The Structural Architecture of an Infectious Mammalian Prion Using Electron Cryomicroscopy

The structure of the infectious prion protein (PrPSc), which is responsible for Creutzfeldt-Jakob disease in humans and bovine spongiform encephalopathy, has escaped all attempts at elucidation due to its insolubility and propensity to aggregate. PrPSc replicates by converting the non-infectious, cellular prion protein (PrPC) into the misfolded, infectious conformer through an unknown mechanism. PrPSc and its N-terminally truncated variant, PrP 27-30, aggregate into amorphous aggregates, 2D crystals, and amyloid fibrils. The structure of these infectious conformers is essential to understanding prion replication and the development of structure-based therapeutic interventions. Here we used the repetitive organization inherent to GPI-anchorless PrP 27-30 amyloid fibrils to analyze their structure via electron cryomicroscopy. Fourier-transform analyses of averaged fibril segments indicate a repeating unit of 19.1 Å. 3D reconstructions of these fibrils revealed two distinct protofilaments, and, together with a molecular volume of 18,990 Å3, predicted the height of each PrP 27-30 molecule as ~17.7 Å. Together, the data indicate a four-rung β-solenoid structure as a key feature for the architecture of infectious mammalian prions. Furthermore, they allow to formulate a molecular mechanism for the replication of prions. Knowledge of the prion structure will provide important insights into the self-propagation mechanisms of protein misfolding

Lifestyle and comorbid conditions as risk factors for community-acquired pneumonia in outpatient adults (NEUMO-ES-RISK project)

Introduction: Information about community-acquired pneumonia (CAP) risk in primary care is limited. We assess different lifestyle and comorbid conditions as risk factors (RF) for CAP in adults in primary care. Methods: A retrospective-observational-controlled study was designed. Adult CAP cases diagnosed at primary care in Spain between 2009 and 2013 were retrieved using the National Surveillance System of Primary Care Data (BiFAP). Age-matched and sex-matched controls were selected by incidence density sampling (ratio 2:1). Associations are presented as percentages and OR. Binomial regression models were constructed to avoid bias effects. Results: 51 139 patients and 102 372 controls were compared. Mean age (SD) was 61.4 (19.9) years. RF more significantly linked to CAP were: HIV (OR [95% CI]: 5.21 [4.35 to 6.27]), chronic obstructive pulmonary disease (COPD) (2.97 [2.84 to 3.12]), asthma (2.16 [2.07,2.26]), smoking (1.96 [1.91 to 2.02]) and poor dental hygiene (1.45 [1.41 to 1.49]). Average prevalence of any RF was 82.2% in cases and 69.2% in controls (2.05 [2.00 to 2.10]). CAP rate increased with the accumulation of RF and age: risk associated with 1RF was 1.42 (1.37 to 1.47) in 18-60-year-old individuals vs 1.57 (1.49 to 1.66) in >60 years of age, with 2RF 1.88 (1.80 to 1.97) vs 2.35 (2.23, 2.48) and with >/= 3 RF 3.11 (2.95, 3.30) vs 4.34 (4.13 to 4.57). Discussion: Prevalence of RF in adult CAP in primary care is high. Main RFs associated are HIV, COPD, asthma, smoking and poor dental hygiene. Our risk stacking results could help clinicians identify patients at higher risk of pneumonia

Increased Serum Levels of sCD14 and sCD163 Indicate a Preponderant Role for Monocytes in COVID-19 Immunopathology

Background: Emerging evidence indicates a potential role for monocytes in COVID-19 immunopathology. We investigated two soluble markers of monocyte activation, sCD14 and sCD163, in COVID-19 patients, with the aim of characterizing their potential role in monocyte-macrophage disease immunopathology. To the best of our knowledge, this is the first study of its kind. Methods: Fifty-nine SARS-Cov-2 positive hospitalized patients, classified according to ICU or non-ICU admission requirement, were prospectively recruited and analyzed by ELISA for levels of sCD14 and sCD163, along with other laboratory parameters, and compared to a healthy control group. Results: sCD14 and sCD163 levels were significantly higher among COVID-19 patients, independently of ICU admission requirement, compared to the control group. We found a significant correlation between sCD14 levels and other inflammatory markers, particularly Interleukin-6, in the non-ICU patients group. sCD163 showed a moderate positive correlation with the time lapsed from admission to sampling, independently of severity group. Treatment with corticoids showed an interference with sCD14 levels, whereas hydroxychloroquine and tocilizumab did not. Conclusions: Monocyte-macrophage activation markers are increased and correlate with other inflammatory markers in SARS-Cov-2 infection, in association to hospital admission. These data suggest a preponderant role for monocyte-macrophage activation in the development of immunopathology of COVID-19 patients

Diagnostic Test Accuracy of a 2-Transcript Host RNA Signature for Discriminating Bacterial vs Viral Infection in Febrile Children.

