Case report: persistently seronegative neuroborreliosis in an immunocompromised patient.

BACKGROUND: Infection with Borrelia burgdorferi sensu lato complex (B. b. sl) spirochetes can cause Lyme borreliosis, manifesting as localized infection (e.g. erythema migrans) or disseminated disease (e.g. Lyme neuroborreliosis). Generally, patients with disseminated Lyme borreliosis will produce an antibody response several weeks post-infection. So far, no case of neuroborreliosis has been described with persistently negative serology one month after infection. CASE PRESENTATION: We present a patient with a history of Mantle cell lymphoma and treatment with R-CHOP (rituximab, doxorubicine, vincristine, cyclofosfamide, prednisone), with a meningo-encephalitis, who was treated for a suspected lymphoma relapse. However, no malignant cells or other signs of malignancy were found, and microbial tests did not reveal any clues, including Borrelia serology. He did not recall being bitten by ticks, and a Borrelia PCR on CSF was negative. After spontaneous improvement of symptoms, he was discharged without definite diagnosis. Several weeks later, he was readmitted with a relapse of symptoms of meningo-encephalitis. This time however, a Borrelia PCR on CSF was positive, confirmed by two independent laboratories, and the patient received ceftriaxone upon which he partially recovered. Interestingly, during the diagnostic process of this exceptionally difficult case, a variety of different serological assays for Borrelia antibodies remained negative. Only P41 (flagellin) IgG was detected by blot and the Liaison IgG became equivocal 2 months after initial testing. CONCLUSIONS: To the best of our knowledge this is the first case of neuroborreliosis that is seronegative on repeated sera and multiple test modalities. This unique case demonstrates the difficulty to diagnose neuroborreliosis in severely immunocompromised patients. In this case, a delay in diagnosis was caused by broad differential diagnosis, an absent known history of tick bites, negative serology and the low sensitivity of PCR on CSF. Therefore, awareness of the diagnostic limitations to detect Borrelia infection in this specific patient category is warranted.

Diagnostic value of cerebrospinal fluid CXCL13 for acute Lyme neuroborreliosis. A systematic review and meta-analysis.

OBJECTIVES: The utility of cerebrospinal fluid (CSF) CXCL13 for diagnosis of acute Lyme neuroborreliosis (LNB) has been debated and the test is not yet routinely performed. This study's aim was to evaluate its overall diagnostic accuracy through meta-analysis. METHODS: Electronic searches in PubMed MEDLINE and Web of Science were performed to identify relevant articles published before January 2018. A summary receiver operating characteristic curve and an optimal cut-off were estimated modelling multiple cut-offs. Publication bias was evaluated using a funnel plot and the associated regression test. RESULTS: A total of 18 studies involving 618 individuals with acute LNB and 2326 individuals with other neurological disorders meeting the eligibility criteria were identified. The pooled sensitivity for CSF CXCL13 was 89% (95% CI 85%-93%) and the pooled specificity was 96% (95% CI 92%-98%), using the identified optimal cut-off value of 162 pg/mL. There was marked heterogeneity between studies, caused by differences in the designs of the study populations and age distribution. The optimal cut-off in the seven studies with a cross-sectional design was 91 pg/mL (sensitivity 96%, 95% CI 92%-98%; specificity 94%, 95% CI 86%-97%) and in the 11 case-control studies it was 164 pg/mL (sensitivity 85%, 95% CI 78%-91%; specificity 95%, 95% CI 90%-98%). CSF CXCL13 values above the optimal cut-off level (determined in this meta-analysis) were also detectable in some other central nervous system disorders, namely neurosyphilis and central nervous system lymphoma. CONCLUSIONS: Our meta-analysis shows that CSF CXCL13 has the potential to become a useful adjunct in the diagnosis of acute LNB.

Pediatric Lyme Neuroborreliosis: Different clinical presentations of the same agent; Single center experience.

