC, FOXP3 and EBI3 was assessed in 33 patients with relapsing-remitting MS and 20 healthy controls. In addition, flow cytometry was performed to assess T lymphocyte numbers. RESULTS: In relapsing-remitting MS, diminished expression of FOXP3 (Treg) was found (p < 0.05), despite normal numbers of CD4(+)CD25(hi)Treg. Immunoregulatory EBI3 and Th2-associated GATA3 ([a-z]+) was also decreased in MS (p < 0.005 and p < 0.05, respectively). Expression of TBX21 (Th1) and RORC (Th17) did not differ between patients and controls. Similar changes were observed when analysing beta-interferon treated (n = 12) or untreated (n = 21) patients. Analysis of transcription factor ratios, comparing TBX21/GATA3 and RORC/FOXP3, revealed an increase in the RORC/FOXP3 ratio in patients with relapsing-remitting MS (p < 0.005). CONCLUSION: Our findings indicate systemic defects at the mRNA level, involving downregulation of beneficial CD4(+)phenotypes. This might play a role in disease development by permitting activation of harmful T cell populations.

Natalizumab treatment in multiple sclerosis: marked decline of chemokines and cytokines in cerebrospinal fluid.

Natalizumab exerts impressive therapeutic effects in patients with multiple sclerosis (MS). The proposed main mode of action is reducing transmigration of leukocytes into the CNS, but other immunological effects may also be operative. Cytokines and chemokines are involved in the regulation of inflammatory responses and may reflect the disease process in MS. The objective of this study was to evaluate the effects of natalizumab treatment on cytokine and chemokine profiles systemically and intrathecally in multiple sclerosis. We used luminex to analyse a panel of cytokines (IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, TNF-alpha, IFN-gamma, GM-CSF) and chemokines (CXCL9, CXCL10, CXCL11, CCL17, CCL22) in blood and cerebrospinal fluid (CSF) from 31 patients with relapsing MS before and after one year of natalizumab treatment. There was a marked decline in CSF levels of cytokines and chemokines, thus including pro-inflammatory cytokines (IL-1beta, IL-6 and IL-8) as well as chemokines associated with both Th1 (CXCL9, CXCL10, CXCL11) and Th2 (CCL22). Circulating plasma levels of some cytokines (GM-CSF, TNF-alpha, IL-6 and IL-10) also decreased after one year of treatment. This is the first study to show that natalizumab treatment is associated with a global decline in cytokine and chemokine levels at a protein level. This finding was most pronounced in CSF, in line with the reduced transmigration of cells into CNS, whereas reduction in plasma levels indicates other possible mechanisms of natalizumab treatment.

Polyneuropathy associated with IgM vs IgG monoclonal gammopathy: comparison between clinical and electrophysiological findings.

OBJECTIVE: The neuropathy associated with IgM monoclonal gammopathy (IgM-MG) is regarded as a sensorimotor, mainly demyelinating neuropathy. It is not fully known whether the neuropathy in IgG-MG is caused by the same mechanisms and shows the same electrophysiological characteristics. We aimed at making a comparison between clinical and neurophysiological findings in these two conditions. PATIENTS AND METHODS: Twenty-seven patients with IgM-associated neuropathy [18 with anti-myelin-associated glycoprotein (anti-MAG) antibodies] were compared with 15 age-matched patients with IgG-associated neuropathy. RESULTS: Patients with IgM-associated neuropathy (especially those with anti-MAG antibodies) had significantly clinically more severe disabilities with involvement of both motor and sensory functions compared with patients with IgG-associated neuropathy in whom clinical sensory disturbances were more prominent than motor dysfunction. Motor and sensory conduction velocities were significantly lower and distal latencies significantly longer in the IgM group than in the IgG group concerning the median, ulnar and peroneal nerves. Fifty-four per cent of the patients in the IgM group did not present a sensory response of the median nerve vs 13% in the IgG group. There was also a significant difference concerning absent responses from the peroneal and sural nerves in the IgM vs IgG group (peroneal: 48% vs 13%, sural: 88% vs 27%). CONCLUSION: Polyneuropathy associated with IgM-MG, especially when associated with anti-MAG antibodies, appears to have more of a demyelinating involvement that meets the criteria for demyelination. This was not as clear in those associated with IgG. The IgG neuropathy showed less and milder deficit in the electrophysiological studies.

In vitro Th2 deviation of myelin-specific peripheral blood lymphocytes from patients with multiple sclerosis.