IMPORTANCE: Because clinical features do not reliably distinguish bacterial from viral infection, many children worldwide receive unnecessary antibiotic treatment, while bacterial infection is missed in others. OBJECTIVE: To identify a blood RNA expression signature that distinguishes bacterial from viral infection in febrile children. DESIGN, SETTING, AND PARTICIPANTS: Febrile children presenting to participating hospitals in the United Kingdom, Spain, the Netherlands, and the United States between 2009-2013 were prospectively recruited, comprising a discovery group and validation group. Each group was classified after microbiological investigation as having definite bacterial infection, definite viral infection, or indeterminate infection. RNA expression signatures distinguishing definite bacterial from viral infection were identified in the discovery group and diagnostic performance assessed in the validation group. Additional validation was undertaken in separate studies of children with meningococcal disease (n = 24) and inflammatory diseases (n = 48) and on published gene expression datasets. EXPOSURES: A 2-transcript RNA expression signature distinguishing bacterial infection from viral infection was evaluated against clinical and microbiological diagnosis. MAIN OUTCOMES AND MEASURES: Definite bacterial and viral infection was confirmed by culture or molecular detection of the pathogens. Performance of the RNA signature was evaluated in the definite bacterial and viral group and in the indeterminate infection group. RESULTS: The discovery group of 240 children (median age, 19 months; 62% male) included 52 with definite bacterial infection, of whom 36 (69%) required intensive care, and 92 with definite viral infection, of whom 32 (35%) required intensive care. Ninety-six children had indeterminate infection. Analysis of RNA expression data identified a 38-transcript signature distinguishing bacterial from viral infection. A smaller (2-transcript) signature (FAM89A and IFI44L) was identified by removing highly correlated transcripts. When this 2-transcript signature was implemented as a disease risk score in the validation group (130 children, with 23 definite bacterial, 28 definite viral, and 79 indeterminate infections; median age, 17 months; 57% male), all 23 patients with microbiologically confirmed definite bacterial infection were classified as bacterial (sensitivity, 100% [95% CI, 100%-100%]) and 27 of 28 patients with definite viral infection were classified as viral (specificity, 96.4% [95% CI, 89.3%-100%]). When applied to additional validation datasets from patients with meningococcal and inflammatory diseases, bacterial infection was identified with a sensitivity of 91.7% (95% CI, 79.2%-100%) and 90.0% (95% CI, 70.0%-100%), respectively, and with specificity of 96.0% (95% CI, 88.0%-100%) and 95.8% (95% CI, 89.6%-100%). Of the children in the indeterminate groups, 46.3% (63/136) were classified as having bacterial infection, although 94.9% (129/136) received antibiotic treatment. CONCLUSIONS AND RELEVANCE: This study provides preliminary data regarding test accuracy of a 2-transcript host RNA signature discriminating bacterial from viral infection in febrile children. Further studies are needed in diverse groups of patients to assess accuracy and clinical utility of this test in different clinical settings

Life-threatening infections in children in Europe (the EUCLIDS Project): a prospective cohort study