OBJECTIVES: Lyme disease is a vector-associated infectious disease, caused by the agent, spirochete Borrelia burgdorferi. Neurologic findings are observed in approximately 12% of the cases and termed Lyme neuroborreliosis (LNB). Lyme neuroborreliosis may manifest with different clinical neurologic manifestations. METHODS: The study was conducted at tertiary training and research hospital. From January 2014 to September 2015, a total of 75 patients diagnosed with encephalitis, ataxia, Guillain Barre Syndrome (GBS), facial paralysis, acute disseminated encephalomyelitis (ADEM), pseudotumorcerebri were evaluated for inclusion to the study. Among these patients whom investigations of B. burgdorferi antibody IgM and/or IgG ELISA and Western Blot (WB) were detected to be positive, were assessed. Epidemiologic data, tick bite histories, duration of symptoms, clinical findings, radiologic findings, treatment durations and prognosis were investigated. RESULTS: Totally 7 patients had been treated with the diagnosis of Lyme neuroborreliosis. The mean age was 9.14±4.91 years; duration of symptoms before admission was 8.0±4.50 days; and the duration of antibiotic use was 2.85±0.89 weeks. All patients had received ceftriaxone and intravenous immunoglobulin (IVIG); 3 patients had received plasmapheresis (42.9%) and one patient had received pulse corticosteroid therapy. While the patient with the diagnosis of encephalomyeloneuritis and atypical GBS had partially improved, the other patients were completely cured. CONCLUSION: In this article, we report pediatric LNB patients, B. burgdorferi should also be considered in patients with atypical or severe neurologic involvement or a history of tick bite; it is known that the prognosis is good with appropriate and early treatment.

The MEK/ERK pathway is the primary conduit for Borrelia burgdorferi-induced inflammation and P53-mediated apoptosis in oligodendrocytes.

Lyme neuroborreliosis (LNB) affects both the central and peripheral nervous systems. In a rhesus macaque model of LNB we had previously shown that brains of rhesus macaques inoculated with Borrelia burgdorferi release inflammatory mediators, and undergo oligodendrocyte and neuronal cell death. In vitro analysis of this phenomenon indicated that while B. burgdorferi can induce inflammation and apoptosis of oligodendrocytes per se, microglia are required for neuronal apoptosis. We hypothesized that the inflammatory milieu elicited by the bacterium in microglia or oligodendrocytes contributes to the apoptosis of neurons and glial cells, respectively, and that downstream signaling events in NFkB and/or MAPK pathways play a role in these phenotypes. To test these hypotheses in oligodendrocytes, several pathway inhibitors were used to determine their effect on inflammation and apoptosis, as induced by B. burgdorferi. In a human oligodendrocyte cell line (MO3.13), inhibition of the ERK pathway in the presence of B. burgdorferi markedly reduced inflammation, followed by the JNK, p38 and NFkB pathway inhibition. In addition to eliciting inflammation, B. burgdorferi also increased total p53 protein levels, and suppression of the ERK pathway mitigated this effect. While inhibition of p53 had a minimal effect in reducing inflammation, suppression of the ERK pathway or p53 reduced apoptosis as measured by active caspase-3 activity and the TUNEL assay. A similar result was seen in primary human oligodendrocytes wherein suppression of ERK or p53 reduced apoptosis. It is possible that inflammation and apoptosis in oligodendrocytes are divergent arms of MAPK pathways, particularly the MEK/ERK pathway.

Seronegative lyme neuroborreliosis in a patient using rituximab.

A 66-year-old woman presented with severe shooting pains throughout her back and legs, followed by progressive deafness, weight loss and headache. She had a history of marginal zone B-cell lymphoma stage IV-B, for which she was successfully treated with immunochemotherapy and rituximab maintenance therapy. A relapse was suspected, but chemotherapy was not administered, since, despite elaborate investigations, malignancy could not be proven. Because of a history of tick bites she was tested for antibodies against Borrelia burgdorferi in serum and cerebrospinal fluid (CSF), which were negative. However, a B burgdorferi PCR on CSF came back positive. The patient was treated for seronegative Lyme neuroborreliosis with ceftriaxone intravenously and dramatically improved. This case presentation demonstrates that, in immunocompromised patients, it is important not to solely rely on antibody testing and to use additional diagnostic tests to avoid missing or delaying the diagnosis.

