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Legionella Infections Molecular detection and typing of Legionella infections Sophie JARRAUD ESCMID eLibrary © by author Disclosure Research Grants, consulting: Diagenode, IDEEX, bioMerieux, Orgentec, Alere ESCMID eLibrary © by author Legionella Legionnaires’ disease (LD) infections Pontiac fever = ü atypical pneumonia (eg. legionellosis ü a mild, flu-like disease extrapulmonary findings) ü without pneumonia ü severe pneumonia ü high attack rate ü Mortality: 10-30% ü short incubation period ü rapid recoveries Extrapulmonary forms ü with or without pneumonia ü especially due to L. non pneumophila ESCMIDü endocarditis, eLibrary arthritis … © by author Legionella associated to LD genus Legionella ~ 25 species: at least once isolated from humans L. pneumophila ~ 91% L. longbeachae ~ 5% cases worldwide ~ 60 L. micdadei ~ 2% species (with isolates available) Other species ~ 2% (Yu et al., J. infect. Dis. 2002) Legionella longbeachae : ~ 30% - 50% of the cases in Australia and New Zealand L. pneumophila is clearly predominant in human infection followed ESCMIDby L.eLibrary longbeachae © by author Legionella associated to LD Legionella pneumophila : ~ 91% of the cases worldwide (Yu et al., J. infect. Dis. 186:127-128) 16 L. pneumophila sg1: ~ 88,6% of cases serogroups ~ 30% of isolates from environment Higher virulence or better niche adaptation of ESCMIDLegionella eLibrary pneumophila Sg 1? © by author What is the use of Legionella PCR in Europe? Beauté J. et al. Legionnaires’ disease in Europe, 2011 to 2015. Eurosurveillance 2017 33,809 laboratory tests 29 reporting countries PCR, 6,8% other tests culture, (serology), 10,8% 1,1% Proportion of cases diagnosed by PCR increased 4.3% (2011) to 10.5% (2015) urinary antigen, ESCMID eLibrary78,2% © by author What are major advantages of Legionella PCR ? 1- main advantage: diagnosis of all Legionella spp. infections – largely unknown and suspected to be under-diagnosed – Legionella sp.: 16S rRNA, 5S rRNA, intergenic spacer region 23S-5S, • Species identification available after sequencing – L. pneumophila: mip gene (or other) ü L. non-pneumophila associated to immunocompromised patients ü Higher mortality due to these patients ESCMID eLibrary © by author 2- qPCR as a pronostic marker? q In respiratory samples Maurin et al., CMI, 2010 (n = 70); Murdoch et al., CID, 2013 (n = 114) • Correlation of bacterial load on admission with • Pneumonia severity index (Fine class or CURB-65) • Need for hospitalization in ICU • Duration of hospitalization • Mortality q limitation: standardization sampling procedure q Sputum and tracheal aspirate provide better results than Bronchial aspirate samples • higher positive rates ESCMID• higher bacterial loads eLibrary © by author 2- qPCR as a pronostic marker? q In respiratory samples Maurin et al., CMI, 2010 (n = 70); Murdoch et al., CID, 2013 (n = 114) • Correlation of bacterial load on admission with • Pneumonia severity index (Fine class or CURB-65) • Need for hospitalization in ICU • Duration of hospitalization • Mortality q In blood samples Lindsay et al., 1994; Murdoch et al., 1996; Murdoch et al., 1999; Murdoch et al., 2000; Matsiota-Bernard et al., 2000; Diederen et al., 2007; Van de Veerdonk et al. 2009; Mentasti et al., 2012 • ESCMIDCorrelation between PCR on serum eLibrary / LD severity not clear-cut © by author 3- Molecular method for detecting antibiotic resistance • resistance to antibiotics seems not a major problem for LD – Only 2 descriptions of resistance to Quinolone ! • 1 isolate from patient (Bruin et al. JAC 2014) • 1 description by targeted NGS (Shadoud et al. EBIOM 2015) – No description (from patient / environment) for macrolides and Rifampicin • Isolates available for only 12% of cases (diagnosed in Europe) • Development of molecular methods to confirm these data and to perform surveillance ESCMID eLibrary © by author 3- Molecular method for detecting antibiotic resistance • Molecular mechanisms associated to resistance determined on in vitro selected resistant mutants (Nielsen et al., 2000; Almahmoud et al., 2009; Descours et al., 2017) PCR and real time PCR assay Digital PCR Targeted sanger sequencing targeting mutations NGS method Resistant Quantification FQ resistance WT 1:1,000 resistant/ susceptible allele ratios Almahmoud et al., 2009 Hennebique et al., 2017 Shadoud et al. 2015 ESCMIDShadoud et al. 2015 eLibrary © by author Targeted Next Generation Sequencing (NGS) ü ability to detect minority subpopulations ü demonstration of in vivo selection of FQ-resistant mutants of Legionella for 2 patients during hospitalisation Shadoud et al. EBIOM (2015) Day of sampling % of gyrA83 mutant (D0= diagnosis) alleles Patient #2 D0 2.9 % D4 94 % Patient #4 D0 1.05 % D3 75 % D5 95 % % ESCMIDof mutated alleles in respiratory sampleseLibrary from