Identification of DAGLA as an autoantibody target in cerebellar ataxia.

BACKGROUND: We aimed to investigate the clinical, imaging and fluid biomarker characteristics in patients with antidiacylglycerol lipase alpha (DAGLA)-autoantibody-associated cerebellitis. METHODS: Serum and cerebrospinal fliud (CSF) samples from four index patients were subjected to comprehensive autoantibody screening by indirect immunofluorescence assay (IIFA). Immunoprecipitation, mass spectrometry and recombinant protein assays were used to identify the autoantigen. Sera from 101 patients with various neurological symptoms and a similar tissue staining pattern as the index patient samples, and 102 healthy donors were analysed in recombinant cell-based IIFA (RC-IIFA) with the identified protein. Epitope characterisation of all positive samples was performed via ELISA, immunoblot, immunoprecipitation and RC-IIFA using different DAGLA fragments. RESULTS: All index patients were relatively young (age: 18-34) and suffered from pronounced gait ataxia, dysarthria and visual impairments. Paraclinical hallmarks in early-stage disease were inflammatory CSF changes and cerebellar cortex hyperintensity in MRI. Severe cerebellar atrophy developed in three of four patients within 6 months. All patient samples showed the same unclassified IgG reactivity with the cerebellar molecular layer. DAGLA was identified as the target antigen and confirmed by competitive inhibition experiments and DAGLA-specific RC-IIFA. In RC-IIFA, serum reactivity against DAGLA was also found in 17/101 disease controls, including patients with different clinical phenotypes than the one of the index patients, and in 1/102 healthy donors. Epitope characterisation revealed that 17/18 anti-DAGLA-positive control sera reacted with a C-terminal intracellular DAGLA 583-1042 fragment, while the CSF samples of the index patients targeted a conformational epitope between amino acid 1 and 157. CONCLUSIONS: We propose that anti-DAGLA autoantibodies detected in CSF, with a characteristic tissue IIFA pattern, represent novel biomarkers for rapidly progressive cerebellitis.

Beta-Trace Protein as a Potential Marker of Acute Kidney Injury: A Pilot Study.

INTRODUCTION: Acute kidney injury (AKI) is a frequent complication among patients in the intensive care unit (ICU). The limitations of serum Cr (sCr) in timely detecting AKI are well known. Beta-trace protein (BTP) is emerging as a novel endogenous glomerular filtration rate marker. The aim of this study was to explore the role of BTP as a marker of AKI. METHODS: Patients admitted to the ICU undergoing surgery were included. BTP, sCr, Cystatin C (CysC), and neutrophil gelatinase-associated lipocalin (NGAL) were measured preoperatively, postoperatively (post-op), and at the first (D1) and second (D2) post-op day. AKI was defined as an increase of sCr to ≥1.5-fold from baseline within 2 days after surgery. RESULTS: Of the 52 patients studied, 10 patients (19%) developed AKI. Patients with AKI were older (69.6 ± 10.7 vs. 58.1 ± 16.7 years, p = 0.043) and had a longer length of ICU stay (13 [IQR 6-49] vs. 6 [IQR 5-8] days, p = 0.032). Between the 2 groups, the evolution of BTP, sCr, CysC, and NGAL over time differed significantly, with overall higher values in the AKI group. ROC analysis for the detection of AKI within 2 days after surgery showed a great accuracy for BTP. The area under the curve (AUC) for BTP post-op; D1; and D2 was, respectively, 0.869 ± 0.049; 0.938 ± 0.035; and 0.943 ± 0.032. The discriminative power of a BTP measurement on D1 was superior in detecting AKI compared to NGAL (adjusted p value = 0.027). We could not detect a significant difference between the AUCs of other biomarkers (NGAL, sCr, and CysC). CONCLUSION: Serum BTP is a promising marker for diagnosing AKI in ICU patients undergoing surgery.

Evaluation of three commercially available ELISA kits for the determination of chromogranin A.