No evidence for the diagnostic value of Borrelia serology in patients with sudden hearing loss.

In this evidence-based case report, we address the following clinical question: What is the predictive value of serological testing for Borrelia for diagnosing neuroborreliosis in patients with sudden sensorineural hearing loss? We searched for relevant articles in PubMed, Embase, and Web of Science. We retrieved 49 unique publications and screened the title and abstract of these articles for relevance. We included 2 of 12 studies initially considered relevant to answer our question. These 2 studies reported a seroprevalence of antibodies against Borrelia of 16% in patients with sudden sensorineural hearing loss (SHL) as compared with 13.5% in the general population, but in neither patients with definite neuroborreliosis were they found. To date, there is no evidence regarding the added value of routine diagnostic serologic testing for Borrelia in diagnosing neuroborreliosis in patients with sudden SHL. Neuroborreliosis seems to be a rare cause of sudden SHL, and routine screening of patients for borrelia antibodies in serum should therefore not be recommended.

Cytotoxic mechanisms may play a role in the local immune response in the central nervous system in neuroborreliosis.

Aiming to investigate the role of cytotoxic mechanisms in neuroborreliosis (NB), the cytokines IL-2, IL-7, IL-10, IL-12p70, IL-15, GM-CSF and the Th17-cytokine IL-17 were analyzed in cerebrospinal fluid (CSF) and plasma from NB-patients. NB-patients showed increased levels in CSF compared to controls of all analyzed cytokines except IL-15 but not in plasma. Blood lymphocytes from three NB-patients showed functional cytotoxicity in response to autologous Borrelia-infected macrophages. The findings support a role for cytotoxic mechanisms in the local immune response in NB and in addition suggest an increase of IL-17.

Expression of Fas receptor on human T lymphocytes under stimulation with Borrelia burgdorferi sensu lato - preliminary results.

PURPOSE: Apoptosis of activated T lymphocytes is essential to immunoregulation and its abnormalities have been observed in immune system disorders and persistent infections. To asses Borrelia burgdorferi influence on the susceptibility of T lymphocytes to apoptosis, we have measured expression of the Fas death receptor on these cells after incubation with live B. burgdorferi. MATERIAL AND METHODS: Peripheral blood mononuclear cells from 23 LD patients (18 with Lyme arthritis, 5 with neuroborreliosis) and 13 healthy controls (C) were incubated for 48 hours with and without live B. burgdorferi spirochetes: B. afzelii, B. garinii or B. burgdorferi sensu stricto. After incubation, Fas expression on CD3+ cells was measured cytometrically with FITC-labeled monoclonal antibody. RESULTS: Median fraction of Fas-expressing T lymphocytes increased under incubation with B. burgdorferi, with more cells expressing Fas after incubation with B. burgdorferi sensu stricto than with B. garinii. There was a tendency for a higher expression of Fas on T lymphocytes from LD patients then from controls, both in unstimulated and B. burgdorferi-stimulated cultures, but it did not reach a level of statistical significance. CONCLUSIONS: B. burgdorferi seems to increase Fas expression on CD3+ T lymphocytes, which may render these cells more susceptible to apoptosis. This effect is stronger for B. burgdorferi s.s. than for B. garinii genospecies.

T-cell epitope mapping of the Borrelia garinii outer surface protein A in lyme neuroborreliosis.