Background Sepsis and severe focal infections represent a substantial disease burden in children admitted to hospital. We aimed to understand the burden of disease and outcomes in children with life-threatening bacterial infections in Europe. Methods The European Union Childhood Life-threatening Infectious Disease Study (EUCLIDS) was a prospective, multicentre, cohort study done in six countries in Europe. Patients aged 1 month to 18 years with sepsis (or suspected sepsis) or severe focal infections, admitted to 98 participating hospitals in the UK, Austria, Germany, Lithuania, Spain, and the Netherlands were prospectively recruited between July 1, 2012, and Dec 31, 2015. To assess disease burden and outcomes, we collected demographic and clinical data using a secured web-based platform and obtained microbiological data using locally available clinical diagnostic procedures. Findings 2844 patients were recruited and included in the analysis. 1512 (53·2%) of 2841 patients were male and median age was 39·1 months (IQR 12·4–93·9). 1229 (43·2%) patients had sepsis and 1615 (56·8%) had severe focal infections. Patients diagnosed with sepsis had a median age of 27·6 months (IQR 9·0–80·2), whereas those diagnosed with severe focal infections had a median age of 46·5 months (15·8–100·4; p<0·0001). Of 2844 patients in the entire cohort, the main clinical syndromes were pneumonia (511 [18·0%] patients), CNS infection (469 [16·5%]), and skin and soft tissue infection (247 [8·7%]). The causal microorganism was identified in 1359 (47·8%) children, with the most prevalent ones being Neisseria meningitidis (in 259 [9·1%] patients), followed by Staphylococcus aureus (in 222 [7·8%]), Streptococcus pneumoniae (in 219 [7·7%]), and group A streptococcus (in 162 [5·7%]). 1070 (37·6%) patients required admission to a paediatric intensive care unit. Of 2469 patients with outcome data, 57 (2·2%) deaths occurred: seven were in patients with severe focal infections and 50 in those with sepsis. Interpretation Mortality in children admitted to hospital for sepsis or severe focal infections is low in Europe. The disease burden is mainly in children younger than 5 years and is largely due to vaccine-preventable meningococcal and pneumococcal infections. Despite the availability and application of clinical procedures for microbiological diagnosis, the causative organism remained unidentified in approximately 50% of patients

Diagnostic Test Accuracy of a 2-Transcript Host RNA Signature for Discriminating Bacterial vs Viral Infection in Febrile Children

Importance: Because clinical features do not reliably distinguish bacterial from viral infection, many children worldwide receive unnecessary antibiotic treatment, while bacterial infection is missed in others. Objective: To identify a blood RNA expression signature that distinguishes bacterial from viral infection in febrile children. Design, Setting, and Participants: Febrile children presenting to participating hospitals in the United Kingdom, Spain, the Netherlands, and the United States between 2009-2013 were prospectively recruited, comprising a discovery group and validation group. Each group was classified after microbiological investigation as having definite bacterial infection, definite viral infection, or indeterminate infection. RNA expression signatures distinguishing definite bacterial from viral infection were identified in the discovery group and diagnostic performance assessed in the validation group. Additional validation was undertaken in separate studies of children with meningococcal disease (n = 24) and inflammatory diseases (n = 48) and on published gene expression datasets. Exposures: A 2-transcript RNA expression signature distinguishing bacterial infection from viral infection was evaluated against clinical and microbiological diagnosis. Main Outcomes and Measures: Definite bacterial and viral infection was confirmed by culture or molecular detection of the pathogens. Performance of the RNA signature was evaluated in the definite bacterial and viral group and in the indeterminate infection group. Results: The discovery group of 240 children (median age, 19 months; 62% male) included 52 with definite bacterial infection, of whom 36 (69%) required intensive care, and 92 with definite viral infection, of whom 32 (35%) required intensive care. Ninety-six children had indeterminate infection. Analysis of RNA expression data identified a 38-transcript signature distinguishing bacterial from viral infection. A smaller (2-transcript) signature (FAM89A and IFI44L) was identified by removing highly correlated transcripts. When this 2-transcript signature was implemented as a disease risk score in the validation group (130 children, with 23 definite bacterial, 28 definite viral, and 79 indeterminate infections; median age, 17 months; 57% male), all 23 patients with microbiologically confirmed definite bacterial infection were classified as bacterial (sensitivity, 100% [95% CI, 85%-100%]) and 27 of 28 patients with definite viral infection were classified as viral (specificity, 96.4% [95% CI, 89.3%-100%]). When applied to additional validation datasets from patients with meningococcal and inflammatory diseases, bacterial infection was identified with a sensitivity of 91.7% (95% CI, 79.2%-100%) and 90.0% (95% CI, 70.0%-100%), respectively, and with specificity of 96.0% (95% CI, 88.0%-100%) and 95.8% (95% CI, 89.6%-100%). Of the children in the indeterminate groups, 46.3% (63/136) were classified as having bacterial infection, although 94.9% (129/136) received antibiotic treatment. Conclusions and Relevance: This study provides preliminary data regarding test accuracy of a 2-transcript host RNA signature discriminating bacterial from viral infection in febrile children. Further studies are needed in diverse groups of patients to assess accuracy and clinical utility of this test in different clinical settings.This work was supported by the Imperial College Comprehensive Biomedical Research Centre (DMPED P26077); National Institute of Health Research (NIHR) Senior Investigator award (Dr Levin); Great Ormond St Hospital Charity (V1401) (Dr Wright); European Union’s Seventh Framework Program (EC-GA 279185) (EUCLIDS) (Dr Herberg); Imperial College-Wellcome Trust Antimicrobial Research Collaborative (ARC) Early Career Fellowship (RSRO 54990) (Dr Kaforou); Spanish Research Program (FIS; PI10/00540 and Intensificación actividad investigadora of National Plan I + D + I and FEDER funds) and Regional Galician funds (Promotion of Research Project 10 PXIB 918 184 PR) (Dr Martinón-Torres); Southampton NIHR Wellcome Trust Clinical Research Facility and NIHR Wessex Local Clinical Research Network; and Academic Medical Centre Amsterdam MD/PhD program 2013 (Ms Barendregt). The UK meningococcal disease cohort was established with grant support from the Meningitis Research Foundation (United Kingdom); the inflammatory disease cohort was supported by a Macklin Foundation grant (Dr Burns), National Institutes of Health grant U54-HL108460 (Dr Burns); and The Hartwell Foundation and The Harold Amos Medical Faculty Development Program/Robert Wood Johnson Foundation (Dr Tremoulet)