Chromogranin A (CgA) is currently the most valuable tumor biomarker for diagnostic work-up, management and follow-up of neuro-endocrine tumors (NET). The aim of our study was to compare three different commercially available CgA ELISA kits and to evaluate their analytical and clinical performance. CgA was measured with three different commercial ELISA kits on leftover sera from 40 patients: Chromoa R assay (Cis), Hu chromogranin A ELISA (Dia), and Neolisa chromogranin A (Euro). Analytical and clinical performance was evaluated by measuring precision, area under the ROC curve, sensitivity, specificity, positive and negative predictive value, correlation coefficients and Passing and Bablok regression analyses. Precision (CV%) was acceptable for all evaluated ELISA's (Cis 10.3%; Dia 9.8%; Euro 14.5%). The area under the curve (AUC) was comparable between the three assays (Cis 0.693; Dia 0.627; Euro 0.721). Sensitivity varied between 41.2% and 64.7%. Specificity ranged between 69.6% and 82.6%. Pairwise comparison revealed significant systematic and proportional differences when comparing Cis versus Dia (Cis = 25.30 + 1.94 Dia) and Euro versus Dia (Euro = 26.54 + 1.92 Dia). Analytical and clinical performance was comparable for the three ELISA's. CgA results obtained with different ELISA's are not interchangeable.AbbreviationsCgA: Chromogranin A; ELISA: Enzyme-linked immunosorbent assay; IRMA: Immunoradiometric assay; NET: Neuroendocrine tumors; PPI: Proton-pump inhibitors; RIA: Radioimmunoassay.

HPV vaccine stimulates cytotoxic activity of killer dendritic cells and natural killer cells against HPV-positive tumour cells.

Cervarix™ is approved as a preventive vaccine against infection with the human papillomavirus (HPV) strains 16 and 18, which are causally related to the development of cervical cancer. We are the first to investigate in vitro the effects of this HPV vaccine on interleukin (IL)-15 dendritic cells (DC) as proxy of a naturally occurring subset of blood DC, and natural killer (NK) cells, two innate immune cell types that play an important role in antitumour immunity. Our results show that exposure of IL-15 DC to the HPV vaccine results in increased expression of phenotypic maturation markers, pro-inflammatory cytokine production and cytotoxic activity against HPV-positive tumour cells. These effects are mediated by the vaccine adjuvant, partly through Toll-like receptor 4 activation. Next, we demonstrate that vaccine-exposed IL-15 DC in turn induce phenotypic activation of NK cells, resulting in a synergistic cytotoxic action against HPV-infected tumour cells. Our study thus identifies a novel mode of action of the HPV vaccine in boosting innate immunity, including killing of HPV-infected cells by DC and NK cells.

Clinical determinants and biomarkers associated with cardiac fibrosis after heart transplantation as assessed by magnetic resonance: Size matters.

Background: Cardiac fibrosis is increasingly recognized as a marker of worse outcomes in long-term follow-up after heart transplantation (HTX). We investigated the clinical determinants and biomarkers of focal and interstitial cardiac fibrosis as assessed with cardiac magnetic resonance (CMR). Methods: Consecutive HTX recipients underwent CMR with late gadolinium enhancement for focal myocardial fibrosis and T1 mapping for interstitial fibrosis. We calculated the correlations of these findings with clinical parameters, history, biomarkers of fibrosis (B-type natriuretic peptide (BNP), growth differentiation factor-15, galectin-3 and soluble ligand ST2) and echocardiography. Results: Forty-eight HTX patients were included: median age 63 ± 13 years, 11 ± 6 years after heart transplantation. Only donor weight (p 0.044) and the rate of a > 30 % mismatch between donor and recipient weight (p 0.02) were significantly different in patients with vs. without late LGE. Extracellular volume (ECV) was correlated with the weight mismatch between donor and recipient (r = 0.32, p 0.04), resulting in a higher ECV for oversized donors. BNP was the only biomarker of the four studied that was correlated with interstitial fibrosis as assessed by ECV (r = 0.35, p 0.04). T1 relaxation time was correlated with treated acute cellular rejection grade ≥ 2 (ISHLT grading) (r = 0.34, p 0.02). Conclusion: Both focal and interstitial fibrosis, as determined by CMR, after heart transplantation are correlated with donor and recipient weight mismatch. BNP was the only biomarker clinically relevant to interstitial cardiac fibrosis.