We studied the T-cell reactivity to overlapping peptides of B. garinii OspA, in order to locate possible immunodominant T-cell epitopes in neuroborreliosis. Cells from cerebrospinal fluid (CSF) and blood from 39 patients with neuroborreliosis and 31 controls were stimulated with 31 overlapping peptides, and interferon-gamma secreting cells were detected by ELISPOT. The peptides OspA(17-36), OspA(49-68), OspA(105-124), OspA(137-156), OspA(193-212) and OspA(233-252) showed the highest frequency of positive responses, being positive in CSF from 38% to 50% of patients with neuroborreliosis. These peptides also elicited higher responses in CSF compared with controls (P = 0.004). CSF cells more often showed positive responses to these peptides than blood cells (P = 0.001), in line with a compartmentalization to the central nervous system. Thus, a set of potential T-cell epitopes were identified in CSF cells from patients with neuroborreliosis. Further studies may reveal whether these epitopes can be used diagnostically and studies involving HLA interactions may show their possible pathogenetic importance.

Persisting atypical and cystic forms of Borrelia burgdorferi and local inflammation in Lyme neuroborreliosis.

BACKGROUND: The long latent stage seen in syphilis, followed by chronic central nervous system infection and inflammation, can be explained by the persistence of atypical cystic and granular forms of Treponema pallidum. We investigated whether a similar situation may occur in Lyme neuroborreliosis. METHOD: Atypical forms of Borrelia burgdorferi spirochetes were induced exposing cultures of Borrelia burgdorferi (strains B31 and ADB1) to such unfavorable conditions as osmotic and heat shock, and exposure to the binding agents Thioflavin S and Congo red. We also analyzed whether these forms may be induced in vitro, following infection of primary chicken and rat neurons, as well as rat and human astrocytes. We further analyzed whether atypical forms similar to those induced in vitro may also occur in vivo, in brains of three patients with Lyme neuroborreliosis. We used immunohistochemical methods to detect evidence of neuroinflammation in the form of reactive microglia and astrocytes. RESULTS: Under these conditions we observed atypical cystic, rolled and granular forms of these spirochetes. We characterized these abnormal forms by histochemical, immunohistochemical, dark field and atomic force microscopy (AFM) methods. The atypical and cystic forms found in the brains of three patients with neuropathologically confirmed Lyme neuroborreliosis were identical to those induced in vitro. We also observed nuclear fragmentation of the infected astrocytes using the TUNEL method. Abundant HLA-DR positive microglia and GFAP positive reactive astrocytes were present in the cerebral cortex. CONCLUSION: The results indicate that atypical extra- and intracellular pleomorphic and cystic forms of Borrelia burgdorferi and local neuroinflammation occur in the brain in chronic Lyme neuroborreliosis. The persistence of these more resistant spirochete forms, and their intracellular location in neurons and glial cells, may explain the long latent stage and persistence of Borrelia infection. The results also suggest that Borrelia burgdorferi may induce cellular dysfunction and apoptosis. The detection and recognition of atypical, cystic and granular forms in infected tissues is essential for the diagnosis and the treatment as they can occur in the absence of the typical spiral Borrelia form.

Lyme meningoradiculitis: prospective evaluation of biological diagnosis methods.

The symptoms of Lyme meningoradiculitis and the value of biological examinations in an endemic area were determined in a prospective study in which data were collected on all patients consecutively hospitalised for Lyme meningoradiculitis at our institution during an 18-month period. Specific antibody titres in the serum and cerebrospinal fluid (CSF) were determined by Vidas enzyme-linked-immunosorbent-assay (IgG + IgM), Dade-Behring enzyme immunoassay (EIA) (IgM; IgG) and Western blot analysis (IgG). We also searched for Borrelia burgdorferi in the CSF by PCR analysis and following culture on a specific medium. A control group was recruited, consisting of 16 consecutive patients who had been referred during the same period with suspected but not confirmed Lyme meningoradiculitis. Eleven patients were included. Borrelia EIA of the serum revealed that 40% of the patients had both elevated specific IgM titres and intrathecal synthesis of specific IgG; 40% of the patients was negative for IgM but had isolated intrathecal synthesis of IgG; 20% of the patients had elevated specific IgM titres without intrathecal synthesis of IgG. PCR analysis and the CSF culture were positive in one case only (B. garinii). The results of this study highlight the importance of systematic serological testing for B. burgdorferi in the CSF in the case of early neuroborreliosis suspicion, even in the absence of IgM serum antibodies, which was the case in 40% of the patients in the present study. Nevertheless, intrathecal anti-B. burgdorferi IgG synthesis, which remains the "gold standard" for the diagnosis of neuroborreliosis, was not detectable in 20% of the patients for whom diagnosis was subsequently confirmed by demonstration of specific serum IgM.