This study aimed at investigating if selective ex vivo immune deviation of myelin-specific cytokine secretion towards Th2 is possible in blood cells from patients with multiple sclerosis (MS). Interleukin (IL)-4 (Th2) and interferon-gamma (Th1) secreting cells were recorded by ELISPOT in 13 MS patients. Deviation was successful in 10 patients. Interleukin-4 alone was most effective in inducing myelin-specific immune deviation in MS patients whereas IL-1 or IL-15 in combination with IL-4 did not improve the results. Further studies and improvements are needed before ex vivo immune deviation can be considered a potential treatment in patients with MS.

Linkage analysis of HLA class II genes in Swedish multiplex families with multiple sclerosis.

We assessed the influence of human leukocyte antigen (HLA) class II as susceptibility genes in multiple sclerosis (MS) by linkage analysis. Other research groups, who have shown negative results in studies on affected sibling pairs, have questioned the influence of the HLA class II genes, although they confirmed the association of the DR15,DQ6,Dw2 haplotype to MS. In this report, we find a significant lod score (>3) when using a 2-point linkage analysis of 49 small Swedish nuclear MS families with at least two affected family members, typed for HLA class II alleles by PCR amplification with sequence-specific primers. We obtained maximum lod scores when we used dominant or intermediate models with low penetrance. We found that the positive effect on the total lod score was not confined to those families carrying the presumed susceptibility HLA-haplotype Dw2. This observation supports the notion of a functional role of the HLA class II molecules themselves in MS. In addition, we observed significant transmission distortion and an intrafamilial association of the MS-associated class II haplotype HLA-Dw2. These results indicate that the HLA class II genes are of clear importance for MS in the studied population.

T lymphocyte subset abnormalities in peripheral blood from patients with the Guillain-Barré syndrome.

T lymphocytes are probably of pathogenic importance in many autoimmune diseases. Recently, deviations of circulating T-helper (CD4+) subpopulations have been noticed. Blood samples from 12 patients with Guillain-Barré syndrome (GBS) were studied with flow cytometry during their disease course to define circulating T cell populations. The proportion of T-helper cells (CD4+) was decreased (mean value 41 +/- 15%, P = 0.01) and the proportion of T cytotoxic/suppressor cells (CD8+) was increased (35 +/- 18%, P = 0.0006) as compared to the control group of healthy blood donors (47 +/- 8% and 26 +/- 7% respectively). The CD4+ population is divided into the helper/inducer (CD4+CD29+) and suppressor/inducer (CD4+CD45RA+) subsets, which normally are equally distributed (mean values in our control group were 45 +/- 15% and 44 +/- 15%, respectively). In patients with GBS, the helper/inducer (CD4+CD29+) subset was increased (54 +/- 10%, P = 0.05) and the suppressor/inducer (CD4+CD45RA+) subset was decreased (31 +/- 9, P = 0.005) compared to the controls. The proportion of activated HLA-DR-expressing T cells was increased (7 +/- 8%, P = 0.005) as compared to controls (3 +/- 3%). The total proportions of T cells (CD2+), B cells (CD19+) and natural killer (NK) cells (CD56+) were similar in patients and controls. The CD4+ and CD8+ populations, as well as the activated HLA-DR+ T cells, normalized during the disease course. The deviations within the CD4+ population also tended to normalize, but even at follow up after 6-33 (mean 23) months, some abnormalities remained. In conclusion, we confirm previous reports of T cell activation in peripheral blood from patients with GBS. A new finding is the deviation of T helper subpopulations with an increased helper/inducer (CD4+CD29+) subset and a decreased suppressor/inducer (CD4+CD45RA+) subset, which indicates a possible autoimmune character of GBS.

Antibodies to peripheral nerve myelin may occur without clinical neuropathy in healthy persons.

To study the role of autoantibodies against peripheral nerve myelin (PNM), sera from 255 healthy blood donors were investigated by enzyme-linked immunosorbent assay (ELISA), and persons with anti-PNM antibodies were further studied clinically and by neurography, 25 blood donors (10%) had anti-PNM antibodies of IgM, IgG or IgA isotype. The 12 blood donors with anti-PNM antibodies of IgM isotype showed only slightly decreased nerve conduction velocities compared to 12 blood donors without anti-PNM antibodies. Two blood donors with anti-PNM antibodies of IgM and IgG isotype respectively showed a clinical, previously undiagnosed polyneuropathy (PN), verified by neurography. However, also one blood donor without anti-PNM antibodies had a clinical and neurography-verified PN. We conclude that antibodies against PNM may occur in healthy persons without evidence of a clinical or subclinical PN.

Ig-secreting cells pass the blood-brain barrier: studies on kappa and lambda light chain secreting cells in plasma cell dyscrasia.