Blood transcriptomics to facilitate diagnosis and stratification in pediatric rheumatic diseases - a proof of concept study.

BACKGROUND: Transcriptome profiling of blood cells is an efficient tool to study the gene expression signatures of rheumatic diseases. This study aims to improve the early diagnosis of pediatric rheumatic diseases by investigating patients' blood gene expression and applying machine learning on the transcriptome data to develop predictive models. METHODS: RNA sequencing was performed on whole blood collected from children with rheumatic diseases. Random Forest classification models were developed based on the transcriptome data of 48 rheumatic patients, 46 children with viral infection, and 35 controls to classify different disease groups. The performance of these classifiers was evaluated by leave-one-out cross-validation. Analyses of differentially expressed genes (DEG), gene ontology (GO), and interferon-stimulated gene (ISG) score were also conducted. RESULTS: Our first classifier could differentiate pediatric rheumatic patients from controls and infection cases with high area-under-the-curve (AUC) values (AUC = 0.8 ± 0.1 and 0.7 ± 0.1, respectively). Three other classifiers could distinguish chronic recurrent multifocal osteomyelitis (CRMO), juvenile idiopathic arthritis (JIA), and interferonopathies (IFN) from control and infection cases with AUC ≥ 0.8. DEG and GO analyses reveal that the pathophysiology of CRMO, IFN, and JIA involves innate immune responses including myeloid leukocyte and granulocyte activation, neutrophil activation and degranulation. IFN is specifically mediated by antibacterial and antifungal defense responses, CRMO by cellular response to cytokine, and JIA by cellular response to chemical stimulus. IFN patients particularly had the highest mean ISG score among all disease groups. CONCLUSION: Our data show that blood transcriptomics combined with machine learning is a promising diagnostic tool for pediatric rheumatic diseases and may assist physicians in making data-driven and patient-specific decisions in clinical practice.

Diagnosis of catheter-related bloodstream infection in neonates: a study on the value of differential time to positivity of paired blood cultures.

OBJECTIVE: Diagnosis of neonatal catheter-related bloodstream infection (CRBSI) is currently based on isolation of identical bacterial species from bloodstream and catheter tip cultures. This requires removal of the catheter followed by the insertion of a new catheter. The objective of this study was to investigate whether differential time to positivity (DTP) of blood cultures drawn from paired peripheral vein and central vascular catheter is useful for diagnosing neonatal CRBSI, avoiding removal of the catheter. DESIGN: Retrospective observational study. SETTING: Neonatal intensive care unit, University Hospital of Antwerp, Belgium. PATIENTS: Neonates with probable and definite nosocomial bloodstream infection. INTERVENTIONS: All episodes of nosocomial bloodstream infection (NBSI) in an approximately 7.5-yr period were identified retrospectively. Definite NBSI episodes in which paired blood cultures were obtained were retained to calculate DTP, to determine the optimal DTP cutoff for the diagnosis of CRBSI, and to assess the validity of DTP for the diagnosis of CRBSI. MEASUREMENTS AND MAIN RESULTS: Of 32 NBSI episodes included in the study, 16 were CRBSI, seven were non-CRBSI, and nine were classified as "diagnosis uncertain." In CRBSI, blood cultures drawn from a central vascular catheter were positive earlier than those drawn from a peripheral vein (median 9.67 hrs vs. 21.58 hrs, p < .01). Median DTP was 10.42 hrs in CRBSI and -0.33 hrs in non-CRBSI (p = .01). The optimal DTP cutoff for the diagnosis of CRBSI was > or =1 hr (area under the receiver operating characteristic curve = 0.84 +/- 0.11), with a sensitivity of 94%, a specificity of 71%, a positive predictive value of 88%, and a negative predictive value of 83%. CONCLUSIONS: Differential time to positivity of paired blood cultures may have some potential in the diagnosis of catheter-related infections in neonatal intensive care unit patients and should be subjected to a prospective study.

Time to positivity of neonatal blood cultures: fast and furious?