Borrelia garinii induces CXCL13 production in human monocytes through Toll-like receptor 2.

Recent studies have suggested an important role for the B-cell-attracting chemokine CXCL13 in the B-cell-dominated cerebrospinal fluid (CSF) infiltrate in patients with neuroborreliosis (NB). High levels of CXCL13 were present in the CSF of NB patients. It has not been clear, however, whether high CSF CXCL13 titers are specific for NB or are a characteristic of other spirochetal diseases as well. Furthermore, the mechanisms leading to the observed CXCL13 expression have not been identified yet. Here we describe similarly elevated CSF CXCL13 levels in patients with neurosyphilis, while pneumococcal meningitis patient CSF do not have high CXCL13 levels. In parallel, challenge of human monocytes in vitro with two of the spirochetal causative organisms, Borrelia garinii (the Borrelia species most frequently found in NB patients) and Treponema pallidum, but not challenge with pneumococci, induced CXCL13 release. This finding implies that a common spirochetal motif is a CXCL13 inducer. Accordingly, we found that the lipid moiety N-palmitoyl-S-(bis[palmitoyloxy]propyl)cystein (Pam(3)C) (three palmitoyl residues bound to N-terminal cysteine) of the spirochetal lipoproteins is critical for the CXCL13 induction in monocytes. As the Pam(3)C motif is known to signal via Toll-like receptor 2 (TLR2) and an anti-TLR2 monoclonal antibody blocked CXCL13 production of human monocytes incubated with B. garinii, this suggests that TLR2 is a major mediator of Borrelia-induced secretion of CXCL13 from human monocytes.

Analysis of cerebrospinal fluid cell populations with monoclonal antibodies.

Sixty-five samples of cerebrospinal fluid (CSF) were evaluated using an automated cytoflow method with the CD-Sapphire hematology analyzer in order to investigate possible relationships between cell population patterns and diagnostic groups and better understand the biology of neurological disease. A basic panel of CD markers, including CD3/4/8/19/138/HLA-DR, was used to analyze CSF samples from clinical and laboratory confirmed cases of multiple sclerosis, neuroborreliosis, viral and bacterial neuroinfective diseases, malignant infiltrations of meninges and scavenger macrophagic reactions of the central nervous system. The principles of immune response and the contribution of cytological 'disease-related patterns' for these nosological entities are described. The distinct patterns of lymphocyte subpopulations in neuroborreliosis appear to be characteristic and could possibly serve as diagnostic indicators. Further verification and research will be necessary to clarify the significance and nature of CD4+ CD8+ positive subset in cerebrospinal fluid.

Immunophenotypic patterns of T-cell activation in neuroinflammatory diseases.

OBJECTIVES: We aimed to gain insights into the pathogen-specific differences in early adaptive immune responses following central nervous system infections with Borrelia burgdorferi and viral pathogens by studying the immunophenotypic patterns of T-cell activation. Moreover, we wished to determine whether the expression of T-cell activation markers reflects disease activity in multiple sclerosis (MS). METHODS: Proportions of cerebrospinal fluid T-cells expressing the markers HLA-DR, CD25 and CD38 were determined in patients with MS (n = 40), acute viral meningomyeloradiculoneuritis (VID, n = 26), early neuroborreliosis (NB, n = 23) and non-inflammatory neurologic diseases (n = 51) by using flow cytometry. In relapsing-remitting MS, disease activity was assessed by clinical examination and magnetic resonance imaging. RESULTS: For each of the surface markers that were examined, significant differences in T cell proportions were found between patient groups. The proportion of HLA-DR+ T cells was higher and that of CD25+ T cells lower in NB compared with VID. These differences were attributable only to the early phase of the disease (< or = 6 days after symptom onset). Among MS patients, there was a trend for higher proportions of T cells expressing activation markers in patients with gadolinium-enhancing lesions. CONCLUSIONS: The decreased CD25 expression in NB may reflect immunomodulatory effects of B. burgdorferi facilitating persistent infection. Larger prospective studies of T-cell activation markers for ascertaining the association between cellular markers and clinical surrogates of disease activity in MS are warranted.