control patients: <0.5% © by author 4- Rapid point of care diagnostic tests for viral and bacterial respiratory tract infections Why does Legionella a good candidate as target ? ü Severe pneumonia: rapid diagnosis ü Several mimics of LD: rapid differential diagnosis ü Potential co-infection: influenza virus / others ? ü Urinary antigen tests detect only Lp1 ESCMID eLibrary © by author ePlex® BIOFIRE® FILMARRAY® Respiratory Pathogen Pneumonia Panel (GenMark Diagnostics) (bioMéreux) Upper respiratory infection Lower respiratory infection Panels available for Legionella pneumophila DiaCORE® Unyvero® HPN Respiratory Panel (Curetis) (STAT Dx) Hospitalized pneumonia ESCMID eLibrary © by author ePlex® nasopharyngeal swabs not adapted to LD Respiratory Pathogen (GenMark Diagnostics) Upper respiratory infection CE-IVD cleared for nasopharyngeal swabs only R. H. T. Nijhuis, J Clin Microbiol. 2017 Analysed 6 LD from sputum = positive result DiaCORE® Respiratory Panel (STAT Dx) nasopharyngeal swabs ESCMIDNo publication eLibrary © by author Authorization seeked from the FDA BIOFIRE® FILMARRAY® Pneumonia Panel ü Sputum - endotracheal aspirate (bioMéreux) ü Bronchoalveolar Lavage ü No publication Lower respiratory infection ü Poster at ECCMID: only few LD tested Unyvero® HPN ü No publication (Curetis) ü Poster at ECCMID: only few LD tested Hospitalized pneumonia ü Incidence of LD is low ü We need also evaluation from National Centers ESCMIDof Legionella eLibrary © by author 5- 16S rRNA targeted metagenomic on clinical samples Which applications for Legionella infections? • Diversity of bacteria in clinical samples from LD – What are the dynamics of the lung microbiome and the pathogen during Legionella infection and antibiotic treatment? – Are there other bacteria needed to treat? • Legionella species identification – Are there several Legionella species in the clinical sample? ESCMIDLung microbiome during LDeLibrary - only very few published studies © by author What are the dynamics of the lung microbiome and the pathogen during Legionella infection and antibiotic treatment? Ana Elena PEREZ COBAS Carmen Buchrieser’s team Christophe Christophe Ginevra Rusniok ESCMID eLibrary © by author Persistent Legionella infection during hospitalisation Thoracic CT scan: chest X-ray a voluminous lung abscess Resection of abscess rapid clinical improvement D4 D5 D14 D24 D33 D42 D54 D62 D72 Erythromycin + Erythromycin + nitroimidazole Levofloxacin levofloxacin + levoflo + imidazole + Rifampicin (3 months) ESCMIDBAL: Legionella positive culture eLibrary © by author METHODS Illumina technology PCR 16S rRNA V3-V4 ITS1 Operational taxonomic units Bioinformatics algorythms ESCMID eLibraryAdapted from Hong et al , 2016 Clin. Microb. Rev. © by author RESULTS 16S rRNA sequencing Antibiotics 100 Actinobacteria Actinomyces Actinobacteria Atopobium Legionella Actinobacteria Corynebacterium Bacteroidetes Alloprevotella Bacteroidetes Prevotella 80 Firmicutes Abiotrophia Firmicutes Enterococcus Firmicutes Gemella Firmicutes Granulicatella Firmicutes Oribacterium Firmicutes Peptostreptococcus 60 Firmicutes Shuttleworthia Firmicutes Staphylococcus Firmicutes Streptococcus Firmicutes Veillonella Firmicutes uc_Lachnospiraceae Fusobacteria Fusobacterium 40 Proteobacteria Haemophilus Proteobacteria Legionella Tenericutes Mycoplasma Others 20 Very high abundance of Legionella in the microbiome during infection (75%) 0 Antibiotic therapy changes the diversity of the microbiome due to resistant species day 5 BAL day day 24 BAL 24 day 33 BAL day BAL 42 day day 14 BAL 14 day day 4 sputum4 day (Prevotella, Alloprevotella, Fusobacterium) ESCMID abscess 42 day eLibrary © by author RESULTS 16S rRNA sequencing 100 Actinobacteria Actinomyces Actinobacteria Atopobium Bacteroidetes Alloprevotella Sputum versus BAL Bacteroidetes Prevotella Firmicutes Abiotrophia High-abundance taxa > 2% (%) 80 Firmicutes Gemella Firmicutes Granulicatella Firmicutes Peptostreptococcus Firmicutes Streptococcus Firmicutes Veillonella Sputum Proteobacteria Legionella 60 Others Streptococcus (43%) abondance Prevotella (13%) Legionella Gemella (13%) 40 Legionella (3%) Relative 20 BAL Legionella (99%) 0 Highly different microbiome composition of BAL and sputum day 5 BAL 5 day sample ESCMIDsputum4 day eLibrary © by author RESULTS 16S rRNA sequencing 100 Actinobacteria Actinomyces Actinobacteria Atopobium Sputum versus BAL Actinobacteria Corynebacterium Actinobacteria Microbacterium Actinobacteria Propionibacterium Low-abundance taxa < 2% Actinobacteria uc_Actinomycetales (%) Bacteroidetes Alloprevotella 80 Bacteroidetes Flavobacterium Bacteroidetes Prevotella Crenarchaeota uc_Thermoprotei Cyanobacteria uc_Cyanobacteria Firmicutes Abiotrophia Firmicutes Atopostipes Firmicutes Enterococcus Firmicutes Exiguobacterium
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