Retrospective Evaluation of Various Serological Assays and Multiple Parameters for Optimal Diagnosis of Lyme Neuroborreliosis in a Routine Clinical Setting.

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.

The Performance of Nine Commercial Serological Screening Assays for the Diagnosis of Lyme Borreliosis: a Multicenter Modified Two-Gate Design Study.

In this retrospective study, the performance of nine serological screening assays for Lyme borreliosis (LB) diagnostics was evaluated using a study population of LB cases and controls. Sera derived from 74 well-defined LB cases and 122 controls were included. The LB cases were diagnosed with erythema migrans (EM; n = 11), Lyme neuroborreliosis (LNB; n = 35), Lyme arthritis (LA; n = 20), or acrodermatitis chronica atrophicans (ACA; n = 8). Controls comprised 74 age- and gender-matched healthy individuals and 48 patients with other diseases with anticipated high rates of cross-reactivity. The assays under evaluation were selected based on a literature review and expected continued availability with CE marking under the new in vitro diagnostic regulation (European Union) 2017/746. The overall sensitivity (IgG and IgM results combined) among LB cases ranged between 54.5% (6 of 11) and 90.9% (10 of 11) for EM patients and between 97.1% (34 of 35) and 100% for patients with LNB, LA, and ACA. The positivity rate ranged between 8.1% (6 of 74) and 29.7% (22 of 74) among the healthy controls and between 22.9% (11 of 48) and 64.6% (31 of 48) among the cross-reactivity controls. The IgM results were more heterogeneous than the IgG and IgM/IgG results and did not contribute to the overall sensitivity but substantially increased the positivity rates among the controls. In conclusion, all evaluated Borrelia serological screening assays performed comparably with respect to early- and late-disseminated LB. The addition of an IgM assay to the screening of Borrelia-specific IgG antibodies had no added value for the diagnosis of Lyme borreliosis. IMPORTANCE Serology plays an important role in the diagnosis of Lyme borreliosis. Guidelines prescribe a two-tier testing algorithm in which a highly sensitive screening assay is used for screening and reactive sera are retested with an immunoblot to reduce false positivity rates. Recently, two commonly used screening assays were discontinued, including the very well-performing C6 Lyme enzyme-linked immunosorbent assay (ELISA) (Immunetics). This study provides an evaluation of the performance of nine different Borrelia serology screening assays, eight with expected future availably and the C6 Lyme ELISA, using a well-defined study panel of Lyme borreliosis patients, healthy population controls, and cross-reactivity controls. Evaluation data on multiple assays aid diagnostic laboratories in their choice for a reliable Borrelia serology screening assay to improve their diagnostic algorithm for Lyme borreliosis.

The Usefulness of Two CXCL13 Assays on Cerebrospinal Fluid for the Diagnosis of Lyme Neuroborreliosis: a Retrospective Study in a Routine Clinical Setting.

Recent studies have shown elevated levels of the B-cell chemokine (C-X-C motif) ligand 13 (CXCL13) in the cerebrospinal fluid (CSF) of patients with early Lyme neuroborreliosis (LNB). In this retrospective study, we evaluated the diagnostic performance of the Quantikine CXCL13 enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Inc., MN, USA) and the recomBead CXCL13 assay (Mikrogen, Neuried, Germany) for the detection of CXCL13 in CSF. All consecutive patients from whom a CSF and a serum sample had been collected between August 2013 and June 2016 were eligible for inclusion. Patients suspected of LNB were classified as definite, possible, or non-LNB according to the guidelines of the European Federation of Neurological Societies (EFNS). Due to the limited number of LNB patients in the predefined study period, additional LNB patients were included from outside this period. In total, 156 patients (150 consecutive patients and 6 additional LNB patients) were included. Seven (4.5%) were classified as definite, eight (5.1%) as possible, and 141 (90.4%) as non-LNB patients. Receiver operating characteristic (ROC) curve analysis comparing definite-LNB patients with non-LNB patients showed a cutoff value of 85.9 pg/ml for the Quantikine CXCL13 ELISA and 252.2 pg/ml for the recomBead CXCL13 assay. The corresponding sensitivity was 100% (95% confidence interval [CI], 100% to 100%) for both, and the corresponding specificities were 98.6% (95% CI, 96.5% to 100%) for the CXCL13 ELISA and 97.2% (95% CI, 93.6% to 100%) for the recomBead CXCL13 assay. This study showed that CXCL13 in CSF can be of additional value for the diagnosis of LNB.

Consequences of the Edge Effect in a Commercial Enzyme-Linked Immunosorbent Assay for the Diagnosis of Lyme Neuroborreliosis.

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.

CD1b presents self and Borrelia burgdorferi diacylglycerols to human T cells.

Lyme disease is a common multisystem disease caused by infection with a tick-transmitted spirochete, Borrelia burgdorferi and related Borrelia species. The monoglycosylated diacylglycerol known as B. burgdorferi glycolipid II (BbGL-II) is a major target of antibodies in sera from infected individuals. Here, we show that CD1b presents BbGL-II to human T cells and that the TCR mediates the recognition. However, we did not detect increased frequency of CD1b-BbGL-II binding T cells in the peripheral blood of Lyme disease patients compared to controls. Unexpectedly, mapping the T cell specificity for BbGL-II-like molecules using tetramers and activation assays revealed a concomitant response to CD1b-expressing APCs in absence of BbGL-II. Further, among all major classes of self-lipid tested, BbGL-II responsive TCRs show strong cross-reactivity to diacylglycerol, a self-lipid antigen with structural similarities to BbGL-II. Extending prior work on MHC and CD1b, CD1c, and CD1d proteins, this study provides evidence for cross-reactive CD1b-restricted T cell responses to bacterial and self-antigens, and identifies chemically defined targets for future discovery of self and foreign antigen cross-reactive T cells.