[Cytokine CXCL13--a possible early CSF marker for neuroborreliosis]. / Zyktokin CXCL13 : Ein früher Liquormarker für die Neuroborreliose?

The definitive diagnosis of acute neuroborreliosis (NB) is based upon the presence of lymphomonocytic CSF pleocytosis and intrathecal Borrelia burgdorferi (B.b.)-specific antibody production (expressed by an antibody index of >2). However, the latter might be absent in early stages of the disease. Now a recently discovered additional CSF marker-the cytokine CXCL13-was found to be positive in every initial CSF sample from patients with NB and therefore could be a valuable tool for early diagnosis and initiation of antibiotic therapy. We report an unusual case of NB in a patient with a history of metastatic carcinoma of the prostate and unilateral polyradiculitis. While no intrathecal B.b.-specific antibody production could be demonstrated initially, the CSF CXCL13 level was high (>500 ng/g vs <1.7 ng/g in healthy controls). During the course of the disease, the antibody index turned positive (4.8) and the patient responded to antibiotic therapy, thus confirming the diagnosis. In this case, measuring CXCL13 in the CSF would have led to earlier diagnosis and treatment of NB.

Innate immune responses in Lyme borreliosis: enhanced tumour necrosis factor-alpha and interleukin-12 in asymptomatic individuals in response to live spirochetes.

Innate immunity is important for early defence against borrelia spirochetes and should play a role in the clinical outcome of the infection. In order to study early cytokine responses, in vitro differentiated dendritic cells (DCs) and whole blood cells from 21 patients with different clinical outcomes of Lyme neuroborreliosis were stimulated with live borrelia spirochetes. The borrelia-induced secretion of interleukin (IL)-4, IL-10, IL-12p70, interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha in DCs and IL-1beta, IL-6, IL-8, IL-10, IL-12p70, TNF-alpha, regulated upon activation normal T cell expressed and secreted (RANTES), monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1alpha, MIP-1beta and eotaxin in whole blood cells was measured by enzyme-linked immunospot (ELISPOT) and multiplex arrays, respectively. We found increased numbers of TNF-alpha-secreting DCs (P = 0.018) in asymptomatic seropositive individuals compared to patients with subacute neuroborreliosis and seronegative controls. Asymptomatic individuals were also found to have elevated levels of IL-12p70 (P = 0.031) in whole blood cell supernatants compared to seronegative controls. These results are in line with previous experiments using cells of the adaptive immune response, indicating that strong T helper type 1 (Th1) proinflammatory responses might be associated with a successful resolution of Lyme disease.

Borrelia-specific interferon-gamma and interleukin-4 secretion in cerebrospinal fluid and blood during Lyme borreliosis in humans: association with clinical outcome.

The Borrelia-specific interferon (IFN)- gamma and interleukin (IL)-4 responses of 113 patients and control subjects were analyzed using the sensitive enzyme-linked immunospot method. Cerebrospinal fluid (CSF) and blood samples were obtained, during the course of disease, from patients with chronic or nonchronic neuroborreliosis (NB) and from control subjects without NB. Blood samples were obtained from patients with Lyme skin manifestations and from healthy blood donors. Early increased secretion of Borrelia-specific IFN- gamma (P<.05) and subsequent up-regulation of IL-4 (P<.05) were detected in the CSF cells of patients with nonchronic NB. In contrast, persistent Borrelia-specific IFN- gamma responses were observed in the CSF cells of patients with chronic NB (P<.05). In patients with erythema migrans, increased IFN- gamma (P<.001) was observed in blood samples obtained early during the course of disease, whereas increased IL-4 (P<.05) was observed after clearance. On the contrary, patients with acrodermatitis chronica atrophicans had Borrelia-specific IFN- gamma (P<.001), but not IL-4, detected in blood samples. The present data suggest that an initial IFN- gamma response, followed by up-regulation of IL-4, is associated with nonchronic manifestations, whereas a persistent IFN- gamma response may lead to chronic Lyme borreliosis.