Osteoarticular Infections in Pediatric Hospitals in Europe: A Prospective Cohort Study From the EUCLIDS Consortium

BACKGROUND: Pediatric osteoarticular infections (POAIs) are serious diseases requiring early diagnosis and treatment. METHODS: In this prospective multicenter cohort study, children with POAIs were selected from the European Union Childhood Life-threatening Infectious Diseases Study (EUCLIDS) database to analyze their demographic, clinical, and microbiological data. RESULTS: A cohort of 380 patients with POAIs, 203 with osteomyelitis (OM), 158 with septic arthritis (SA), and 19 with both OM and SA, was analyzed. Thirty-five patients were admitted to the Pediatric Intensive Care Unit; out of these, six suffered from shock, one needed an amputation of the right foot and of four left toes, and two had skin transplantation. According to the Pediatric Overall Performance Score, 36 (10.5%) showed a mild overall disability, 3 (0.8%) a moderate, and 1 (0.2%) a severe overall disability at discharge. A causative organism was detected in 65% (247/380) of patients. Staphylococcus aureus (S. aureus) was identified in 57.1% (141/247) of microbiological confirmed cases, including 1 (0.7%) methicillin-resistant S. aureus (MRSA) and 6 (4.2%) Panton-Valentine leukocidin (PVL)-producing S. aureus, followed by Group A Streptococcus (18.2%) and Kingella kingae (8.9%). K. kingae and PVL production in S. aureus were less frequently reported than expected from the literature. CONCLUSION: POAIs are associated with a substantial morbidity in European children, with S. aureus being the major detected pathogen. In one-third of patients, no causative organism is identified. Our observations show an urgent need for the development of a vaccine against S. aureus and for the development of new microbiologic diagnostic guidelines for POAIs in European pediatric hospitals

Identification of regulatory variants associated with genetic susceptibility to meningococcal disease

Non-coding genetic variants play an important role in driving susceptibility to complex diseases but their characterization remains challenging. Here, we employed a novel approach to interrogate the genetic risk of such polymorphisms in a more systematic way by targeting specific regulatory regions relevant for the phenotype studied. We applied this method to meningococcal disease susceptibility, using the DNA binding pattern of RELA - a NF-kB subunit, master regulator of the response to infection - under bacterial stimuli in nasopharyngeal epithelial cells. We designed a custom panel to cover these RELA binding sites and used it for targeted sequencing in cases and controls. Variant calling and association analysis were performed followed by validation of candidate polymorphisms by genotyping in three independent cohorts. We identified two new polymorphisms, rs4823231 and rs11913168, showing signs of association with meningococcal disease susceptibility. In addition, using our genomic data as well as publicly available resources, we found evidences for these SNPs to have potential regulatory effects on ATXN10 and LIF genes respectively. The variants and related candidate genes are relevant for infectious diseases and may have important contribution for meningococcal disease pathology. Finally, we described a novel genetic association approach that could be applied to other phenotypes

Bacteremia in Childhood Life-Threatening Infections in Urban Gambia: EUCLIDS in West Africa

Background: The limited availability of microbiology services in sub-Saharan Africa impedes accurate diagnosis of bacterial pathogens and understanding of trends in prevalence and antibiotic sensitivities. We aimed to characterize bacteremia among hospitalized children in The Gambia and to identify factors associated with bacteremia and mortality. Methods: We prospectively studied children presenting wit