Positive predictive value of ELISpot in BAL and pleural fluid from patients with suspected pulmonary tuberculosis.

BACKGROUND: The aim of this study was to evaluate the positive predictive value (PPV) of ELISpot in bronchoalveolar lavage (BAL) and pleural fluid for the diagnosis of active tuberculosis (TB) in real-life clinical practice, together with the added value of a cut-off >1.0 for the ratio between the extra-sanguineous and systemic interferon-gamma responses in positive samples. METHODS: A retrospective, single-centre study was performed. Patients with positive ELISpot in BAL and pleural fluid were included. RESULTS: The PPV for TB in patients with positive ELISpot in BAL (n = 40) was 64.9%, which increased to 82.6% for the ESAT-6 panel and 71.4% for the CFP-10 panel after the introduction of a cut-off >1.0 for the ratio between the BAL and blood interferon-gamma responses. In patients with positive ELISpot in pleural fluid (n = 16), the PPV for TB was 85.7%, which increased to 91.7% for the ESAT-6 panel and 92.3% for the CFP-10 panel after the introduction of a cut-off >1.0 for the ratio between the pleural fluid and blood interferon-gamma responses. CONCLUSIONS: This report describes the PPV of ELISpot in BAL and pleural fluid for the diagnosis of active TB in real-life clinical practice. The results indicate the possibility of an increase of the PPV using a cut-off >1.0 for the ratio between the extra-sanguineous and systemic interferon-gamma responses. Further studies are needed to underline this ratio-approach and to evaluate the full diagnostic accuracy of ELISpot in extra-sanguineous fluids like BAL and pleural fluid.

Changes in the genomic content of circulating Bordetella pertussis strains isolated from the Netherlands, Sweden, Japan and Australia: adaptive evolution or drift?

BACKGROUND: Bordetella pertussis is the causative agent of human whooping cough (pertussis) and is particularly severe in infants. Despite worldwide vaccinations, whooping cough remains a public health problem. A significant increase in the incidence of whooping cough has been observed in many countries since the 1990s. Several reasons for the re-emergence of this highly contagious disease have been suggested. A particularly intriguing possibility is based on evidence indicating that pathogen adaptation may play a role in this process. In an attempt to gain insight into the genomic make-up of B. pertussis over the last 60 years, we used an oligonucleotide DNA microarray to compare the genomic contents of a collection of 171 strains of B. pertussis isolates from different countries. RESULTS: The CGH microarray analysis estimated the core genome of B. pertussis, to consist of 3,281 CDSs that are conserved among all B. pertussis strains, and represent 84.8% of all CDSs found in the 171 B. pertussis strains. A total of 64 regions of difference consisting of one or more contiguous CDSs were identified among the variable genes. CGH data also revealed that the genome size of B. pertussis strains is decreasing progressively over the past 60 years. Phylogenetic analysis of microarray data generated a minimum spanning tree that depicted the phylogenetic structure of the strains. B. pertussis strains with the same gene content were found in several different countries. However, geographic specificity of the B. pertussis strains was not observed. The gene content was determined to highly correlate with the ptxP-type of the strains. CONCLUSIONS: An overview of genomic contents of a large collection of isolates from different countries allowed us to derive a core genome and a phylogenetic structure of B. pertussis. Our results show that B. pertussis is a dynamic organism that continues to evolve.

Comparative genomic profiling of Dutch clinical Bordetella pertussis isolates using DNA microarrays: identification of genes absent from epidemic strains.

BACKGROUND: Whooping cough caused by Bordetella pertussis in humans, is re-emerging in many countries despite vaccination. Several studies have shown that significant shifts have occurred in the B. pertussis population resulting in antigenic divergence between vaccine strains and circulating strains and suggesting pathogen adaptation. In the Netherlands, the resurgence of pertussis is associated with the rise of B. pertussis strains with an altered promoter region for pertussis toxin (ptxP3). RESULTS: We used Multi-Locus Sequence Typing (MLST), Multiple-Locus Variable Number of Tandem Repeat Analysis (MLVA) and microarray-based comparative genomic hybridization (CGH) to characterize the ptxP3 strains associated with the Dutch epidemic. For CGH analysis, we developed an oligonucleotide (70-mers) microarray consisting of 3,581 oligonucleotides representing 94% of the gene repertoire of the B. pertussis strain Tohama I. Nine different MLST profiles and 38 different MLVA types were found in the period 1993 to 2004. Forty-three Dutch clinical isolates were analyzed with CGH, 98 genes were found to be absent in at least one of the B. pertussis strains tested, these genes were clustered in 8 distinct regions of difference. CONCLUSION: The presented MLST, MLVA and CGH-analysis identified distinctive characteristics of ptxP3 B. pertussis strains -the most prominent of which was a genomic deletion removing about 23,000 bp. We propose a model for the emergence of ptxP3 strains.