Spinal cord involvement in the nonhuman primate model of Lyme disease.

Lyme borreliosis is a multisystemic disease caused by infection with various genospecies of the spirochete Borrelia burgdorferi. The organs most often affected are the skin, joints, the heart, and the central and peripheral nervous systems. Multiple neurological complications can occur, including aseptic meningitis, encephalopathy, facial nerve palsy, radiculitis, myelitis, and peripheral neuropathy. To investigate spinal cord involvement in the nonhuman primate (NHP) model of Lyme borreliosis, we inoculated 25 adult Macaca mulatta with B. burgdorferi sensu strictu strains N40 by needle (N=9) or by tick (N=4) or 297 by needle (N=2), or with B. burgdorferi genospecies garinii strains Pbi (N=4), 793 (N=2), or Pli (N=4) by needle. Immunosuppression either transiently (TISP) or permanently (IS) was used to facilitate establishment of infection. Tissues and fluids were collected at necropsy 7-24 weeks later. Hematoxylin and eosin staining was used to study inflammation, and immunohistochemistry and digital image analysis to measure inflammation and localize spirochetes. The spirochetal load and C1q expression were measured by TaqMan RT-PCR. The results showed meningoradiculitis developed in only one of the 25 NHP's examined, TISP NHP 321 inoculated with B. garinii strain Pbi. Inflammation was localized to nerve roots, dorsal root ganglia, and leptomeninges but rarely to the spinal cord parenchyma itself. T cells and plasma cells were the predominant inflammatory cells. Significantly increased amounts of IgG, IgM, and C1q were found in inflamed spinal cord. Taqman RT-PCR found spirochetes in the spinal cord only in IS-NHP's, mostly in nerve roots and ganglia rather than in the cord parenchyma. C1q mRNA expression was significantly increased in inflamed spinal cord. This is the first comprehensive study of spinal cord involvement in Lyme borreliosis.

Diagnosis of Lyme neuroborreliosis with antibodies to recombinant proteins DbpA, BBK32, and OspC, and VlsE IR6 peptide.

Three recombinant antigens, decorin binding protein A (DbpA), BBK32, and outer surface protein C (OspC), and IR(6) peptide of borrelial VlsE protein, were evaluated for the diagnosis of neuroborreliosis (NB), using cerebrospinal fluid (CSF) and serum samples from 89 patients. Their performances in enzyme-linked immunosorbent assay (ELISA) were compared with that of commercial flagella antigen. IgG ELISAs were performed with three variants of each recombinant antigen originating from Borrelia burgdorferi sensu stricto, B. afzelii and B. garinii, and with the IR(6) peptide. IgM antibodies were analysed against OspC and flagella. Of the patients whose CSF contained elevated anti-flagella IgG antibodies, 93% were positive for at least three of the new antigens. Of those with negative or borderline CSF anti-flagella antibodies, 51% were positive for three new antigens. Antibodies to BBK32 were detectable mainly in early disease. Antibodies to DbpA and IR(6) were observed in early and late NB. The use of the new antigens at presentation of the disease improved the laboratory diagnosis of NB. In IgG ELISAs, the diagnostic sensitivity of assays with the new antigens was between 75 and 88%, but was only 52% with the flagella antigen. The discriminatory power between patient and control samples appeared better in the CSF than in the serum. We suggest that assessment of CSF antibodies to at least two antigens, using either flagella and one of the new antigens or two of the new antigens, would improve the current diagnostic yield of NB.