The aim of this study was to determine the time to positivity (TTP) of neonatal blood cultures, to investigate differences between early onset versus late-onset sepsis, and non-proven versus proven sepsis, and to examine differences in TTP by organism type using a retrospective observational study at the Neonatal Intensive Care Unit, Antwerp University Hospital, Belgium. The subjects were 1828 neonates with suspected sepsis who were treated with antimicrobials for at least 3 days. The TTP was recorded for all episodes of suspected sepsis in an approximately 6.5 year period. A total of 2916 blood cultures were collected, of which 437 (15%) became positive. The overall TTP was 21.33 h (Q1-Q3 13.17-32.46). The difference between the median TTP in early onset versus late-onset sepsis was 0.83 h (22.00 versus 21.17 h, P=0.75). The median TTP for Gram-negative organisms was 11.17 h (Q1-Q3 8.84-15.67), whereas the median TTP for Gram-positive organisms was 23.59 h (Q1-Q3 15.29-34.58, P<0.001). In Gram-positive isolates, the median TTP for coagulase-negative staphylococci (CNS) was 26.67 h (Q1-Q3 19.00-38.17), whereas the median TTP for non-CNS was 12.83 h (Q1-Q3 10.50-18.17, P<0.001). The median TTP in proven sepsis was 20.17 h (Q1-Q3 13.00-30.37), whereas it was 29.67 h (Q1-Q3 21.17-50.63, P<0.001) in non-proven sepsis. TTP of neonatal blood cultures was significantly shorter for Gram-negative organisms. We suggest shortening the total incubation time of neonatal blood cultures to a maximum of 3 days. However, blood cultures collected in infants<72 h of age might require a longer incubation time. According to our results, it may be safe to narrow the antimicrobial spectrum to solely target Gram-positive bacteria when the culture is still negative after 48 h, and to cease antimicrobial therapy when the culture is still negative after 72 h in clinically well infants.

Multicentre evaluation of a new point-of-care test for the determination of NT-proBNP in whole blood.

BACKGROUND: The Roche CARDIAC proBNP point-of-care (POC) test is the first test intended for the quantitative determination of N-terminal pro-brain natriuretic peptide (NT-proBNP) in whole blood as an aid in the diagnosis of suspected congestive heart failure, in the monitoring of patients with compensated left-ventricular dysfunction and in the risk stratification of patients with acute coronary syndromes. METHODS: A multicentre evaluation was carried out to assess the analytical performance of the POC NT-proBNP test at seven different sites. RESULTS: The majority of all coefficients of variation (CVs) obtained for within-series imprecision using native blood samples was below 10% for both 52 samples measured ten times and for 674 samples measured in duplicate. Using quality control material, the majority of CV values for day-to-day imprecision were below 14% for the low control level and below 13% for the high control level. In method comparisons for four lots of the POC NT-proBNP test with the laboratory reference method (Elecsys proBNP), the slope ranged from 0.93 to 1.10 and the intercept ranged from 1.8 to 6.9. The bias found between venous and arterial blood with the POC NT-proBNP method was < or =5%. All four lots of the POC NT-proBNP test investigated showed excellent agreement, with mean differences of between -5% and +4%. No significant interference was observed with lipaemic blood (triglyceride concentrations up to 6.3 mmol/L), icteric blood (bilirubin concentrations up to 582 micromol/L), haemolytic blood (haemoglobin concentrations up to 62 mg/L), biotin (up to 10 mg/L), rheumatoid factor (up to 42 IU/mL), or with 50 out of 52 standard or cardiological drugs in therapeutic concentrations. With bisoprolol and BNP, somewhat higher bias in the low NT-proBNP concentration range (<175 ng/L) was found. Haematocrit values between 28% and 58% had no influence on the test result. Interference may be caused by human anti-mouse antibodies (HAMA) types 1 and 2. No significant influence on the results with POC NT-proBNP was found using volumes of 140-165 muL. High NT-proBNP concentrations above the measuring range of the POC NT-proBNP test did not lead to false low results due to a potential high-dose hook effect. CONCLUSIONS: The POC NT-proBNP test showed good analytical performance and excellent agreement with the laboratory method. The POC NT-proBNP assay is therefore suitable in the POC setting.