To study if immunoglobulin (Ig)-secreting cells actively pass the blood-brain barrier (BBB), 15 patients with monoclonal gammopathy underwent bonemarrow (BM) iliac crest aspiration biopsy, peripheral blood (PB) sampling and cerebrospinal fluid (CSF) analysis. With an enzyme-linked immunospot assay we investigated the number and ratio of mononuclear cells secreting Ig with kappa and lambda light chains in the three different compartments. A statistically significant (P < 0.05) correlation between the ratio of Ig kappa/lambda-secreting cells in CSF, PB and BM was found. The frequency of kappa and lambda (i.e. Ig in total) secreting mononuclear cells, of the same Ig class as the paraprotein, per 10(4) mononuclear cells was higher in BM (median 2.16%, range 0.43-9.28%) compared to CSF (median 0.44%, range 0.05-9.25%) and in CSF compared to PB (median 0.12%, range 0.02-0.96%). The proportion of all mononuclear cells with Ig kappa and lambda light chain (i.e. Ig) secretion was on average 5-fold greater in CSF compared to PB and 11-fold greater in BM compared to PB. The present study indicates that paraprotein-secreting cells preferentially pass from PB to CSF.

Susceptibility to demyelinating polyneuropathy in plasma cell dyscrasia may be influenced by amino acid position 9 of the HLA-DR beta chain.

Fifty-five patients with plasma cell dyscrasias were investigated by genomic typing for HLA-DR and -DQ genes by restriction fragment length polymorphism, neurophysiology and for presence of anti-myelin-associated glycoprotein (MAG) antibodies. In 26 patients, a polyneuropathy (PN) of demyelinating type was established. Among these individuals, an association was found with the presence of a tryptophan amino acid residue at position 9 of the DR beta chain (P < 0.01). This position is part of the first hypervariable region of the DR beta chain, and may be of importance in determining preferential peptide-binding capacity of the HLA-DR molecule. The presence of anti-MAG antibodies in 15 out of 17 patients with an IgM M-component and demyelinating PN (14 of these 15 individuals carrying a tryptophan at position 9) supports the pathogenic role of an autoimmune response against MAG. The finding of an HLA class II association may indicate a pathogenic role of T cell immunity in this condition.

Compartmentalization of antigen specific cytokine responses to the central nervous system in CNS borreliosis: secretion of IFN-gamma predominates over IL-4 secretion in response to outer surface proteins of Lyme disease Borrelia spirochetes.

The neurological manifestations of Lyme disease have been proposed to be partly due to cytokine-mediated immunopathological mechanisms. In this study, the number of Borrelia-specific cells secreting interferon-gamma and interleukin-4 was determined in blood and cerebrospinal fluid from patients with CNS borreliosis (n = 23), other neurological diseases (n = 20), and in blood from healthy controls (n = 10), utilizing an ELISPOT-assay. Elevated specific secretion of IFN-gamma was found in CNS borreliosis, most pronounced in cerebrospinal fluid, whereas secretion of IL-4 was strikingly low. This may indicate that symptoms are due to side effects of the immune response, since IFN-gamma secretion in the absence of corresponding levels of IL-4 may be associated with tissue destruction.

The HLA-Dw2 haplotype segregates closely with multiple sclerosis in multiplex families.

Multiple sclerosis (MS) is associated with the HLA class II specificity Dw2, but the importance of its influence has been questioned, since sib-pair analysis has failed to show linkage with this haplotype. However, the use of 'identity by descent' (IBD) analysis may not be ideal, since it does not make use of the facts that (i) the Dw2-haplotype is the only haplotype with a confirmed role in MS, and (ii) it performs its influence in a dominant manner. We have investigated nine Swedish multiplex MS families. In eight of the families, the Dw2 haplotype occurred in MS patients. Within these families, Dw2 was shared by all 17 individuals with MS. In a compilation of 48 published multiplex MS families in which at least one patient carried Dw2, only three of 107 individuals with MS did not carry the Dw2 haplotype. This indicates that the Dw2 haplotype, when present in familial MS, may confer a stronger influence in MS susceptibility than generally recognized.

Lyme borreliosis in Sweden--diagnostic performance of five commercial Borrelia serology kits using sera from well-defined patient groups.

Five commercial Borrelia serology kits available in Sweden were evaluated and compared for their diagnostic performance in sera from clinically well-characterized patient groups. With the clinically defined groups as the gold standard, i.e. without knowledge of antibody status in serum and cerebrospinal fluid, the diagnostic performance of the kits was compared and important differences in diagnostic usefulness were found. The kits from Abbot and DAKO, that often predict clinically relevant Borrelia infection and do not detect antibodies in sera from patients without strong suspicion of Borrelia infection, were considered the most useful in the population studied. This kind of validation study is an important part of good laboratory practice and should be performed by laboratories serving patient populations with varying endemicity of Borrelia.